研究者情報

マスター

アカウント（マスター）

• 氏名

  西村　紳一郎(ニシムラ　シンイチロウ), ニシムラ　シンイチロウ

所属（マスター）

• 先端生命科学研究院　先端融合科学研究部門　新薬探索研究分野

所属（マスター）

• 先端生命科学研究院　先端融合科学研究部門　新薬探索研究分野

独自項目

syllabus

• 2021, 生命機能制御科学特論, Functional and Regulatory Life Science, 修士課程, 生命科学院, 糖鎖生物学、糖鎖工学、糖鎖構造解析法、相互作用解析法、機能解明法、抗体医薬
• 2021, ソフトマター科学概論, Soft Matter Science, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, 生命融合科学概論, Transdisciplinary Life Science, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, Drug discovery, design, screening, developing and sales
• 2021, 応用生物有機化学, Applied Biological Organic Chemistry, 学士課程, 理学部, 生体有機化合物、炭水化物、糖、オリゴ糖、多糖、アミノ酸、ペプチド、タンパク質、触媒作用、分子内反応、酵素触媒反応、ビタミンと補酵素、補酵素と酸化還元反応、代謝（解糖系を中心として）、脂質、テルペン、ヌクレオシド、ヌクレオチド、核酸、医薬品

researchmap

プロフィール情報

所属

• 遠友ファーマ株式会社

学位

• 理学博士（1987年06月 北海道大学）

プロフィール情報

• プロフィール

  複合糖質の構造と機能の解明を通して新しい医薬品開発に貢献できればと考えております。

• 姓

  西村, ニシムラ

• 名

  紳一郎, シンイチロウ

• ID各種

  200901058006212901

対象リソース

http://altair.sci.hokudai.ac.jp/g4/index.html

所属

• 遠友ファーマ株式会社

業績リスト

研究キーワード

• 糖鎖医学   複合糖質   抗体   がん特異的抗原   有機化学   エピトープ   医化学   ケミカルバイオロジー   糖鎖工学   生物有機化学   

研究分野

• ナノテク・材料 / ケミカルバイオロジー / ナノソーム、エクソソーム、ドラッグデリバリーシステム（DDS）
• ナノテク・材料 / 生物分子化学 / ナノソーム、エクソソーム、ドラッグデリバリーシステム（DDS）
• ナノテク・材料 / 構造有機化学、物理有機化学 / ナノソーム、エクソソーム、ドラッグデリバリーシステム（DDS）
• ナノテク・材料 / ナノバイオサイエンス / ナノソーム、エクソソーム、ドラッグデリバリーシステム（DDS）
• ライフサイエンス / ゲノム生物学 / 糖鎖工学、医化学、エピトープ工学
• ライフサイエンス / 構造生物化学 / 糖鎖工学、医化学、エピトープ工学
• ライフサイエンス / 生物有機化学 / 糖鎖工学、医化学、エピトープ工学
• ライフサイエンス / 応用生物化学 / 糖鎖工学、医化学、エピトープ工学

経歴

• 2019年12月 - 現在 遠友ファーマ株式会社
• 2006年04月 - 現在 北海道大学 大学院先端生命科学研究院 新薬探索研究分野 教授
• 2012年08月 - 2022年03月 医化学創薬株式会社
• 2010年11月 - 2012年08月 医化学創薬合同会社
• 2002年06月 - 2010年03月 産業技術総合研究所 北海道センター 招聘研究員
• 1995年04月 - 2006年03月 北海道大学 大学院理学研究科 生物科学専攻 生体高分子設計学講座 教授
• 1993年04月 - 1995年03月 北海道大学 理学部生物科学科 教授
• 1993年07月 - 1994年04月 Johns Hopkins University 文部省在外研究員
• 1992年04月 - 1993年03月 北海道大学 理学部高分子学科 講師
• 1989年04月 - 1992年03月 成蹊大学 工学部工業化学科 助手
• 1988年04月 - 1989年03月 特定国立研究開発法人理化学研究所 生物有機化学研究室 特別研究生
• 1987年07月 - 1988年03月 特定国立研究開発法人理化学研究所 日本学術振興会特別研究員（PD）
• 1986年04月 - 1987年06月 北海道大学 日本学術振興会特別研究員（DC）

学歴

• 1984年04月 - 1987年06月   北海道大学   大学院理学研究科 高分子学専攻 博士後期課程
• 1982年04月 - 1984年03月   北海道大学   大学院理学研究科 高分子学専攻 修士課程
• 1978年04月 - 1982年03月   北海道大学   理学部高分子学科

受賞

• 2024年03月 公益社団法人高分子学会 令和5年度高分子科学功績賞
   複合糖質の高分子科学研究 

  受賞者: 西村 紳一郎
• 2022年02月 北海道大学 令和3年度北海道大学教育研究総長表彰
   

  受賞者: 西村紳一郎
• 2020年04月 文部科学省 令和2年度 科学技術分野の文部科学大臣表彰（科学技術賞）
   革新的創薬イノベーションを目指す糖鎖研究 

  受賞者: 西村紳一郎
• 2009年06月 内閣府 第7回産学官連携功労者表彰 日本学術会議会長賞
   大規模糖鎖解析装置及び疾患マーカー探索技術の開発 

  受賞者: 西村紳一郎
• 2008年11月 北海道新聞社 第62回北海道新聞文化賞
   

  受賞者: 西村紳一郎
• 2006年03月 公益社団法人 新化学技術推進協会 第５回グリーン・サスティナブル・ケミストリー賞 経済産業大臣賞
   人工ゴルジ装置による複合糖質の自動合成法 

  受賞者: 西村紳一郎;塩野義製薬株式会社;東洋紡績株式会社;株式会社日立ハイテクノロジーズ
• 2005年09月 科学技術振興機構、新エネルギー・産業技術総合開発機構 イノベーションジャパン2005 UBSスペシャルアワード優秀賞
   複合糖質糖鎖精製キット S-Bio BlotGlyco 

  受賞者: 西村 紳一郎
• 2004年05月 高分子学会 平成15年度 高分子学会賞
   複合糖質の合成研究 

  受賞者: 西村紳一郎
• 1999年03月 日本化学会 日本化学会 第16回学術賞
   効率的糖鎖構築法の創製と複合糖質の機能解明への応用 

  受賞者: 西村 紳一郎
• 1998年07月 日本糖質学会 第1回日本糖質学会奨励賞
   複合糖質の機能解明とその応用 

  受賞者: 西村 紳一郎
• 1997年05月 ジョンズ・ホプキンズ大学 Membership of the Johns Hopkins Society of Scholars
   西村紳一郎

論文

• Smart Nanomedicine Targeting Endocytosis Mediated by Cancer Cell Surface Neuraminidase‐1

  Ken Murakami, Daiki Kambe, Yasuhiro Yokoi, Hajime Wakui, Shun Hayakawa, Nozomi Hirane, Ryosuke Koide, Michiru Otaki, Noriko Nagahori, Shin-Ichiro Nishimura

  Advanced NanoBiomed Research 3 12 2023年11月08日 

  Human neuraminidase‐1 (NEU1) plays a much more profound function in human cancers than previously considered. It is demonstrated that cancer cell surface NEU1 is a desired gatekeeper for an innovative anticancer therapeutic nanomedicine enabling active drug‐targeting delivery by specific endocytosis into the cytoplasm. Nanosome, an antiadhesive nanoparticular shuttle, carrying multiple suicide substrates for NEU1 confers potent and universal inhibitory effects on the proliferation of human cancer cells, such as hepatocellular carcinoma (HCC) (HepG2, IC₅₀ = 13.5 nM), lung cancer (A549, IC₅₀ = 9.57 nM), and colon cancer (HT‐29, IC₅₀ = 11.1 nM), in which irreversible inactivation of cell surface NEU1 is essential for the intracellular trafficking and subsequent lysosomal membrane permeabilization by nanosomal aggregation due to the formation of “sialidase corona” through irreversible inactivation of NEU1–NEU4 residing in lysosome. Nanomedicine targeting membrane‐tethered NEU1 allows efficient delivery of hydrophobic sorafenib (Nexavar), a RAF family kinase inhibitor for the treatment of advanced renal cell carcinoma and unresectable HCC at the recommended dose of 400 mg orally twice daily, into endolysosome, resulting in a potent and sustainable inhibition (IC₅₀ = 3.1–6.2 nM at 24–96 h after coincubation) against HepG2 cell growth.
• Altering the Modular Architecture of Galectins Affects its Binding with Synthetic α‐Dystroglycan O‐Mannosylated Core M1 Glycoconjugates In situ

  Lareno L. Villones, Anna-Kristin Ludwig, Seiya Kikuchi, Rika Ochi, Shin-Ichiro Nishimura, Hans-Joachim Gabius, Herbert Kaltner, Hiroshi Hinou

  ChemBioChem 2023年03月09日 [査読有り]
  
• Structural and molecular insight into antibody recognition of dynamic neoepitopes in membrane tethered MUC1 of pancreatic cancer cells and secreted exosomes

  Hajime Wakui, Yasuhiro Yokoi, Chieko Horidome, Toyoyuki Ose, Min Yao, Yoshikazu Tanaka, Hiroshi Hinou, Shin-Ichiro Nishimura

  RSC Chemical Biology 2023年 [査読有り][通常論文]
   

  We unveil for the first time that pancreatic cancer cells (PANC-1) and secreted exosomes express MUC1 bearing cancer-relevant dynamic epitopes recognized specifically by an anti-MUC1 antibody (SN-131).
• Mouse tissue glycome atlas 2022 highlights inter-organ variation in major N-glycan profiles

  Michiru Otaki, Nozomi Hirane, Yayoi Natsume-Kitatani, Mari Nogami Itoh, Masanori Shindo, Yoichi Kurebayashi, Shin-Ichiro Nishimura

  Scientific Reports 12 1 17804  2022年10月24日 [査読有り][通常論文]
   

  Abstract This study presents “mouse tissue glycome atlas” representing the profiles of major N-glycans of mouse glycoproteins that may define their essential functions in the surface glycocalyx of mouse organs/tissues and serum-derived extracellular vesicles (exosomes). Cell surface glycocalyx composed of a variety of N-glycans attached covalently to the membrane proteins, notably characteristic “N-glycosylation patterns” of the glycocalyx, plays a critical role for the regulation of cell differentiation, cell adhesion, homeostatic immune response, and biodistribution of secreted exosomes. Given that the integrity of cell surface glycocalyx correlates significantly with maintenance of the cellular morphology and homeostatic immune functions, dynamic alterations of N-glycosylation patterns in the normal glycocalyx caused by cellular abnormalities may serve as highly sensitive and promising biomarkers. Although it is believed that inter-organs variations in N-glycosylation patterns exist, information of the glycan diversity in mouse organs/tissues remains to be elusive. Here we communicate for the first-time N-glycosylation patterns of 16 mouse organs/tissues, serum, and serum-derived exosomes of Slc:ddY mice using an established solid-phase glycoblotting platform for the rapid, easy, and high throughput MALDI-TOFMS-based quantitative glycomics. The present results elicited occurrence of the organ/tissue-characteristic N-glycosylation patterns that can be discriminated to each other. Basic machine learning analysis using this N-glycome dataset enabled classification between 16 mouse organs/tissues with the highest F1 score (69.7–100%) when neural network algorithm was used. A preliminary examination demonstrated that machine learning analysis of mouse lung N-glycome dataset by random forest algorithm allows for the discrimination of lungs among the different mouse strains such as the outbred mouse Slc:ddY, inbred mouse DBA/2Crslc, and systemic lupus erythematosus model mouse MRL-lpr/lpr with the highest F1 score (74.5–83.8%). Our results strongly implicate importance of “human organ/tissue glycome atlas” for understanding the crucial and diversified roles of glycocalyx determined by the organ/tissue-characteristic N-glycosylation patterns and the discovery research for N-glycome-based disease-specific biomarkers and therapeutic targets.
• Exploring the In situ pairing of human galectins toward synthetic O-mannosylated core M1 glycopeptides of α-dystroglycan

  Lareno L. Villones, Anna-Kristin Ludwig, Hiroyuki Kumeta, Seiya Kikuchi, Rika Ochi, Tomoyasu Aizawa, Shin-Ichiro Nishimura, Hans-Joachim Gabius, Hiroshi Hinou

  Scientific Reports 12 1 17800  2022年10月23日 [査読有り]
   

  Abstract Dystroglycan (DG), which constitutes a part of the dystrophin–glycoprotein complex, connects the extracellular matrix to the cytoskeleton. The matriglycans presented by the extracellular α-DG serve as a contact point with extracellular matrix proteins (ECM) containing laminin G-like domains, providing cellular stability. However, it remains unknown whether core M1 (GlcNAcβ1-2Man) structures can serve as ligands among the various O-Mannosylated glycans. Therefore, based on the presence of N-acetylLactosamine (LacNAc) in this glycan following the core extension, the binding interactions with adhesion/growth-regulatory galectins were explored. To elucidate this process, the interaction between galectin (Gal)-1, -3, -4 and -9 with α-DG fragment ³⁷²TRGAIIQTPTLGPIQPTRV³⁹⁰ core M1-based glycopeptide library were profiled, using glycan microarray and nuclear magnetic resonance studies. The binding of galectins was revealed irrespective of its modular architecture, adding galectins to the list of possible binding partners of α-DG core M1 glycoconjugates by cis-binding (via peptide- and carbohydrate-protein interactions), which can be abrogated by α2,3-sialylation of the LacNAc units. The LacNAc-terminated α-DG glycopeptide interact simultaneously with both the S- and F-faces of Gal-1, thereby inducing oligomerization. Furthermore, Gal-1 can trans-bridge α-DG core M1 structures and laminins, which proposed a possible mechanism by which Gal-1 ameliorates muscular dystrophies; however, this proposal warrants further investigation.
• Generation 6 polyamidoamine dendrimer provides an ideal nanoparticular platform for enzyme-assisted synthesis of glycopeptides having bulky and complex glycans

  Takahiko Matsushita, Hiroshi Hinou, Shin-Ichiro Nishimura

  Chemistry Letters 51 1044 - 1048 2022年09月16日 [査読有り]
  
• Selective reaction monitoring approach using structure-defined synthetic glycopeptides for validating glycopeptide biomarkers pre-determined by bottom-up glycoproteomics.

  Kouta Shiratori, Yasuhiro Yokoi, Hajime Wakui, Nozomi Hirane, Michiru Otaki, Hiroshi Hinou, Tohru Yoneyama, Shingo Hatakeyama, Satoshi Kimura, Chikara Ohyama, Shin-Ichiro Nishimura

  RSC advances 12 33 21385 - 21393 2022年07月21日 [査読有り]
   

  Clusterin is a heavily glycosylated protein that is upregulated in various cancer and neurological diseases. The findings by the Hancock and Iliopoulos group that levels of the tryptic glycopeptide derived from plasma clusterin, 372Leu-Ala-Asn-Leu-Thr-Gln-Gly-Glu-Asp-Gln-Tyr-Tyr-Leu-Arg385 with a biantennary disialyl N-glycan (A2G2S2 or FA2G2S2) at Asn374 differed significantly prior to and after curative nephrectomy for clear cell renal cell carcinoma (RCC) patients motivated us to verify the feasibility of this glycopeptide as a novel biomarker of RCC. To determine the precise N-glycan structure attached to Asn374, whether A2G2S2 is composed of the Neu5Acα2,3Gal or/and the Neu5Acα2,6Gal moiety, we synthesized key glycopeptides having one of the two putative isomers. Selective reaction monitoring assay using synthetic glycopeptides as calibration standards allowed "top-down glycopeptidomics" for the absolute quantitation of targeted label-free glycopeptides in a range from 313.3 to 697.5 nM in the complex tryptic digests derived from serum samples of RCC patients and healthy controls. Our results provided evidence that the Asn374 residue of human clusterin is modified dominantly with the Neu5Acα2,6Gal structure and the levels of clusterin bearing an A2G2S2 with homo Neu5Acα2,6Gal terminals at Asn374 decrease significantly in RCC patients as compared with healthy controls. The present study elicits that a new strategy integrating the bottom-up glycoproteomics with top-down glycopeptidomics using structure-defined synthetic glycopeptides enables the confident identification and quantitation of the glycopeptide targets pre-determined by the existing methods for intact glycopeptide profiling.
• Clinical utility of a serum glycome analysis in patients with colorectal cancer

  Takei D, Harada K, Nouso K, MiyaharaK, Dohi C, Matsushita H, Kinugasa H, Hiraoka S, Nishimura S-I, Okada H

  J. Gastroen. Hepatol. 37 4 727 - 733 2022年 [査読有り][通常論文]
   

  BACKGROUND AND AIM: Serum glycans are known to be good markers for the early diagnosis and prognostic prediction in many cancers. The aims of this study were to reveal the serum glycan changes comprehensively during the process of carcinogenesis from colorectal adenoma (CRA) to colorectal cancer (CRC) and to evaluate the usefulness of the glycan profiles as clinical markers for CRC. METHODS: Serum samples were obtained from 80 histologically proven CRC and 36 CRA cases. The levels of glycans in the serum were examined with a comprehensive, quantitative, high-throughput unique glycome analysis, and their diagnostic and prognostic abilities were evaluated. RESULTS: Among 34 stably detected glycans, nine were differentially expressed between CRC and CRA. Serum levels of hybrid type glycans were increased in patients with CRC compared with those with CRA (P < 0.001), and both hybrid-type and multi-antennary glycans were significantly increased in advanced cancer cases. The glycan, m/z 1914, showed the highest diagnostic value among the decreased glycans, whereas m/z 1708 showed the highest among the increased glycans. The glycan ratio m/z 1708/1914 showed a higher area under the receiver operating characteristic curve (0.889) than any other single glycan or conventional tumor marker, such as carcinoembryonic antigen (0.766, P = 0.040) and carbohydrate antigen 19-9 (0.615, P < 0.001). High m/z 1708/1914 was also correlated with an advanced cancer stage and short overall survival. CONCLUSION: Serum glycans, especially the m/z 1708/1914 ratio, were useful for the diagnosis, staging, and prognosis prediction of CRC.
• Antiadhesive nanosome elicits role of glycocalyx of tumor cell-derived exosomes in the organotropic cancer metastasis

  Koidea R, Hirane N, Kambea D, Yoko Y, Otakia M, Nishimura S-I

  Biomaterials 280 121314  2022年01月 [査読有り][通常論文]
  
• Erratum to “Cluster binding studies with two anti-Thomsen-Friedenreich (anti-core-1, CD176) antibodies: Evidence for a quadruple epitope” [Int. Immunopharmacol. 72 (2019) 186–194] (International Immunopharmacology (2019) 72 (186–194), (S1567576918308750), (10.1016/j.intimp.2019.03.058))

  A. Flechner, G. Butschak, A. Löffler, J. Rühmann, S. I. Nishimura, R. Dölling, B. Purfürst, S. Goletz, A. Danielczyk, U. Karsten

  International Immunopharmacology 93 2021年04月01日 

  The publisher regrets that Table 2 was incompletely published in the original article. The correct version of Table 2 is presented below: Table 2. Synthetic glycopeptides used as antigens. A. Synthetic glycopeptides related to anti-freeze glycoprotein (AFGP). Three sets were synthesized: unlabeled (I), labeled (II), and Thr replaced by Ser, also labeled (III). Each set consists of glycopeptides of different length (n = 1–6 or 1–5). [Table presneted] B. Synthetic glycopeptides corresponding to two sections of the extracellular asialoglycophorin (aGP) domain [12]. All are biotinylated at the C-terminus. [Table presneted]C. Synthetic glycopeptides modeling TF clusters of the extracellular domain of aGP. Numbers in brackets are synthesis numbers of Biosyntan. All are biotinylated at the C-terminus. [Table presneted] The publisher would like to apologise for any inconvenience caused.
• A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes (vol 11, pg 4999, 2020)

  Hajime Wakui, Yoshikazu Tanaka, Toyoyuki Ose, Isamu Matsumoto, Koji Kato, Yao Min, Taro Tachibana, Masaharu Sato, Kentaro Naruchi, Fayna Garcia Martin, Hiroshi Hinou, Shin-Ichiro Nishimura

  CHEMICAL SCIENCE 11 46 12588 - 12589 2020年12月 [査読有り]
   

  Correction for 'A straightforward approach to antibodies recognising cancer specific glycopeptidic neoepitopes' by Hajime Wakui et al., Chem. Sci., 2020, 11, 4999-5006, DOI: ; 10.1039/D0SC00317D.
• Analysis of the correlation between alterations in N‑glycans and invasiveness in liver cancer cell lines

  Hidenori Takahashi, Toshiya Kamiyama, Nozomi Hirane, Nozomi Kobayashi, Takeshi Aiyama, Akihisa Nagatsu, Shingo Shimada, Tatsuya Orimo, Tatsuhiko Kakisaka, Moto Fukai, Hideki Yokoo, Hirofumi Kamachi, Shin‐Ichiro Nishimura, Akinobu Taketomi

  Oncology Reports 44 6 2757 - 2769 2020年10月08日 [査読有り][通常論文]
   

  The N‑glycoforms of glycoproteins modify protein function and control a number of biological pathways. The aim of the present study was to investigate the correlation between alterations in N‑glycans and cancer aggressiveness in terms of cancer cell invasion ability. The expression of urokinase‑type plasminogen activator (uPA) and N‑acetylglucosaminyltransferase V (GnT‑V) in liver cancer cell lines was analyzed by western blotting. Cell invasiveness was analyzed by Matrigel invasion assays. uPA and GnT‑V expression in liver cancer cell lines was knocked down by RNA interference. Furthermore, uPA was overexpressed in liver cancer cells using lentiviral vectors, and a mutant strain of HepG2 cells overexpressing uPA deficient in N‑glycans was established. A glycoblotting‑assisted matrix‑assisted laser desorption/ionization‑time‑of‑flight/mass spectrometry‑based quantitative analysis of liver cancer cell lines was performed, in which invasiveness was altered by modifying the expression of uPA and GnT‑V. N‑glycan profiles were found to differ between the highly invasive liver cancer cell line HLE and the less invasive cell line HepG2. The expression of several N‑glycans, including a form with m/z=1892, was changed according to invasiveness controlled by knockdown and overexpression of uPA. The invasiveness of HepG2 cells with mutant uPA did not increase regardless of the level of expression of uPA. Following GnT‑V knockdown and N‑glycan alteration, uPA expression did not change, whereas cell invasiveness decreased. One N‑glycan (m/z=1892) was common among N‑glycans in the comparative analysis between HLE and HepG2, HLE and uPA knockdown HLE, HepG2 and uPA‑overexpressing HepG2, and HLE and GnT‑V knockdown HLE cells and among N‑glycan profiles in human uPA. Therefore, N‑glycosylation is an important factor controlling invasiveness of liver cancer cells, and a specific N‑glycan (m/z=1892) associated with the invasion of liver cancer cells via uPA was identified in the present study.
• Effect of Site‐Specific O ‐Glycosylation on the Structural Behavior of NOTCH1 Receptor Extracellular EGF‐like Domains 11 and 10

  Yasuhiro Yokoi, Shin‐Ichiro Nishimura

  Chemistry – A European Journal 26 54 12363 - 12372 2020年09月25日 [査読有り][通常論文]
  
• Amplified Detection of Breast Cancer Autoantibodies Using MUC1-Based Tn Antigen Mimics

  Pablo A. Guillen-Poza, Elena M. Sánchez-Fernández, Gerard Artigas, José Manuel García Fernández, Hiroshi Hinou, Carmen Ortiz Mellet, Shin-Ichiro Nishimura, Fayna Garcia-Martin

  Journal of Medicinal Chemistry 63 15 8524 - 8533 2020年08月13日 [査読有り][通常論文]
   

  In many human carcinomas, mucin-1 (MUC1) is overexpressed and aberrantly glycosylated, resulting in the exposure of previously hidden antigens. This generates new patient antibody profiles that can be used in cancer diagnosis. In the present study, we focused on the MUC1-associated Tn antigen (α-O-GalNAc-Ser/Thr) and substituted the GalNAc monosaccharide by a glycomimic to identify MUC1-based glycopeptides with increased antigenicity. Two different glycopeptide libraries presenting the natural Tn antigen or the sp2-iminosugar analogue were synthesized and evaluated with anti-MUC1 monoclonal antibodies in a microarray platform. The most promising candidates were tested with healthy and breast cancer sera aiming for potential autoantibody-based biomarkers. The suitability of sp2-iminosugar glycopeptides to detect anti-MUC1 antibodies was demonstrated, and serological experiments showed stage I breast cancer autoantibodies binding with a specific unnatural glycopeptide with almost no healthy serum interaction. These results will promote further studies on their capabilities as early cancer biomarkers.
• Impaired O-Glycosylation at Consecutive Threonine TTX Motifs in Mucins Generates Conformationally Restricted Cancer Neoepitopes.

  Shun Hayakawa, Takahiko Matsushita, Yasuhiro Yokoi, Hajime Wakui, Fayna Garcia-Martin, Hiroshi Hinou, Koji Matsuoka, Kazuhiro Nouso, Toshiya Kamiyama, Akinobu Taketomi, Shin-Ichiro Nishimura

  Biochemistry 59 12 1221 - 1241 2020年03月31日 [査読有り][通常論文]
   

  Autoantibody signatures of circulating mucin fragments stem from cancer tissues, and microenvironments are promising biomarkers for cancer diagnosis and therapy. This study highlights dynamic epitopes generated by aberrantly truncated immature O-glycosylation at consecutive threonine motifs (TTX) found in mucins and intrinsically disordered proteins (IDPs). NMR analysis of synthetic mucin models having glycosylated TTX motifs and colonic MUC2 tandem repeats (TRs) containing TTP and TTL moieties unveils a general principle that O-glycosylation at TTX motifs generates a highly extended and rigid conformation in IDPs. We demonstrate that the specific conformation of glycosylated TTX motifs in MUC2 TRs is rationally rearranged by concerted motions of multiple dihedral angles and noncovalent interactions between the carbohydrate and peptide region. Importantly, this canonical conformation of glycosylated TTX motifs minimizes steric crowding of glycans attached to threonine residues, in which O-glycans possess restricted orientations permitting further sugar extension. An antiadhesive microarray displaying synthetic MUC2 derivatives elicited the presence of natural autoantibodies to MUC2 with impaired O-glycosylation at TTX motifs in sera of healthy volunteers and patients diagnosed with early stage colorectal cancer (CRC). Interestingly, autoantibody levels in sera of the late stage CRC patients were distinctly lower than those of early stage CRC and normal individuals, indicating that the anti-MUC2 humoral response to MUC2 neoepitopes correlates inversely with the CRC stage of patients. Our results uncovered the structural basis of the creation of dynamic epitopes by immature O-glycosylation at TTX motifs in mucins that facilitates the identification of high-potential targets for cancer diagnosis and therapy.
• Serum N-glycan profiling can predict biopsy-proven graft rejection after living kidney transplantation.

  Osamu Soma, Shingo Hatakeyama, Tohru Yoneyama, Mitsuru Saito, Hideo Sasaki, Yuki Tobisawa, Daisuke Noro, Yuichiro Suzuki, Masakazu Tanaka, Shin-Ichiro Nishimura, Hiroshi Harada, Hideki Ishida, Kazunari Tanabe, Shigeru Satoh, Chikara Ohyama

  Clinical and experimental nephrology 24 2 174 - 184 2020年02月 [査読有り][通常論文]
   

  BACKGROUND: To evaluate whether serum N-glycan profile can be used as a diagnostic marker of graft rejection after living-donor kidney transplants (KT). METHODS: We retrospectively examined 174 KT recipients at five medical centers. N-Glycan levels were analyzed in postoperative serum samples using glycoblotting combined with mass spectrometry. We developed an integrated score to predict graft rejection based on a combination of age, gender, immunological risk factors, and serum N-glycan levels at post-KT day D1 and D7. Rejection-free survival rates stratified by the sum of integrated scores (D1 + D7) were evaluated using Kaplan-Meier curves. RESULTS: Of 174, 52 showed graft rejection (Rejection-pos. group) and 122 recipients did not show graft rejection (Rejection-neg. group). The integrated scores were significantly higher in the Rejection-pos. group than those of the Rejection-neg. group. Area-under-curve (AUC) value of integrated scores at post-KT D1, and D7 were 0.84 and 0.84, respectively. The sum of integrated scores (D1 + D7) ≥ 0.50 identified graft rejection with 81% sensitivity and 80% specificity; with an AUC value of 0.87. Recipients with higher sum of integrated scores (D1 + D7 ≥ 0.5) had significantly shorter rejection-free survival than those with lower scores. CONCLUSION: Evaluation of serum N-glycosylation profiles can identify recipients who are prone to rejection.
• Synthesis, conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment

  Iris A. Bermejo, Claudio D. Navo, Jorge Castro-López, Ana Guerreiro, Ester Jiménez-Moreno, Elena M. Sánchez Fernández, Fayna García-Martín, Hiroshi Hinou, Shin-Ichiro Nishimura, José M. García Fernández, Carmen Ortiz Mellet, Alberto Avenoza, Jesús H. Busto, Gonçalo J. L. Bernardes, Ramón Hurtado-Guerrero, Jesús M. Peregrina, Francisco Corzana

  Chemical Science 11 15 3996 - 4006 2020年 [査読有り][通常論文]
   

  An anti-cancer vaccine based on an unnatural antigen with an sp²-iminosugar fragment.

• Serum N-glycan profiling is a potential biomarker for castration-resistant prostate cancer

  Teppei Matsumoto, Shingo Hatakeyama, Tohru Yoneyama, Yuki Tobisawa, Yusuke Ishibashi, Hayato Yamamoto, Takahiro Yoneyama, Yasuhiro Hashimoto, Hiroyuki Ito, Shin-Ichiro Nishimura, Chikara Ohyama

  Scientific Reports 9 1 2019年12月 [査読有り][通常論文]
  
• Antiadhesive Nanosomes Facilitate Targeting of the Lysosomal GlcNAc Salvage Pathway through Derailed Cancer Endocytosis

  Ryosuke Koide, Shin‐Ichiro Nishimura

  Angewandte Chemie International Edition 58 41 14513 - 14518 2019年10月07日 [査読有り][通常論文]
  
• Glycan-immobilized dual-channel field effect transistor biosensor for the rapid identification of pandemic influenza viral particles.

  Hideshima S, Hayashi H, Hinou H, Nambuya S, Kuroiwa S, Nakanishi T, Momma T, Nishimura SI, Sakoda Y, Osaka T

  Scientific reports 9 1 11616 - 11616 2019年08月12日 [査読有り][通常論文]
   

  Pandemic influenza, triggered by the mutation of a highly pathogenic avian influenza virus (IFV), has caused considerable damage to public health. In order to identify such pandemic IFVs, antibodies that specifically recognize viral surface proteins have been widely used. However, since the analysis of a newly discovered virus is time consuming, this delays the availability of suitable detection antibodies, making this approach unsuitable for the early identification of pandemic IFVs. Here we propose a label-free semiconductor-based biosensor functionalized with sialic-acid-containing glycans for the rapid identification of the pandemic IFVs present in biological fluids. Specific glycans are able to recognize wild-type human and avian IFVs, suggesting that they are useful in discovering pandemic IFVs at the early stages of an outbreak. We successfully demonstrated that a dual-channel integrated FET biosensing system, which were modified with 6'-sialyllactose and 3'-sialyllactose for each gate area, can directly and specifically detect human H1N1 and avian H5N1 IFV particles, respectively, present in nasal mucus. Furthermore, to examine the possibility of identifying pandemic IFVs, the signal attributed to the detection of Newcastle disease virus (NDV) particles, which was selected as a prime model of a pandemic IFV, was clearly observed from both sensing gates. Our findings suggest that the proposed glycan-immobilized sensing system could be useful in identifying new pandemic IFVs at the source of an outbreak.
• Cluster binding studies with two anti-Thomsen-Friedenreich (anti-core-1, CD176, TF) antibodies: Evidence for a multiple TF epitope

  A. Flechner, G. Butschak, A. Löffler, J. Rühmann, S.-I. Nishimura, R. Dölling, B. Purfürst, S. Goletz, A. Danielczyk, U. Karsten

  International Immunopharmacology 72 186 - 194 2019年07月 [査読有り][通常論文]
  
• Synthetic glycopeptides reveal specific binding pattern and conformational change at O-mannosylated position of alpha-dystroglycan by POMGnT1 catalyzed GlcNAc modification

  Hinou Hiroshi, Kikuchi Seiya, Ochi Rika, Igarashi Kota, Takada Wataru, Nishimura Shin-Ichiro

  BIOORGANIC & MEDICINAL CHEMISTRY 27 13 2822 - 2831 2019年07月01日 [査読有り][通常論文]
  
• Exploring serum and immunoglobulin G N-glycome as diagnostic biomarkers for early detection of breast cancer in Ethiopian women.

  Gebrehiwot AG, Melka DS, Kassaye YM, Gemechu T, Lako W, Hinou H, Nishimura SI

  BMC cancer 19 1 588  2019年06月 [査読有り][通常論文]
  
• Glycoblotting of Egg White Reveals Diverse N-Glycan Expression in Quail Species.

  Sanes JT, Hinou H, Lee YC, Nishimura SI

  Journal of agricultural and food chemistry 67 1 531 - 540 2019年01月 [査読有り][通常論文]
   

  The glycan part of glycoproteins is known to be involved in the structure and modulatory functions of glycoproteins, serving as ligands for cell-to-cell interactions, and as specific ligands for cell-to-microbe interactions. It is believed that intraspecies and interspecies variations in glycosylation exist. As an approach to better understand glycan diversity, egg whites (EW) from four different quail species are studied by the well-established glycoblotting procedure, a glycan enrichment and analysis method. N-Glycans were classified and the profiles were established for quail egg white samples which showed 21 relevant glycan peaks; 18 peaks were expressed significantly, and 10 glycan peaks are found to be abundant in certain species. The result establishes glycan profiles for Blue Scaled, Bobwhite, Japanese, and Mountain Quail egg whites and shows a unique difference among glycan expressions, particularly, high mannose in Japanese Quail and tetra-antennary glycan structure for other quail species.
• Synthetic Glycopeptides Allow for the Quantitation of Scarce Nonfucosylated IgG Fc N-Glycans of Therapeutic Antibody.

  Hammura K, Ishikawa A, H V RK, Miyoshi R, Yokoi Y, Tanaka M, Hinou H, Nishimura SI

  ACS medicinal chemistry letters 9 9 889 - 894 2018年09月 [査読有り][通常論文]
  
• Synthetic glycopeptides as a designated standard in focused glycoproteomics to discover serum cancer biomarkers.

  Yogesh KV, Kamiyama T, Ohyama C, Yoneyama T, Nouso K, Kimura S, Hinou H, Nishimura SI

  MedChemComm 9 8 1351 - 1358 2018年08月 [査読有り][通常論文]
   

  Previous studies on the large-scale glycomics of more than 3500 human serum samples revealed that the serum glycoproteins of cancer patients often have more dominant and specific glycoforms, namely, branched tri- and tetra-antennary N-glycans, most cancer patient groups than normal control groups. We herein established an efficient synthetic protocol of glycopeptides having highly complicated N-glycan structures that may be generated by direct tryptic digestion of serum glycoproteins. A preliminary selected reaction monitoring (SRM) assay using the synthetic model glycopeptide 1, (40)Ser-Val-Gln-Glu-Ile-Gln-Ala-Thr-Phe-Phe-Tyr-Phe-Thr-Pro-Asn-Lys-Thr-Glu-Asp-Thr-Ile-Phe-Leu-Arg(63) having an asialo tri-antennary N-glycan at the Asn54 residue as a designated calibration standard allowed for the rapid and absolute quantitation of the tryptic fragment derived from the serum alpha 1-acid glycoprotein carrying a focused N-glycoform of cancer patients and healthy controls in a range between 200 and 1600 fmole mu L-1 without any enrichment process for the target glycoprotein.
• 網羅的糖鎖解析による肝細胞癌の浸潤能と糖鎖異常の検討

  高橋 秀徳, 神山 俊哉, 柿坂 達彦, 相山 健, 若山 顕治, 折茂 達也, 蒲池 浩文, 横尾 英樹, 西村 紳一郎, 武冨 紹信

  日本消化器外科学会総会 73回 685 - 685 (一社)日本消化器外科学会 2018年07月
• New biomarker for castration-resistant prostate cancer: A glycobiological perspective

  Shingo Hatakeyama, Tohru Yoneyama, Hayato Yamamoto, Yuki Tobisawa, Shin-Ichiro Nishimura, Chikara Ohyama

  Hormone Therapy and Castration Resistance of Prostate Cancer 307 - 315 2018年05月11日 

  Cancer-associated glycan aberrations are frequently observed in tumors. Glycan alterations are reported to have potential as cancer biomarkers. An aberrant glycosylation and glycosyl epitope have been known to be tumor-associated antigens. In addition, changes in glycosyltransferase could be associated with the signal pathways. Detecting in aberrant glycosylation on prostate-specific antigen (α2,3-linked sialylation) may improve specificity in detection of prostate cancer. However, a practical procedure to analyze a large number of glycan samples quickly is not available for serum due to methodological problems. Recent progress in mass spectrometry has led to new challenges in glycan analysis including a chemo-selective glycan enrichment technology called glycoblotting to purify oligosaccharides from a crude glycoprotein mixture. Previous study suggested that triand tetra-antennary N-glycans were significantly higher in CRPC patients than in non-CRPC patients, and the expression of N-glycan branching enzyme genes was significantly upregulated in CRPC cell lines. These results suggest that the overexpression of triand tetra-antennary N-glycans may be associated with the castration-resistant status in prostate cancer and may be a potential predictive biomarker for CRPC. The incorporation of glycan biomarkers appears to be a promising approach for improving CRPC detection.
• CLEC10A Is a specific marker for human CD1c+ dendritic cells and enhances their toll-like receptor 7/8-Induced cytokine secretion

  Lukas Heger, Silke Balk, Jennifer J. Lühr, Gordon F. Heidkamp, Christian H.K. Lehmann, Lukas Hatscher, Ariawan Purbojo, Arndt Hartmann, Fayna Garcia-Martin, Shin-Ichiro Nishimura, Robert Cesnjevar, Falk Nimmerjahn, Diana Dudziak

  Frontiers in Immunology 9 744  2018年04月27日 [査読有り][通常論文]
   

  Dendritic cells (DCs) are major players for the induction of immune responses. Apart from plasmacytoid DCs (pDCs), human DCs can be categorized into two types of conventional DCs: CD141+ DCs (cDC1) and CD1c+ DCs (cDC2). Defining uniquely expressed surface markers on human immune cells is not only important for the identification of DC subpopulations but also a prerequisite for harnessing the DC subset-specific potential in immunomodulatory approaches, such as antibody-mediated antigen targeting. Although others identified CLEC9A as a specific endocytic receptor for CD141+ DCs, such a receptor for CD1c+ DCs has not been discovered, yet. By performing transcriptomic and flow cytometric analyses on human DC subpopulations from different lymphohematopoietic tissues, we identified CLEC10A (CD301, macrophage galactose-type C-type lectin) as a specific marker for human CD1c+ DCs. We further demonstrate that CLEC10A rapidly internalizes into human CD1c+ DCs upon binding of a monoclonal antibody directed against CLEC10A. The binding of a CLEC10A-specific bivalent ligand (the MUC-1 peptide glycosylated with N-acetylgalactosamine) is limited to CD1c+ DCs and enhances the cytokine secretion (namely TNFα, IL-8, and IL-10) induced by TLR 7/8 stimulation. Thus, CLEC10A represents not only a candidate to better define CD1c+ DCs-due to its high endocytic potential-CLEC10A also exhibits an interesting candidate receptor for future antigen-targeting approaches.
• Healthy human serum N-glycan profiling reveals the influence of ethnic variation on the identified cancer-relevant glycan biomarkers.

  Gebrehiwot AG, Melka DS, Kassaye YM, Rehan IF, Rangappa S, Hinou H, Kamiyama T, Nishimura SI

  PloS one 13 12 e0209515  2018年 [査読有り][通常論文]
  
• Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells.

  Ishihara S, Aoki K, Mizutani T, Amano M, Nishimura SI, Haga H

  Cell structure and function 43 2 177 - 185 2018年 [査読有り][通常論文]
  
• Aberrant N-Glycosylation Profile of Serum Immunoglobulins is a Diagnostic Biomarker of Urothelial Carcinomas.

  Toshikazu Tanaka, Tohru Yoneyama, Daisuke Noro, Kengo Imanishi, Yuta Kojima, Shingo Hatakeyama, Yuki Tobisawa, Kazuyuki Mori, Hayato Yamamoto, Atsushi Imai, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Masakazu Tanaka, Shin-Ichiro Nishimura, Shizuka Kurauchi, Ippei Takahashi, Chikara Ohyama

  International journal of molecular sciences 18 12 2017年12月06日 [査読有り][通常論文]
   

  The aim of this study to determine whether the aberrant N-glycosylated serum immunoglobulins (Igs) can be applied as a diagnostic marker of urothelial carcinoma (UC). Between 2009 and 2016, we randomly obtained serum available from 237 UC and also 96 prostate cancer as other cancer controls from our serum bank and also obtained-from 339 healthy volunteers (HV)-controls obtained from community-dwelling volunteers in Iwaki Health Promotion Project. A total of 32 types of N-glycan levels on Igs were determined by high-throughput N-glycomics and analyzed by multivariable discriminant analysis. We found five UC-associated aberrant N-glycans changes on Igs and also found that asialo-bisecting GlcNAc type N-glycan on Igs were significantly accumulated in UC patients. The diagnostic N-glycan Score (dNGScore) established by combination of five N-glycans on Igs discriminated UC patients from HV and prostate cancer (PC) patients with 92.8% sensitivity and 97.2% specificity. The area under the curve (AUC) for of the dNGScore was 0.969 for UC detection that was much superior to that of urine cytology (AUC, 0.707) and hematuria (AUC, 0.892). Furthermore, dNGScore can detect hematuria and urine cytology negative patients. The dNGscore based on aberrant N-glycosylation signatures of Igs were found to be promising diagnostic biomarkers of UCs.
• The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer

  Victor J. Somovilla, Iris A. Bermejo, Ines S. Albuquerque, Nuria Martinez-Saez, Jorge Castro-Lopez, Fayna Garcia-Martin, Ismael Companon, Hiroshi Hinou, Shin-Ichiro Nishimura, Jesus Jimenez-Barbero, Juan L. Asensio, Alberto Avenoza, Jesus H. Busto, Ramon Hurtado-Guerrero, Jesus M. Peregrina, Goncalo J. L. Bernardes, Francisco Corzana

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 139 50 18255 - 18261 2017年12月 [査読有り][通常論文]
   

  A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-L-proline or 4,4-difluoro-L-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues' stabilize the antigen-antibody complex by enhancing key CH/pi interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.
• Glycopeptides as Targets for Dendritic Cells: Exploring MUC1 Glycopeptides Binding Profile toward Macrophage Galactose-Type Lectin (MGL) Orthologs

  Gerard Artigas, Joao T. Monteiro, Hiroshi Hinou, Shin-Ichiro Nishimura, Bernd Lepenies, Fayna Garcia-Martin

  JOURNAL OF MEDICINAL CHEMISTRY 60 21 9012 - 9021 2017年11月 [査読有り][通常論文]
   

  The macrophage galactose-type lectin (MGL) recognizes glycan moieties exposed by pathogens. and malignant cells. Particularly, mucin-1 (MUC1) glycoprotein presents an altered glycosylation in several cancers. To estimate the ability of distinct MGL orthologs to recognize aberrant glycan cores in mucins, we applied evanescent-field detection to a versatile MUC1-like glycopeptide microarray platform. Here, as binding was sequence-dependent, we demonstrated that not only sugars but also peptide region impact the recognition of murine MGL1 (raMGL1). In addition, we observed for all three MGL orthologs that divalent glycan presentation increased the binding. To assess the utility of the glycopeptide binders of the MGL orthologs for MGL targeting, we performed uptake assays with fluorescein-MUC1 using murine dendritic cells. A diglycosylated MUC1 peptide was preferentially internalized in an MGL-dependent fashion, thus showing the utility for divalent MGL targeting. These findings may be relevant to a rational design of antitumor vaccines targeting dendritic cells via MGL.
• An Efficient Glycoblotting-Based Analysis of Oxidized Lipids in Liposomes and a Lipoprotein

  Takayuki Furukawa, Hiroshi Hinou, Seiji Takeda, Hitoshi Chiba, Shin-Ichiro Nishimura, Shu-Ping Hui

  CHEMBIOCHEM 18 19 1903 - 1909 2017年10月 [査読有り][通常論文]
   

  Although widely occurring lipid oxidation, which is triggered by reactive oxygen species (ROS), produces a variety of oxidized lipids, practical methods to efficiently analyze oxidized lipids remain elusive. Herein, it is shown that the glycoblotting platform can be used to analyze oxidized lipids. Analysis is based on the principle that lipid aldehydes, one of the oxidized lipid species, can be captured selectively, enriched, and detected. Moreover, 3-methyl-1-p-tolyltriazene (MTT) methylates phosphoric and carboxylic acids, and this MTT-mediated methylation is, in combination with conventional tandem mass spectrometry (MS/MS) analysis, an effective method for the structural analysis of oxidized lipids. By using three classes of standards, liposomes, and a lipoprotein, it is demonstrated that glycoblotting represents a powerful approach for focused lipidomics, even in complex macromolecules.
• Chemical Synthesis Demonstrates That Dynamic O-Glycosylation Regulates the Folding and Functional Conformation of a Pivotal EGF12 Domain of the Human NOTCH1 Receptor

  Shun Hayakawa, Yasuhiro Yokoi, Hiroshi Hinou, Shin-Ichiro Nishimura

  BIOCHEMISTRY 56 33 4379 - 4391 2017年08月 [査読有り][通常論文]
   

  The interaction of the human NOTCH1 receptor and its ligands is a crucial step in initiating the intracellular signal transductions, in which O-glycosylation of the extracellular EGF-like domain strongly affects multiple aspects of cell differentiation, development, and, cancer biology. However, consequences of biosynthetic O-glycosylation processes in the endoplasmic reticulum: (ER) and Golgi I on the folding of EGF domains remain unclear. Synthetic I human NOTCH1 EGF12 modules allow for new insight into, the crucial roles of O-glycosylation in the folding and conformation of this pivotal domain. Here, we show - for the, first time that predominant O-glucosylation at Ser458 facilitates proper folding of the EGF12 domain in the presence of calcium. ion, while the nonglycosylated linear EGF12 peptide affords large amounts of misfolded products (>50%) during in vitro oxidative folding. Strikingly, O-fucosylation at Thr466 prior to O-glucosylation at Ser458 totally impedes folding of EGF12 independent of calcium ion, whereas modification of the Fucal -> moiety with beta-linked GlcNAC dramatically. enhances folding effitiency. In addition,. we elicit that extension of the Glc beta 1 -> moiety with xyloses is a negative-regrilation mechanism in the folding of EGF12 when synthesis, of a. trisaccharide (Xylal -> 3Xyla1- 3G1c beta 1 ->) dominates over the posttrarislational modification at Thr466. Comprehensive nuclear magnetic resonance studies of correctly folded EGF12 modules demonstrate that noncovalently bonded bridges, between sugars and, peptide moieties, namely sugar bridges, contribute independently to the stabilization of the antiparallel beta-sheet in the ligand-binding region. Our results provide evidence that the dynamic O-glycosylation status of the EGF12 domain elaborated in the ER and Golgi strongly affects folding and trafficking of the human. NOTCH1 receptor.
• Serum Aberrant N-Glycan Profile as a Marker Associated with Early Antibody-Mediated Rejection in Patients Receiving a Living Donor Kidney Transplant

  Daisuke Noro, Tohru Yoneyama, Shingo Hatakeyama, Yuki Tobisawa, Kazuyuki Mori, Yasuhiro Hashimoto, Takuya Koie, Masakazu Tanaka, Shin-Ichiro Nishimura, Hideo Sasaki, Mitsuru Saito, Hiroshi Harada, Tatsuya Chikaraishi, Hideki Ishida, Kazunari Tanabe, Shigeru Satoh, Chikara Ohyama

  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 18 8 2017年08月 [査読有り][通常論文]
   

  We determined if the serum N-glycan profile can be used as a diagnostic marker of antibody-mediated rejection (ABMR) in living donor kidney transplant (LKTx) recipients. Glycoblotting, combined with mass spectrometry, was used to retrospectively examine N-glycan levels in the postoperative sera of 197 LKTx recipients of whom 16 recipients had ABMR with or without T-cell-mediated rejection (TCMR), 40 recipients had TCMR, and 141 recipients had no adverse events. Multivariate discriminant analysis for prediction of ABMR was performed by inputting an ABMR event as an explanatory variable and sex, age, and serum N-glycan level as objective variables. The N-glycan score was calculated by multiplying the level of candidate objective variables by objective function values. The ABMR predictive performance of the N-glycan score was assessed by receiver operator characteristic curve and Kaplan-Meier curve analyses. The N-glycan score discriminated ABMR with 81.25% sensitivity, 87.85% specificity, and an area under the curve (AUC) of 0.892 that was far superior to that of preformed donor-specific antibody status (AUC, 0.761). Recipients with N-glycan-positive scores >0.8770 had significantly shorter ABMR survival than that of recipients with N-glycan-negative scores. Although the limitations of our study includ its small sample size and retrospective nature, the serum N-glycan score may contribute to prediction of ABMR.
• Comparative Glycomics of Fat Globule Membrane Glycoconjugates from Buffalo (Bubalus bubalis) Milk and Colostrum

  Nagaraju Brijesha, Shin-Ichiro Nishimura, Huligerepura Sosalegowda Aparna

  Journal of Agricultural and Food Chemistry 65 8 1496 - 1506 2017年03月01日 [査読有り][通常論文]
   

  The health-promoting effects of milk fat globule membrane (MFGM) glycoconjugates has attracted curiosity especially with regard to the challenges encountered to unravel the glycan complexities of MFGM glycoproteins and glycosphingolipids. In this context, we characterized glycans present in buffalo milk and colostrum fat globule membranes by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis by adopting chemoselective glycoblotting technique. Unlike human and bovine MFGM glycoproteins, the variations were obvious with respect to their number, size, heterogeneity, and abundance among the samples analyzed. Among N-linked glycans, mono-, di-, and trisialyl glycans were apparent in colostrum, while MFGM predominantly contained mono- and disialyl glycans, in addition to neutral and high-mannose glycoforms. The structural assignments of major glycans were confirmed by TOF/TOF analysis. Core 1 O-glycans were more common in both samples, and the major glycosphingolipids were GM3 and GD3 irrespective of the samples analyzed. The colostrum N-glycans, being effective antibacterials against human pathogens, established the structure-function relationship of oligosaccharides in early milk in providing innate protection to the newborn.
• Generation of Novel Anti-MUC1 Monoclonal Antibodies with Designed Carbohydrate Specificities Using MUC1 Glycopeptide Library

  Shoichi Naito, Tatsuya Takahashi, Junji Onoda, Shoko Uemura, Naoki Ohyabu, Hiroshi Takemoto, Shoji Yamane, Ikuo Fujii, Shin-Ichiro Nishimura, Yoshito Numata

  ACS Omega 2 11 7493 - 7505 2017年 [査読有り][通常論文]
   

  Numerous anti-mucin 1 (anti-MUC1) antibodies that recognize O-glycan core structures have already been developed. However, most of them show low specificities toward O-glycan structures and/or low affinity toward a monovalent epitope. In this study, using an MUC1 glycopeptide library, we established two novel anti-MUC1 monoclonal antibodies (1B2 and 12D10) with designed carbohydrate specificities. Compared with previously reported anti-MUC1 antibodies, 1B2 and 12D10 showed quite different features regarding their specificities, affinities, and reactivity profiles to various cell lines. Both antibodies recognized specific O-glycan structures at the PDT∗R motif (the asterisk represents an O-glycosylation site). 1B2 recognized O-glycans with an unsubstituted O-6 position of the GalNAc residue (Tn, T, and 23ST), whereas 12D10 recognized Neu5Ac at the same position (STn, 26ST, and dST). Neither of them bound to glycopeptides with core 2 O-glycans that have GlcNAc at the O-6 position of the GalNAc residue. Furthermore, 1B2 and 12D10 showed a strong binding to not only native MUC1 but also 20-mer glycopeptide with a monovalent epitope. These anti-MUC1 antibodies should thus become powerful tools for biological studies on MUC1 O-glycan structures. Furthermore, the strategy of using glycopeptide libraries should enable the development of novel antibodies with predesigned O-glycan specificities.
• Synthetic Mucin-Like Glycopeptides as Versatile Tools to Measure Effects of Glycan Structure/Density/Position on the Interaction with Adhesion/Growth-Regulatory Galectins in Arrays

  Gerard Artigas, Hiroshi Hinou, Fayna Garcia-Martin, Hans-Joachim Gabius, Shin-Ichiro Nishimura

  CHEMISTRY-AN ASIAN JOURNAL 12 1 159 - 167 2017年01月 [査読有り][通常論文]
   

  Functional pairing of cellular glycoconjugates with tissue lectins is a highly selective process, whose determinative factors have not yet been fully delineated. Glycan structure and modes of presentation, that is, its position and density, can contribute to binding, as different members of a lectin family can regulate degrees of responsiveness to these factors. Using a peptide repeat sequence motif of the glycoprotein mucin-1, the principle of introducing synthetic (glyco) peptides with distinct variations in these three parameters to an array-based screening of tissue lectins is illustrated. Interaction profiles of seven adhesion/growth-regulatory galectins cover the range from intense signals with core 2 pentasaccharides and core 1 binding for galectins-3 and -5 to a lack of binding for galectin-1 and also the galectin-related protein, which was included as a negative control. Remarkably, the two tandem-repeat-type galectins-4 and -8 were distinguished by core 1 sialylation, as the two separated domains were. These results encourage further synthetic elaboration of the glycopeptide library and testing of the network of natural galectins and rationally engineered variants of the lectins.
• Glycoblotting method allows for rapid and efficient glycome profiling of human Alzheimer's disease brain, serum and cerebrospinal fluid towards potential biomarker discovery

  Solomon T. Gizaw, Tetsu Ohashi, Masakazu Tanaka, Hiroshi Hinou, Shin-Ichiro Nishimura

  BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1860 8 1716 - 1727 2016年08月 [査読有り][通常論文]
   

  Background: Understanding of the significance of posttranslational glycosylation in Alzheimer's disease (AD) is of growing importance for the investigation of the pathogenesis of AD as well as discovery research of the disease specific serum biomarkers. Methods: We designed a standard protocol for the glycoblotting combined with MALDI-TOFMS to perform rapid and quantitative profiling of the glycan parts of glycoproteins (N-glycans) and glycosphingolipids (GSLs) using human AD's post-mortem samples such as brain tissues (dissected cerebral cortices such as frontal, parietal, occipital, and temporal domains), serum and cerebrospinal fluid (CSF). Results: The structural profiles of the major N-glycans released from glycoproteins and the total expression levels of the glycans were found to be mostly similar between the brain tissues of the AD patients and those of the normal control group. In contrast, the expression levels of the serum and CSF protein N-glycans such as bisect-type and multiply branched glycoforms were increased significantly in AD patient group. In addition, the levels of some gangliosides such as GM1, GM2 and GM3 appeared to alter in the AD patient brain and serum samples when compared with the normal control groups. Conclusion: Alteration of the expression levels of major N- and GSL-glycans in human brain tissues, serum and CSF of AD patients can be monitored quantitatively by means of the glycoblotting-based standard protocols. General significance: The changes in the expression levels of the glycans derived from the human post-mortem samples uncovered by the standardized glycoblotting method provides potential serum biomarkers in central nervous system disorders and can contribute to the insight into the molecular mechanisms in the pathogenesis of neurodegenerative diseases and future drug discovery. Most importantly, the present preliminary trials using human post-mortem samples of AD patients suggest that large-scale serum glycomics cohort by means of various-types of human AD patients as well as the normal control sera can facilitate the discovery research of highly sensitive and reliable serum biomarkers for an early diagnosis of AD.. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. (C) 2016 Elsevier B.V. All rights reserved.
• N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice

  Kaneko Kentaro, Takamatsu Takeshi, Inomata Takuya, Oikawa Kazusato, Itoh Kimiko, Hirose Kazuko, Amano Maho, Nishimura Shin-Ichiro, Toyooka Kiminori, Matsuoka Ken, Pozueta-Romero Javier, Mitsui Toshiaki

  PLANT AND CELL PHYSIOLOGY 57 8 1610 - 1628 2016年08月 [査読有り][通常論文]
   

  Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6 Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.
• Inhibitory potential of Buffalo (Bubalus bubalis) colostrum immunoglobulin G on Klebsiella pneumoniae

  Mamatha Bhanu L.S., S.-I. Nishimura, Aparna H.S.

  International Journal of Biological Macromolecules 88 138 - 145 2016年07月 [査読有り][通常論文]
  
• Convergent Solid-Phase Synthesis of Macromolecular MUC1 Models Truly Mimicking Serum Glycoprotein Biomarkers of Interstitial Lung Diseases

  Naoki Ohyabu, Kiyoshi Kakiya, Yasuhiro Yokoi, Hiroshi Hinou, Shin-Ichiro Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 138 27 8392 - 8395 2016年07月 [査読有り][通常論文]
   

  Synthetic macromolecular MUC1 glycopeptides have been used to unravel molecular mechanisms in antibody recognition of disease-specific epitopes. We have established a novel synthetic strategy for MUC1 tandem repeats having complex O-glycosylation states at each repeating unit based on convergent solid-phase fragment condensation under microwave irradiation. We have accomplished the synthesis of 77 amino acid MUC1 glycopeptides (MW = 12 759) having three major antigenic O-glycoforms [Tn, core 1 (T), and core 2 structures] at 10 designated positions out of 19 potential O-glycosylation sites. We demonstrate that the macro molecular MUC1 glycopeptide displaying the essential glycopeptidic neoepitope Pro-Asp-Thr(sialyl-T)-Arg-Pro-Ala-Pro at two different tandem repeats is an excellent serum MUC1 model showing ideal stoichiometric binding with anti-KL6/MUC1 antibody in the sandwich ELISA to quantify human serum KL6/MUC1 levels as a critical biomarker of interstitial lung diseases.
• Short Communication: Increase of Sialylated N-Glycansin Eyes with Neovascular Glaucoma Secondary to Proliferative Diabetic Retinopathy.

  Inafuku S, Noda K, Amano M, Nishimura S, Ishida S

  Current eye research 41 5 721 - 724 2016年05月 [査読有り][通常論文]
   

  PURPOSE: To investigate the alteration of N-glycans in the vitreous fluid of patients with neovascular glaucoma (NVG) secondary to proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples were collected from 18 patients with PDR (including 7 with NVG and 11 without NVG), and 17 patients without diabetes. Profiles of N-glycans were analyzed by glycoblotting-based high throughput protocol, which we recently developed. Protein levels of vascular endothelial growth factor (VEGF)-A were measured by ELISA. RESULTS: The concentration of total N-glycans and the concentration of N-glycans with sialic acids were significantly higher in NVG group compared with those in non-NVG group or control group, whereas there was no significant difference in concentrations of high-mannose N-glycans among three groups. There was a moderate correlation between the concentrations of sialylated N-glycans and VEGF-A. CONCLUSIONS: Our data demonstrate the distinct changes of N-glycan profile and the increase of sialylated N-glycans in eyes with NVG secondary to PDR.
• Fetal Environment and Glycosylation Status in Neonatal Cord Blood

  Ryosuke Sato, Kenji J. Tsuchiya, Hideo Matsuzaki, Nori Takei, Hiroaki Itoh, Naohiro Kanayama, Takafumi Suda, Hiroshi Watanabe, Tetsu Ohashi, Masakazu Tanaka, Shin-Ichiro Nishimura, Masato Maekawa

  Medicine (United States) 95 14 2016年04月01日 [査読有り][通常論文]
   

  Fetal environment is known to be a major predictive factor of type 2 diabetes and cardiovascular disease. However, associations of fetal environment and cord blood glycoforms are uncertain. In this study, we aimed to determine whether glycosylation status in neonatal cord blood is associated with perinatal outcomes reflecting a poor fetal environment. Thirty-six low birth weight (LBW) infants and 120 normal birth weight infants were recruited from a longitudinal birth cohort. We conducted a comprehensive cord blood N-glycan analysis using matrix-Assisted laser-desorption/ionization time-of-flight mass spectrometry. Associations of N-glycans with perinatal outcomes, including LBW, small for gestational age, and levels of cord blood leptin and adiponectin, were evaluated using logistic or multiple regression. We also prospectively explored correlations between N-glycans and 6 or 18-month rapid weight gain (> 0.67 SD score). A total of 35 N-glycans were detected (m/z value 1362.481-3865.407). Of these, abundance levels of G3414 (m/z value 3414.238) were inversely correlated with LBW and small for gestational age. Abundance levels of G1915 (m/z value 1914.698), G2744 (m/z value 2743.994), G3049 (m/z value 3049.105), and G3719 (m/z value 3719.349) were inversely related to LBW. The total N-glycan abundance levels were strongly positively correlated with levels of leptin and adiponectin in cord blood. In a prospective exploratory analysis, the 5 LBW-related N-glycans (G1915, G2744, G3049, G3414, and G3719) were all inversely associated with 6 or 18-month rapid weight gain. These N-glycans are structurally categorized into 2 different categories: fucosylated bi or tri-Antennary N-glycans and tri or tetra-Antennary N-glycans without fucosylation. In conclusion, mass spectrometry-based cord blood glycosylation analysis shows that 5 types of N-glycans are potential predictors of a poor fetal environment.
• Synthetic Human NOTCH1 EGF Modules Unraveled Molecular Mechanisms for the Structural and Functional Roles of Calcium Ions and O-Glycans in the Ligand-Binding Region

  Shun Hayakawa, Ryosuke Koide, Hiroshi Hinou, Shin-Ichiro Nishimura

  BIOCHEMISTRY 55 5 776 - 787 2016年02月 [査読有り][通常論文]
   

  The Notch signaling pathway is an evolutionarily highly conserved mechanism that operates across multicellular organisms and is critical for cell-fate decisions during development and homeostasis in most tissues. Notch signaling is modified by posttranslational glycosylations of the Notch extracellular EGF-like domain. To evaluate the structural and functional roles of various glycoforms at multiple EGF domains in the human Notch transmembrane receptor, we established a universal method for the construction of NOTCH1 EGF modules displaying the desired O-glycans at the designated glycosylation sites. The versatility of this strategy was demonstrated by the rapid and highly efficient synthesis of NOTCH1 EGF12 concurrently having a beta-D-glucopyranose-initiated glycan (Xyl alpha 1 -> 3Xyl alpha 1 -> 3Glc beta 1 ->) at Ser458 and alpha-L-fucopyranose-initiated glycan (Neu5Ac alpha 2 -> 3Gal beta 1 -> 4GlcNA beta 1 -> 3Fuc alpha 1 ->) at Thr466. The efficiency of the proper folding of the glycosylated EGF12 was markedly enhanced in the presence of 5 mM CaCl2. A nuclear magnetic resonance study revealed the existence of strong nuclear Overhauser effects between key sugar moieties and neighboring amino acid residues, indicating that both O-glycans contribute independently to the intramolecular stabilization of the antiparallel beta-sheet structure in the ligand-binding region of EGF12. A preliminary test using synthetic human NOTCH1 EGF modules showed significant inhibitory effects on the proliferation and adhesiveness of human breast cancer cell line MCF-7 and lung adenocarcinoma epithelial cell line A549, demonstrating for the first time evidence that exogenously applied synthetic EGF modules have the ability to interact with intrinsic Notch ligands on the surface of cancer cells.
• Solid-phase synthesis of C-mannosylated glycopeptide on WSXWS motif of human erythropoietin receptor

  Hiroshi Hinou, Yuya Abe, Shun Hayakawa, Kentaro Naruchi, Naoki Fujitani, Shin-Ichiro Nishimura

  TETRAHEDRON LETTERS 57 7 791 - 795 2016年02月 [査読有り][通常論文]
   

  Protein C-mannosylation is a widely conserved posttranslational modification in multicellular organisms although the function of this modification is yet to be clarified. To evaluate the effect of this modification, a C-mannosyltryptophan containing glycopeptide of human erythropoietin receptor (EPOR), which includes WSXWS motif conserved in cytokine receptor superfamily, was synthesized by microwave assisted solid-phase synthesis using per-O-benzyl protected amino acids and C-mannosyltryptophan on Sieber amide resin. Comparative NMR analysis of the C-mannosyl glycopeptide and a corresponding naked peptide revealed that the C-mannosylation enhances the NOE signal between the peptide main chain and the aromatic side chain to suggest enhancement of conformational stability of the WSXWS motif and its surrounding regions. (C) 2016 Elsevier Ltd. All rights reserved.
• Novel β-galactosynthase–β-mannosynthase dual activity of β-galactosidase from Aspergillus oryzae uncovered using monomer sugar substrates

  R. S. Tan, H. Hinou, S.-I. Nishimura

  RSC Advances 6 56 50833 - 50836 2016年 [査読有り][通常論文]
   

  We uncovered β-galactosynthase–β-mannosynthase dual-activity of β-galactosidase (A. oryzae) that could revolutionize chemoenzymatic glycan and NDOs syntheses.

• Effects of the multiple O-glycosylation states on antibody recognition of the immunodominant motif in MUC1 extracellular tandem repeats

  Shobith Rangappa, Gerard Artigas, Risho Miyoshi, Yasuhiro Yokoi, Shun Hayakawa, Fayna Garcia-Martin, Hiroshi Hinou, Shin-Ichiro Nishimura

  MEDCHEMCOMM 7 6 1102 - 1122 2016年 [査読有り][通常論文]
   

  Antibodies that react with human epithelial cell membrane MUC1 glycoprotein with aberrant glycoforms are promising diagnostic and therapeutic reagents in various cancers and interstitial lung diseases. However, the precise epitopes for anti-MUC1 antibodies have remained unclear. Although the MUC1 extracellular domain has multiple O-glycosylation sites within the tandem repeats, there have been few systematic approaches to determine the effects of the multiple O-glycosylation states, in the context of the disease-relevant epitope regions, on antibody recognition. In this study, we established a comprehensive approach for the characterization of anti-MUC1 antibodies by combining microarray-based epitope profiling and NMR-based conformational analysis of synthetic MUC1 glycopeptides. Epitope mapping analysis using a microarray that displayed 23 synthetic MUC1 glycopeptides revealed that anti-KL6/MUC1 monoclonal antibody (anti-KL6 mAb) has absolute binding specificity with an essential epitope, Pro-Asp-Thr[Neu5Ac alpha(2 -> 3)Gal beta(1 -> 3)GalNAc alpha 1 ->]-Arg-Pro-Ala-Pro, in an ultimately glycoform-specific manner when compared with the other well-studied anti-MUC1 mAbs DF3 and SM3, which are directed against the same Pro-Asp-Thr-Arg (PDTR) motif in the tandem repeats. Multiple O-glycosylations at the neighbouring Ser/Thr residues did not disturb this specific recognition by anti-KL6 mAb, even when modified by sterically hindered core 2-type pentasaccharide moieties (SC2). To our surprise, both DF3 and SM3 exhibited a drastic decrease in binding ability with putative MUC1 fragments with an immunodominant PDTR motif when other glycosylation sites were occupied by Tn antigen (GalNAc alpha 1 ->) or T antigen [Gal beta(1 -> 3)GalNAc alpha 1 ->]. However, modification at the two adjacent Ser residues by O-glycans that contained ST antigen [Neu5Ac alpha(2 -> 3)Gal beta( 1 -> 3)GalNAc alpha 1 ->] resulted, exceptionally, in a substantial enhancement of the affinity of DF3 for the PDTR region. These results demonstrated for the first time that the O-glycosylation states around the immunodominant PDTR motif strongly influence the binding potency and profile of DF3 and SM3. NMR studies of the synthetic MUC1 fragments discovered the molecular mechanisms by which multiple O-glycosylations at the adjacent Ser/Thr residues induce significant conformational alterations in the PDTR motif in a glycoform-dependent manner. Anti-KL6 mAb was proved to be the only anti-MUC1 mAb that can recognise a unique glycopeptidic neo-epitope generated via site-specific posttranslational modification by ST antigen independently from O-glycosylation states at the adjacent Ser/Thr residues within the MUC1 tandem repeats.
• Alteration of serum N-glycan profile in patients with autoimmune pancreatitis.

  Tomoda T, Nouso K, Kato H, Miyahara K, Dohi C, Morimoto Y, Kinugasa H, Akimoto Y, Matsumoto K, Yamamoto N, Noma Y, Horiguchi S, Tsutsumi K, Amano M, Nishimura SI, Yamamoto K

  Pancreatology 16 1 44 - 51 2016年 [査読有り][通常論文]
  
• Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator

  Kitatsuji, C, Izumi, K, Nambu, S, Kurogochi, M, Uchida, T, Nishimura, S.-I, Iwai, K, O’Brian, M. R, Ikeda-Saito, M, Ishimori, K

  Scientific Reports 6 18703 - 18703 Nature Publishing Group 2016年01月 [査読有り][通常論文]
   

  The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O-2 to form highly reactive oxygen species (ROS) with the "active site conversion" from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O-2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel "two-step self-oxidative modification" mechanism, during which O-2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as OH at the non-heme iron site in the His-cluster region formed by the active site conversion.
• Large-scale Glycomics of Livestock: Discovery of Highly Sensitive Serum Biomarkers Indicating an Environmental Stress Affecting Immune Responses and Productivity of Holstein Dairy Cows

  Rehan IF, Ueda K, Mitani T, Amano M, Hinou H, Ohashi T, Kondo S, Nishimura SI

  J. Agricul. Food Chem. 63 48 10578 - 10590 2015年12月 [査読有り][通常論文]
   

  Because various stresses strongly influence the food productivity of livestock, biomarkers to indicate unmeasurable environmental stress in domestic animals are of increasing importance. Thermal comfort is one of the basic principles of dairy cow welfare that enhances productivity. To discover sensitive biomarkers that monitor such environmental stresses in dairy cows, we herein performed, for the first time, large-scale glycomics on 336 lactating Holstein cow serum samples over 9 months between February and October. Glycoblotting combined with MALDI-TOF/MS and DMB/HPLC allowed for comprehensive glycomics of whole serum glycoproteins. The results obtained revealed seasonal alterations in serum N-glycan levels and their structural characteristics, such as an increase in high-mannose type N-glycans in spring, the occurrence of di/triantennary complex type N-glycans terminating with two or three Neu5Gc residues in summer and autumn, and N-glycans in winter dominantly displaying Neu5Ac. A multivariate analysis revealed a correlation between the serum expression levels of these season-specific glycoforms and productivity.
• Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells

  Udono M, Fujii K, Harada G, Tsuzuki Y, Kadooka K, Zhang P, Fujii H, Amano M, Nishimura S-I, Tashiro K, Kuhara S, Katakura Y

  Scientific Reports 5 17342 - 17342 2015年12月 [査読有り][通常論文]
   

  Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program.
• The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR.

  Coelho H, Matsushita T, Artigas G, Hinou H, Cañada FJ, Lo-Man R, Leclerc C, Cabrita EJ, Jiménez-Barbero J, Nishimura S, Garcia-Martín F, Marcelo F

  Journal of the American Chemical Society 137 39 12438 - 12441 2015年10月 [査読有り][通常論文]
  
• A comprehensive glycome profiling of Huntington's disease transgenic mice.

  Gizaw ST, Koda T, Amano M, Kamimura K, Ohashi T, Hinou H, Nishimura S

  Biochimica et biophysica acta 1850 9 1704 - 1718 2015年09月 [査読有り][通常論文]
  
• Glycome characterization of immunoglobulin G from buffalo (Bubalus bubalis ) colostrum

  Mamatha Bhanu L.S, Amano M, Nishimura S-I, Aparna H.S

  Glycoconj. J. 32 625 - 634 2015年08月 [査読有り][通常論文]
  
• Alteration of N-Glycan Profiles in Diabetic Retinopathy.

  Inafuku S, Noda K, Amano M, Ohashi T, Yoshizawa C, Saito W, Murata M, Kanda A, Nishimura S, Ishida S

  Investigative ophthalmology & visual science 56 9 5316 - 5322 2015年08月 [査読有り][通常論文]
   

  PURPOSE: To investigate the alteration of vitreal N-glycans in patients with proliferative diabetic retinopathy (PDR). METHODS: Plasma and vitreous samples were collected from 17 patients (10 females and 7 males) with PDR (PDR group) and 17 nondiabetic patients (8 females and 9 males) with epiretinal membrane (ERM) and idiopathic macular hole (MH) (non-diabetes mellitus [DM] group). Profiles of N-glycans were analyzed by a glycoblotting-based high-throughput protocol that we recently developed. Human retinal microvascular endothelial cells (HRMECs) were cultivated with culture media containing either low glucose (5 mM) or high glucose (25 mM), and expression levels of sialyltransferases were analyzed by real-time PCR and ELISA. RESULTS: Amount of N-glycans in the vitreous fluid of the PDR group was significantly higher than that of the non-DM group (495.5 ± 37.4 vs. 142.7 ± 30.8 pmol/100 μg protein, P < 0.005), whereas there was no significant difference in the plasma samples between the PDR and the non-DM group. In addition, profile analysis showed that N-glycans with sialic acids increased in the vitreous of the PDR group (328.4 ± 25.8 pmol/100 μg protein) compared to the non-DM group (92.1 ± 21.2 pmol/100 μg protein, P < 0.0005). Expression levels of sialyltransferases ST3GAL1 and ST3GAL4 were upregulated in the HRMECs after high-glucose stimulation. Consistent with the real-time PCR data, high-glucose stimulation elevated the protein levels of ST3GAL1 (117.4 ± 14.9 pg/mg, P < 0.01) and ST3GAL4 (6.1 ± 0.9 pg/mg, P < 0.05) in the HRMECs compared with the cells cultured with low-glucose culture media (ST3GAL1, 64.4 ± 5.8 pg/mg; ST3GAL4, 3.8 ± 0.3 pg/mg). CONCLUSIONS: Our data demonstrate distinct changes in the N-glycan profile and an increase in sialylated N-glycans in eyes with PDR.
• Serum N-glycan profiles in patients with intraductal papillary mucinous neoplasms of the pancreas.

  Akimoto Y, Nouso K, Kato H, Miyahara K, Dohi C, Morimoto Y, Kinugasa H, Tomoda T, Yamamoto N, Tsutsumi K, Kuwaki K, Onishi H, Ikeda F, Nakamura S, Shiraha H, Takaki A, Okada H, Amano M, Nishimura S, Yamamoto K

  Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] 15 4 432 - 438 2015年07月 [査読有り][通常論文]
  
• Rapid endolysosomal escape and controlled intracellular trafficking of cell surface mimetic quantum dots-anchored peptides and glycopeptides.

  Tan RS, Naruchi K, Amano M, Hinou H, Nishimura SI

  ACS Chemical Biology 10 9 2073 - 2086 2015年07月 [査読有り][通常論文]
   

  A novel strategy for the development of a high performance nanoparticules platform was established by means of cell surface mimetic quantum-dots (QDs)-anchored peptides/glycopeptides, which was developed as a model system for nanopartide-based drug delivery (NDD) vehicles with defined functions helping the specific intracellular trafficking after initial endocytosis. In this paper, we proposed a standardized protocol for the preparation of multifunctional QDs that allows for efficient cellular uptake and rapid escaping from the endolysosomal system and subsequent cytoplasmic molecular delivery to the target cellular compartment. Chemoselective ligation of the ketone-functionalized hexahistidine derivative facilitated both efficient endocytic entry and rapid endolysosomal escape of the aminooxy/phosphorylcholine self-assembled monolayer-coated QDs (AO/PCSAM-QDs) to the cytosol in various cell lines such as human normal and cancer cells, while modifications of these QDs with cell-penetrating arginine-rich peptides showed poor cellular uptake and induced self-aggregation of AO/PCSAM-QDs. Combined use of hexahistidylated AO/PCSAM-QDs with serglycine-like glycopeptides, namely synthetic proteoglycan initiators (PGIs), elicited the entry and controlled intracellular trafficking, Golgi localization, and also excretion of these nanoparticles, which suggested that the present approach would provide an ideal platform for the design of high performance NDD systems.
• Prognostic value of altered N-glycosylation of circulating glycoproteins in patients with unresectable pancreatic cancer treated with gemcitabine.

  Miyahara K, Nouso K, Morimoto Y, Kinugasa H, Kato H, Yamamoto N, Tsutsumi K, Kuwaki K, Onishi H, Ikeda F, Nakamura S, Shiraha H, Takaki A, Nakahara T, Miura Y, Asada H, Amano M, Nishimura S, Yamamoto K

  Pancreas 44 4 551 - 556 2015年05月 [査読有り][通常論文]
   

  OBJECTIVES: The objectives of this study were to examine the whole-serum N-glycan profile of patients with unresectable pancreatic cancer and to evaluate the ability of glycans to predict gemcitabine treatment efficacy and patient survival. METHODS: We collected serum from 52 patients with advanced pancreatic cancer before they began gemcitabine monotherapy. The serum glycan profile was measured through comprehensive quantitative high-throughput glycome analysis and compared with the treatment efficacy and patient survival. RESULTS: Of the 61 glycans detected, the serum levels of glycan 4310 (molecular weight [m/z] 1549.566), 6301 (m/z 2032.724), and 9200 (m/z 2010.692) were high in patients with a short time to tumor progression (TTP). Multivariate analysis revealed that a high glycan 9200 concentration was an independent risk factor for shorter TTP (hazard ratio, 2.11; 95% confidence interval, 1.07-4.17) and poor overall survival (hazard ratio, 2.56; 95% confidence interval, 1.08-6.19). The median TTP of patients with up-regulation of 9200 after gemcitabine treatment was shorter than for the remaining patients (91 vs 301 days; P = 0.0005). A similar relationship was observed for overall survival (median, 181 vs 561 days; P = 0.001). CONCLUSIONS: Glycan 9200 is a possible biomarker predicting gemcitabine efficacy survival in patients with unresectable pancreatic cancer.
• Use of non-invasive serum glycan markers to distinguish non-alcoholic steatohepatitis from simple steatosis.

  Yamasaki Y, Nouso K, Miyahara K, Wada N, Dohi C, Morimoto Y, Kinugasa H, Takeuchi Y, Yasunaka T, Kuwaki K, Onishi H, Ikeda F, Miyake Y, Nakamura S, Shiraha H, Takaki A, Iwasaki Y, Amano M, Nishimura S, Yamamoto K

  Journal of gastroenterology and hepatology 30 3 528 - 534 3 2015年03月 [査読有り][通常論文]
   

  BACKGROUND AND AIMS: Serum glycans have been reported to be promising diagnostic markers for many inflammatory diseases and cancers. The aims of this study were to investigate whole glycan expression in patients with non-alcoholic fatty liver diseases and to evaluate the potential use of glycan profiles as new clinical biomarkers to distinguish non-alcoholic steatohepatitis (NASH) from simple steatosis (SS). METHODS: We collected sera from 42 histologically proven NASH and 15 SS patients prior to treatment. Serum glycan profiles were measured by comprehensive, quantitative, high-throughput glycome analysis, and diagnostic values of serum glycans for NASH prediction were examined. RESULTS: Among the 41 serum glycans examined, the expression levels of 8 glycans in NASH were significantly higher than those of SS. Out of these eight glycans, three glycans (m/z 1955, 2032, and 2584) showed high areas under the receiver operating characteristic curve (0.833, 0.863, and 0.866, respectively) for distinguishing NASH from SS. In multivariate analyses with clinical parameters and serum glycans, these three glycans were significant predictive factors for distinguishing NASH from SS. The odds ratio of m/z 1955, 2032, and 2584 were 48.5, 6.46, and 11.8, respectively. These glycans also correlated significantly with lobular inflammation, ballooning, and fibrosis, but not with steatosis. CONCLUSION: We clearly demonstrated whole-serum glycan profiles in NASH patients, and the feasibility of serum glycans (m/z 1955, 2032, and 2584) as new noninvasive biomarkers for distinguishing NASH from SS.
• Application of glyco-blotting for identification of structures of polysaccharides causing membrane fouling in a pilot-scale membrane bioreactor treating municipal wastewater

  Kimura Katsuki, Nishimura Shin-Ichiro, Miyoshi Risho, Hoque Asiful, Miyoshi Taro, Watanabe Yoshimasa

  BIORESOURCE TECHNOLOGY 179 180 - 186 2015年03月 [査読有り][通常論文]
  
• Nutriglycomics in Mice Fed High-Fat or Low-Protein Diets

  Watanabe N, Hara Y, Kono A, Ohashi T, Amano M, Nishimura S-I

  Journal of Nutrition & Metabolism 1 4 - 8 2014年12月 [査読有り][通常論文]
  
• Delineating Binding Modes of Gal/GalNAc and Structural Elements of the Molecular Recognition of Tumor-Associated Mucin Glycopeptides by the Human Macrophage Galactose-Type Lectin

  Filipa Marcelo, Fayna Garcia-Martin, Takahiko Matsushita, Joao Sardinha, Helena Coelho, Anneloes Oude-Vrielink, Christiane Koller, Sabine Andre, Eurico J. Cabrita, Hans-Joachim Gabius, Shin-Ichiro Nishimura, Jesus Jimenez-Barbero, F. Javier Canada

  CHEMISTRY-A EUROPEAN JOURNAL 20 49 16147 - 16155 2014年12月 [査読有り][通常論文]
   

  The human macrophage galactose-type lectin (MGL) is a key physiological receptor for the carcinoma-associated Tn antigen (GalNAc--1-O-Ser/Thr) in mucins. NMR and modeling-based data on the molecular recognition features of synthetic Tn-bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non-glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca2+. NMR data were complemented with molecular dynamics simulations and Corcema-ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn-presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH- contacts involving exclusively the NHAc moiety.
• Alteration of N-glycan profiles in patients with chronic hepatitis and hepatocellular carcinoma.

  Miyahara K, Nouso K, Dohi C, Morimoto Y, Kinugasa H, Wada N, Takeuchi Y, Kuwaki K, Onishi H, Ikeda F, Miyake Y, Nakamura S, Shiraha H, Takaki A, Amano M, Nishimura SI, Yamamoto K

  Hepatology research : the official journal of the Japan Society of Hepatology 45 9 986 - 993 2014年12月 [査読有り][通常論文]
   

  AIM: Most of the modification of N-glycosylation reported in cancers including hepatocellular carcinoma (HCC) were based on the examinations of a small number of patients or particular proteins. The aim of this study is to reveal changes in whole serum N-glycan profiles systematically during the process of hepatocarcinogenesis and to elucidate their clinical application. METHODS: We analyzed sera from 105 patients with chronic hepatitis/liver cirrhosis (CH/LC) and age-/sex-matched healthy volunteers (HLT), as well as from 114 patients with HCC. Serum N-glycan profiles were measured comprehensively by a new, quantitative, high-throughput method and compared with clinical parameters. RESULTS: The total amount of N-glycan expression was significantly higher in patients with CH/LC than in HLT; however, no differences were observed between CH/LC and HCC patients. In HCC patients, multi-antennary glycans with fucose residues, particularly m/z 3195, were increased compared with CH/LC patients. The expression of m/z 3195 was high, especially in patients with a high number of intrahepatic lesions (>3), large tumor size (>3 cm), macroscopic vascular invasion or metastasis. The ratio of pairs of glycans on the same path of the biosynthesis pathway (m/z 3195/1914) showed a higher area under the receiver-operator curve of 0.810 than any other single glycan to distinguish HCC from CH/LC. CONCLUSION: We demonstrate the full spectrum of the alterations of serum N-glycans comprehensively in patients with liver disease, and elucidate the possible use of glycans as novel biomarkers of liver disease progression.
• Fast Epitope Mapping for the Anti-MUC1 Monoclonal Antibody by Combining a One-Bead-One-Glycopeptide Library and a Microarray Platform

  Fayna Garcia-Martin, Takahiko Matsushita, Hiroshi Hinou, Shin-Ichiro Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 20 48 15891 - 15902 2014年11月 [査読有り][通常論文]
   

  Anti-MUC1 monoclonal antibodies (mAbs) are powerful tools that can be used to recognize cancer-related MUC1 molecules, the O-glycosylation status of which is believed to affect binding affinity. We demonstrate the feasibility of using a rapid screening methodology to elucidate those effects. The approach involves i) " one-bead-one-compound"- based preparation of bilayer resins carrying glycopeptides on the shell and mass-tag tripeptides coding Oglycan patterns in the core, ii) on-resin screening with an anti-MUC1 mAb, iii) separating positive resins by utilizing secondary antibody conjugation with magnetic beads, and (iv) decoding the mass-tag that is detached from the positive resins pool by using mass spectrometric analysis. We tested a small library consisting of 27 MUC1 glycopeptides with different O-glycosylations against anti-MUC1 mAb clone VU-3C6. Qualitative mass-tag analysis showed that increasing the number of glycans leads to an increase in the binding affinity. Six glycopeptides selected from the library were validated by using a microarray-based assay. Our screening provides valuable information on O-glycosylations of epitopes leading to high affinity with mAb.
• SerumTri- and Tetra-Antennary N-Glycan is a Potential Predictive Biomarker for Castration-Resistant Prostate Cancer

  Yusuke Ishibashi, Yuki Tobisawa, Shingo Hatakeyama, Tetsu Ohashi, Masakazu Tanaka, Shintaro Narita, Takuya Koie, Tomonori Habuchi, Shin-Ichiro Nishimura, Chikara Ohyama, Tohru Yoneyama

  PROSTATE 74 15 1521 - 1529 2014年11月 [査読有り][通常論文]
   

  BACKGROUND. The U.S. FDA has approved several novel systemic agents including abiraterone acetate and taxoid cabazitaxel for metastatic castration-resistant prostate cancer (CRPC) result in a complicated decision-making while selecting an appropriate treatment. Therefore, a predictive biomarker for CRPC would provide useful information to physicians. The aim of this study is to evaluate the diagnostic potential of serum N-glycan profiling in CRPC. METHODS. Serum N-glycomics was performed in 80 healthy volunteers and 286 benign prostatic hyperplasia, 258 early-stage PC, 46 PC with androgen deprivation therapy (ADT), and 68 CRPC patients using the glycoblotting method. A total of 36 types of N-glycan levels in each patient were analyzed using logistic regression analysis and receiver operating characteristic curves. We also examined the expression of N-glycan branching enzyme genes in PC cell lines using quantitative RT-PCR. RESULTS. We observed that tri- and tetra-antennary N-glycans were significantly higher in CRPC patients than in any other groups. The longitudinal follow-up of tri- and tetra-antennary N-glycan levels revealed that one PC with ADT patient showed an increase that was more than the cut-off level and two consecutive increases in tri- and tetra-antennary N-glycan levels 3 months apart; resulted in biochemical recurrence despite the castrate level of testosterone, and the patient was defined as CRPC. Expression of N-glycan branching enzyme genes were significantly upregulated in CRPC cell lines. CONCLUSIONS. These results suggest that the overexpression of tri- and tetra-antennary N-glycan may be associated with the castration-resistant status in PC and may be a potential predictive biomarker for CRPC. (C) 2014 Wiley Periodicals, Inc.
• Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation

  Michiyo Terashima, Maho Amano, Tomohiro Onodera, Shin-Ichiro Nishimura, Norimasa Iwasaki

  STEM CELL RESEARCH 13 3 454 - 464 2014年11月 [査読有り][通常論文]
   

  Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs. (C) 2014 The Authors. Published by Elsevier B.V.
• A comparison of N-glycan profiles in human plasma and vitreous fluid.

  Inafuku S, Noda K, Amano M, Ohashi T, Yoshizawa C, Saito W, Kanda A, Nishimura S, Ishida S

  Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie 252 8 1235 - 1243 2014年08月 [査読有り][通常論文]
  
• Serum N-Glycan Alteration Associated with Renal Cell Carcinoma Detected by High Throughput Glycan Analysis

  Shingo Hatakeyama, Maho Amano, Yuki Tobisawa, Tohru Yoneyama, Norihiko Tsuchiya, Tomonori Habuchi, Shin-Ichiro Nishimura, Chikara Ohyama

  JOURNAL OF UROLOGY 191 3 805 - 813 2014年03月 [査読有り][通常論文]
   

  Purpose: Biomarkers for the early detection and prediction of survival in patients with renal cell carcinoma have not been established. We developed what is to our knowledge a novel glycoblotting method that allows high throughput, comprehensive, quantitative analysis of glycans in human serum. In this study we identified alterations in serum N-glycans associated with renal cell carcinoma. Materials and Methods: We performed a comprehensive N-glycan structural analysis of serum from 64 patients with renal cell carcinoma and 34 age matched, healthy volunteers using glycoblotting methods and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The peak intensity of N-glycan was analyzed using logistic regression analysis and ROCs were used to select candidate N-glycans. Candidate N-glycans with a statistically significant relationship to renal cell carcinoma or overall survival were independently evaluated using a Cox regression model to determine superiority compared to other conventional renal cell carcinoma biomarkers. Results: We identified 56 types of N-glycans in serum from healthy volunteers and patients with renal cell carcinoma. Peaks 40 and 43 were significantly more intense in patients than in volunteers. Peak 19 intensity was significantly higher and peak 49 intensity was significantly lower in patients with renal cell carcinoma who survived for a longer period. Multivariate analysis revealed that peaks 19 and 49 were independent predictors of overall survival. Conclusions: Serum N-glycan analysis is a promising approach to discovering new biomarkers for renal cell carcinoma. Further study is warranted to validate our results.
• A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides

  Takahiko Matsushita, Wataru Takada, Kota Igarashi, Kentaro Naruchi, Risho Miyoshi, Fayna Garcia-Martin, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura

  BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1840 3 1105 - 1116 2014年03月 [査読有り][通常論文]
   

  Background: Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear. Methods: A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies. Results: Selective imine-coupling between aminooxy-functionalized methaciylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Ac alpha 2,3Gal beta 1,3GalNAc alpha 1 ->) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUD monoclonal antibodies such as VU-3D, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUD tandem repeats. Conclusion: We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption. General significance: The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes. (C) 2013 Elsevier B.V. All rights reserved.
• Synthesis of neoglycosphingolipid from methoxyamino-functionalized ceramide

  Junya Ishida, Hiroshi Hinou, Kentaro Naruchi, Shin-Ichiro Nishimura

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 24 4 1197 - 1200 2014年02月 [査読有り][通常論文]
   

  An efficient approach for the synthesis of a methoxyamino- functionalized ceramide was established from phytosphingosine using specific Nb -> Na acyl migration of the octadecanoyl group during the removal of Na-Fmoc protective group. One step glycoblotting reaction of the ceramide mimic with lactose afforded a neoglycosphingolipid showing potent inhibitory activity against recombinant endoglycoceramidase II from Rhodococcus sp. (C) 2014 Elsevier Ltd. All rights reserved.
• Serum Glycan as a Prognostic Marker in Patients With Advanced Hepatocellular Carcinoma Treated With Sorafenib

  Koji Miyahara, Kazuhiro Nouso, Yasuhiro Miyake, Shinichiro Nakamura, Shuntaro Obi, Maho Amano, Kazuko Hirose, Shin-ichiro Nishimura, Kazuhide Yamamoto

  HEPATOLOGY 59 1 355 - 356 2014年01月 [査読有り][通常論文]
  
• Allying with armored snails: the complete genome of gammaproteobacterial endosymbiont

  Satoshi Nakagawa, Shigeru Shimamura, Yoshihiro Takaki, Yohey Suzuki, Shun-ichi Murakami, Tamaki Watanabe, So Fujiyoshi, Sayaka Mino, Tomoo Sawabe, Takahiro Maeda, Hiroko Makita, Suguru Nemoto, Shin-Ichiro Nishimura, Hiromi Watanabe, Tomo-O Watsuji, Ken Takai

  ISME JOURNAL 8 1 40 - 51 2014年01月 [査読有り][通常論文]
   

  Deep-sea vents harbor dense populations of various animals that have their specific symbiotic bacteria. Scaly-foot gastropods, which are snails with mineralized scales covering the sides of its foot, have a gammaproteobacterial endosymbiont in their enlarged esophageal glands and diverse epibionts on the surface of their scales. In this study, we report the complete genome sequencing of gammaproteobacterial endosymbiont. The endosymbiont genome displays features consistent with ongoing genome reduction such as large proportions of pseudogenes and insertion elements. The genome encodes functions commonly found in deep-sea vent chemoautotrophs such as sulfur oxidation and carbon fixation. Stable carbon isotope (C-13)-labeling experiments confirmed the endosymbiont chemoautotrophy. The genome also includes an intact hydrogenase gene cluster that potentially has been horizontally transferred from phylogenetically distant bacteria. Notable findings include the presence and transcription of genes for flagellar assembly, through which proteins are potentially exported from bacterium to the host. Symbionts of snail individuals exhibited extreme genetic homogeneity, showing only two synonymous changes in 19 different genes (13 810 positions in total) determined for 32 individual gastropods collected from a single colony at one time. The extremely low genetic individuality in endosymbionts probably reflects that the stringent symbiont selection by host prevents the random genetic drift in the small population of horizontally transmitted symbiont. This study is the first complete genome analysis of gastropod endosymbiont and offers an opportunity to study genome evolution in a recently evolved endosymbiont.
• Potential usage for in vivo lectin screening in live animals utilizing cell surface mimetic glyco-nanoparticles, phosphorylcholine-coated quantum dots (PC-QDs)

  Maho Amano, Hiroshi Hinou, Risho Miyoshi, Shin-Ichiro Nishimura

  Methods in Molecular Biology 1200 361 - 369 2014年 [査読有り][招待有り]
   

  Utilizing glycosylated derivatives as a tag, we are able to explore novel counter-receptor of endogenous lectins or lectin-like molecules in vivo. We have established the standardized methodology including preparation of glycosylated derivatives and construction of a platform for tracing the molecules in vivo at first. Combined use of an aminooxy-terminated thiol derivative and a phosphorylcholine (PC) derivative provides quantum dots (QDs) with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific protein adsorption concurrently. In order to track the derivatives in vivo, near-infrared (NIR) fluorescence imaging of QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse can be performed. It has revealed that distinct long-term delocalization over 2 h can be observed depending on the species of glycans ligated to PC-QDs at least in the liver. Until today we have performed live animal imaging utilizing various kinds of sialyl glyco-PC-QDs. They are still retained stably in whole body after 2 h while they showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution, which should be equivalent to the distribution of sialic acid-recognizing lectins. Here we describe a standardized protocol using ligand-displayed PC-QDs for live cell/animal imaging by versatile NIR fluorescence photometry without influence of size-dependent accumulation/excretion pathway for nanoparticles (e.g., viruses)∈> ∈10 nm in hydrodynamic diameter by the liver. © 2014 Springer Science+Business Media New York.
• Discovery of novel differentiation markers in the early stage of chondrogenesis by glycoform-focused reverse proteomics and genomics

  Takeshi Ishihara, Kiyoshi Kakiya, Koji Takahashi, Hiroto Miwa, Masatomo Rokushima, Tomoyo Yoshinaga, Yoshikazu Tanaka, Takaomi Ito, Hiroko Togame, Hiroshi Takemoto, Maho Amano, Norimasa Iwasaki, Akio Minami, Shin-Ichiro Nishimura

  BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1840 1 645 - 655 2014年01月 [査読有り][通常論文]
   

  Background: Osteoarthritis (OA) is one of the most common chronic diseases among adults, especially the elderly, which is characterized by destruction of the articular cartilage. Despite affecting more than 100 million individuals all over the world, therapy is currently limited to treating pain, which is a principal symptom of OA New approaches to the treatment of OA that induce regeneration and repair of cartilage are strongly needed. Methods: To discover potent markers for chondrogenic differentiation, glycoform-focused reverse proteomics and genomics were performed on the basis of glycoblotting-based comprehensive approach. Results: Expression levels of high-mannose type N-glycans were up-regulated significantly at the late stage of differentiation of the mouse chondroprogenitor cells. Among 246 glycoproteins carrying this glycotype identified by ConA affinity chromatography and LC/MS, it was demonstrated that 52% are classified as cell surface glycoproteins. Gene expression levels indicated that mRNAs for 15 glycoproteins increased distinctly in the earlier stages during differentiation compared with Type II collagen. The feasibility of mouse chondrocyte markers in human chondrogenesis model was demonstrated by testing gene expression levels of these 15 glycoproteins during differentiation in human mesenchymal stem cells. Conclusion: The results showed clearly an evidence of up-regulation of 5 genes, ectonucleotide pyrophosphatase/ phosphodiesterase family member 1, collagen alpha-1(III) chain, collagen alpha-1(XI) chain, aquaporin-1, and netrin receptor UNC5B, in the early stages of differentiation. General significance: These cell surface 5 glycoproteins become highly sensitive differentiation markers of human chondrocytes that contribute to regenerative therapies, and development of novel therapeutic reagents. (C) 2013 Published by Elsevier B.V.
• A new approach for the synthesis of hyperbranched N-glycan core structures from locust bean gum.

  Ravi Kumar HV, Naruchi K, Miyoshi R, Hinou H, Nishimura S

  Organic letters 15 24 6278 - 6281 2013年12月20日 [査読有り][通常論文]
  
• Serum Glycan Markers for Evaluation of Disease Activity and Prediction of Clinical Course in Patients with Ulcerative Colitis

  Koji Miyahara, Kazuhiro Nouso, Shunsuke Saito, Sakiko Hiraoka, Keita Harada, Sakuma Takahashi, Yuki Morimoto, Sayo Kobayashi, Fusao Ikeda, Yasuhiro Miyake, Hidenori Shiraha, Akinobu Takaki, Hiroyuki Okada, Maho Amano, Kazuko Hirose, Shin-Ichiro Nishimura, Kazuhide Yamamoto

  PLoS ONE 8 10 e74861  2013年10月07日 [査読有り][通常論文]
   

  Background:The aims of this study were to determine the change of whole-serum N-glycan profile in ulcerative colitis (UC) patients and to investigate its clinical utility.Methods:We collected serum from 75 UC patients at the time of admission and the same number of age/sex-matched healthy volunteers. Serum glycan profile was measured by comprehensive quantitative high-throughput glycome analysis and was compared with disease activity and prognosis.Results:Out of 61 glycans detected, 24 were differentially expressed in UC patients. Pathway analysis demonstrated that highly sialylated multi-branched glycans and agalactosyl bi-antennary glycans were elevated in UC patients in addition, the glycan ratio m/z 2378/1914, which also increased in UC, showed the highest Area under Receiver Operating Characteristic curve (0.923) for the diagnosis of UC. Highly sialylated multi-branched glycans and the glycan ratio m/z 2378/1914 were higher in the patients with total colitis, Clinical Activity Index > 10, Mayo endoscopic score 3, or a steroid-refractory status. In particular, the glycan ratio m/z 2378/1914 (above median) was an independent prognostic factor for the need for an operation (hazard ratio, 2.67 95% confidence interval, 1.04-7.84).Conclusions:Whole-serum glycan profiles revealed that the glycan ratio m/z 2378/1914 and highly sialylated multi-branched glycans increase in UC patients, and are correlated with disease activity. The glycan ratio m/z 2378/1914 was an independent predictive factor of the prognosis of UC. © 2013 Miyahara et al.
• Clinical utility of high-throughput glycome analysis in patients with pancreatic cancer

  Kazuhiro Nouso, Maho Amano, Yoichi M. Ito, Koji Miyahara, Yuki Morimoto, Hironari Kato, Koichiro Tsutsumi, Takeshi Tomoda, Naoki Yamamoto, Shinichiro Nakamura, Sayo Kobayashi, Kenji Kuwaki, Hiroaki Hagihara, Hideki Onishi, Yasuhiro Miyake, Fusao Ikeda, Hidenori Shiraha, Akinobu Takaki, Taku Nakahara, Shin-Ichiro Nishimura, Kazuhide Yamamoto

  JOURNAL OF GASTROENTEROLOGY 48 10 1171 - 1179 2013年10月 [査読有り][通常論文]
   

  Most of the glycan changes reported in cancers were based on the examinations of a small number of patients or particular proteins. The aim of this study was to determine the changes of the serum N-glycan profile comprehensively in a large number of pancreatic cancer patients and investigate its clinical utility. Glycan levels in the serum of 92 pancreatic cancer patients and 243 healthy volunteers (HLT) were examined by comprehensive quantitative high-throughput glycome analysis and were compared with clinical parameters. Out of 66 glycans detected, 15 were differentially expressed in pancreatic cancer, and 10 out of the 15 glycans were significantly up-regulated in cases with distant metastasis. There was a clear increase in overall expression of serum glycans, especially highly-branched glycans with fucose moieties, in pancreatic cancer. Among these 15 glycans, a tri-antennary complex type glycan (m/z 3195) showed the highest area under the receiver operating characteristic curve (AUROC = 0.799) for the diagnosis of pancreatic cancer. The ratio of pairs of glycans on the same path of the biosynthesis pathway (m/z 3195/1914) was found to be significantly higher in pancreatic cancer than in HLT (median = 1.11 and 0.41, respectively; p < 0.0001, AUROC = 0.831). For this pair ratio, the hazard ratio for survival (2.60, 95 % CI = 1.44-4.79) was higher than that of any single glycan and 1-year survival of patients with a high and low ratio was 36.9 and 69.2 %, respectively, (p = 0.001). Comprehensive glycome analysis can be used to know the presence of pancreatic cancer, distant metastasis, and patient prognosis, simultaneously.
• A novel approach for the parallel synthesis of glycopeptides by combining solid-phase peptide synthesis and dendrimer-supported enzymatic modifications

  MATSUSHITA Takahiko, HANDA Seiji, NARUCHI Kentaro, GARCIA-MARTIN Fayna, HINOU Hiroshi, NISHIMURA Shin-Ichiro

  Polymer journal 45 8 854 - 862 Nature Publishing Group 2013年08月 [査読無し][招待有り]
  
• 血清中糖鎖の網羅的解析による肝細胞癌新規バイオマーカーの開発

  神山 俊哉, 柿坂 達彦, 横尾 英樹, 蒲池 浩文, 若山 顕治, 敦賀 陽介, 三浦 信明, 西村 紳一郎, 藤堂 省, 武冨 紹信

  日本消化器外科学会総会 68回 O - 5 (一社)日本消化器外科学会 2013年07月
• Identification of novel serum biomarkers of hepatocellular carcinoma using glycomic analysis

  Toshiya Kamiyama, Hideki Yokoo, Jun-Ichi Furukawa, Masaki Kurogochi, Tomoaki Togashi, Nobuaki Miura, Kazuaki Nakanishi, Hirofumi Kamachi, Tatsuhiko Kakisaka, Yosuke Tsuruga, Masato Fujiyoshi, Akinobu Taketomi, Shin-Ichiro Nishimura, Satoru Todo

  HEPATOLOGY 57 6 2314 - 2325 2013年06月 [査読有り][通常論文]
   

  The altered N-glycosylation of glycoproteins has been suggested to play an important role in the behavior of malignant cells. Using glycomics technology, we attempted to determine the specific and detailed N-glycan profile for hepatocellular carcinoma (HCC) and investigate the prognostic capabilities. From 1999 to 2011, 369 patients underwent primary curative hepatectomy in our facility and were followed up for a median of 60.7 months. As normal controls, 26 living Japanese related liver transplantation donors were selected not infected by hepatitis B and C virus. Their mean age was 40.0 and 15 (57.7%) were male. We used a glycoblotting method to purify N-glycans from preoperative blood samples from this cohort (10 L serum) which were then identified and quantified using mass spectrometry (MS). Correlations between the N-glycan levels and the clinicopathologic characteristics and outcomes for these patients were evaluated. Our analysis of the relative areas of all the sugar peaks identified by MS, totaling 67 N-glycans, revealed that a proportion had higher relative areas in the HCC cases compared with the normal controls. Fourteen of these molecules had an area under the curve of greater than 0.80. Analysis of the correlation between these 14 N-glycans and surgical outcomes by univariate and multivariate analysis identified G2890 (m/z value, 2890.052) as a significant recurrence factor and G3560 (m/z value, 3560.295) as a significant prognostic factor. G2890 and G3560 were found to be strongly correlated with tumor number, size, and vascular invasion. Conclusion: Quantitative glycoblotting based on whole serum N-glycan profiling is an effective approach to screening for new biomarkers. The G2890 and G3560 N-glycans determined by tumor glycomics appear to be promising biomarkers for malignant behavior in HCCs. (HEPATOLOGY 2013;)
• Attomolar Detection of Influenza A Virus Hemagglutinin Human H1 and Avian H5 Using Glycan-Blotted Field Effect Transistor Biosensor

  Sho Hideshima, Hiroshi Hinou, Daisuke Ebihara, Ryosuke Sato, Shigeki Kuroiwa, Takuya Nakanishi, Shin-Ichiro Nishimura, Tetsuya Osaka

  ANALYTICAL CHEMISTRY 85 12 5641 - 5644 2013年06月 [査読有り][通常論文]
   

  Influenza virus, through cell invasion and propagation with the interaction between hemagglutinin (HA) present on its surface and glycans on the host cell, causes a rapidly spreading infection throughout the world. In the present investigation, we succeeded for the first time in the attomolar-level sensing and discrimination of influenza A viral HA molecules H1 and H5 by using a glycan-immobilized field effect transistor (FET) biosensor. The small ligand glycans immobilized on the FET device, which make effective use of the charge-detectable region for FET-based detection in terms of Debye length, gave an advantage in the highly sensitive detection of the proteins. Two kinds of trisaccharides receptors terminating in sialic acid-alpha 2,6-galactose (6'-sialyllactose) and in sialic acid-alpha 2,3-galactose (3'-sialyllactose) were conjugated directly with the SiO2 surface of FET devices by a simple glycoblotting method using the self-assembled monolayer (SAM) of aminooxy terminated silane-coupling reagent, 3-aminooxypropyltriethoxysilane. Furthermore, it was demonstrated that the FETs with densely immobilized glycans, which possess the high capture ability by achieving the glycoside cluster effect, clearly distinguish HA molecules between their subtypes H1 (human) and H5 (avian) at the attomolar level, while the conventional method based on HA antibodies achieves only picomolar-level detection. Our findings indicate that the glycan-immobilized FET is a promising device to detect various pathogenic bacteria and viruses through glycan-protein interaction found ubiquitously in many infectious diseases.
• 潰瘍性大腸炎の活動性・予後予測における血清糖鎖マーカーの有用性

  宮原 孝治, 能祖 一裕, 平岡 佐規子, 森元 裕貴, 高橋 索真, 小林 沙代, 斎藤 俊介, 原田 馨太, 山本 和秀, 天野 麻穂, 西村 紳一郎

  日本臨床分子医学会学術総会プログラム・抄録集 50回 76 - 76 日本臨床分子医学会 2013年04月
• Microwave-Assisted Solid-Phase Synthesis of Antifreeze Glycopeptides

  Ryukou Izumi, Takahiko Matsushita, Naoki Fujitani, Kentaro Naruchi, Hiroki Shimizu, Sakae Tsuda, Hiroshi Hinou, Shin-Ichiro Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 19 12 3913 - 3920 2013年03月 [査読有り][通常論文]
   

  Microwave-assisted solid-phase synthesis allows for the rapid and large-scale preparation and structureactivity characterization of tandem repeating glycopeptides, namely monodispersed synthetic antifreeze glycopeptides (syAFGPs, H-[Ala-Thr(Gal1,3GalNAc1)-Ala]n-OH, n=26). By employing novel AFGP analogues, we have demonstrated that of the monodispersed syAFGPn (n=26, degree of polymerization, DP=26, Mw=12573690Da), syAFGP5 (DP=5, Mw=3082Da) and syAFGP6 (DP=6, Mw=3690Da) exhibit the ability to form typical hexagonal bipyramidal ice crystals and satisfactory thermal hysteresis activity. Structural characterization by NMR and CD spectroscopy revealed that syAFGP6 forms a typical poly-L-proline typeII helix-like structure in aqueous solution whereas enzymatic modification by sialic acid of the residues at the C-3 positions of the nonreducing Gal residues disturbs this conformation and eliminates the antifreeze activity.
• Glycoblotting-based high throughput protocol for the structural characterization of hyaluronan degradation products during enzymatic fragmentation

  Takayuki Furukawa, Misaki Arai, Fayna Garcia-Martin, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura

  GLYCOCONJUGATE JOURNAL 30 2 171 - 182 2013年02月 [査読有り][通常論文]
   

  Increasing interests have been focused on the functional roles of hyaluronan degradation products, namely hyaluronan oligosaccharides, as signal molecules regulating cell growth, differentiation, malignancy, and inflammatory responses. It is clear that molecular size of hyaluronan oligosaccharides might be crucial for defining possible and dynamic roles in supporting and suppressing homeostatic cellular processes. The present paper communicates a facile and efficient approach based on glycoblotting method for the characterization of hyaluronan fragments liberated from three different sources of hyaluronan (rooster comb, bovine vitreous humor, and Streptococcus) by in vitro degradation using two typical hyaluronidases of bovine testicular (EC 3.2.1.35) and Streptomyces hyalurolyticus (EC 4.2.2.1). It was demonstrated that glycoblotting method allows for high throughput and quantitative analysis of hyaluronan fragments within a wide dynamic range (1 similar to 1,000 pmole) when 5 mu g of hyaluronan digests were applied for this enrichment protocol. Molecular size and distribution of hyaluronan fragments were proved to be influenced strongly by conditions and hyaluronidases employed while source of hyaluronan did not affect the degradation profiles. Strikingly, the present method uncovered the existence of the smallest and unusual hyaluronan degradation fragments such as a disaccharide GlcA beta 1-3GlcNAc during the digestion by bovine hyaluronidase and a trisaccharide GlcA beta 1-3GlcNAc beta 1-4GlcA derivative by Streptomyces hyaluronidase. Bovine testis hyaluronidases afforded hyaluronan tetra- and hexasaccharides as major products. On the other hand, it was demonstrated that Streptomyces hyaluronidase can produce odd number fragments from three to nine sugar residues while even number fragments from four to fourteen sugar residues were major products.
• Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses

  Masatoshi Okamatsu, Fei Feng, Tatsuya Ohyanagi, Noriko Nagahori, Kazuhiko Someya, Yoshihiro Sakoda, Nobuaki Miura, Shin-Ichiro Nishimura, Hiroshi Kida

  JOURNAL OF VIROLOGICAL METHODS 187 2 390 - 394 2013年02月 [査読有り][通常論文]
   

  Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in alpha-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with alpha-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus. (C) 2012 Elsevier B.V. All rights reserved.
• Novel thiosialosides tethered to metal nanoparticles as potent influenza A virus haemagglutinin blockers.

  Feng F, Sakoda Y, Ohyanagi T, Nagahori N, Shibuya H, Okamastu M, Miura N, Kida H, Nishimura SI

  Antiviral therapy 2013年02月 [査読有り][通常論文]
  
• Effect of Ganglioside GM3 Synthase Gene Knockout on the Glycoprotein N-Glycan Profile of Mouse Embryonic Fibroblast

  Noriko Nagahori, Tadashi Yamashita, Maho Amano, Shin-Ichiro Nishimura

  CHEMBIOCHEM 14 1 73 - 82 2013年01月 [査読有り][通常論文]
   

  The structural and clinical significance of cellular glycoproteins and glycosphingolipids (GSLs) are often separately discussed. Considering the biosynthetic pathway of glycoconjugates, glycans of cell-surface glycoproteins and GSLs might partially share functions in maintaining cellular homeostatis. The purpose of this study is to establish a general and comprehensive glycomics protocol for cellular GSLs and N-glycans of glycoproteins. To test the feasibility of a glycoblotting-based protocol, whole glycans released both from GSLs and glycoproteins were profiled concurrently by using GM3 synthase-deficient mouse embryonic fibroblast GM3(-/-). GM3(-/-) cells did not synthesize GM3 or any downstream product of GM3 synthase. Instead, expression levels of o-series gangliosides involving GM1-b and GD1-alpha increased dramatically, whereas a-/b-series gangliosides were predominantly detected in wild-type (WT) cells. We also discovered that glycoprotein N-glycan profiles of GM3(-/-) cells are significantly altered as compared to WT cells, although GM3 synthase is responsible only for GSLs synthesis and is not associated with glycoprotein N-glycan biosynthesis. The present approach allows for high-throughput profiling of cellular glycomes enriched by different classes of glycoconjugates, and our results demonstrated that gene knockout of the enzymes responsible for GSL biosynthesis significantly influences the N-glycans of glycoproteins.
• Macrocyclic Mechanism-Based Inhibitor for Neuraminidases

  Hirokazu Kai, Hiroshi Hinou, Kentaro Naruchi, Takahiko Matsushita, Shin-Ichiro Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 19 4 1364 - 1372 2013年01月 [査読有り][通常論文]
   

  A macrocyclic mechanism-based inhibitor for neuraminidases (NAs) bearing a 2-difluoromethylphenyl aglycone and a linker between the aglycone and C-9 positions of sialic acid was synthesized and evaluated. The macrocyclic structure was designed to keep the aglycone moiety in the active site of the neuraminidase after cleavage of the glycoside bond. When Vibrio chorelae neuraminidase (VCNA) was treated with a similar acyclic derivative in the presence of detergent, the irreversible inhibition property was disabled. In contrast, this macrocyclic compound acted as an irreversible inhibitor for VCNA in the presence of detergent. Inhibition assay for various NAs using this macrocyclic compound revealed that the irreversible inhibition property depends on the kcat of the neuraminidase treated. NAs having small kcat values, such as Influenza viruses, Clostridium, Trypanosoma cruzi, and Human, were also inhibited irreversibly. However, Salmonella typhimurium NA, which has an extremely high kcat, was not affected irreversibly by the inhibitor. Interestingly, in contrast to common kcat inhibitors, the irreversibility of inhibition by this macrocyclic compound is inversely proportional to the kcat of the target neuraminidase.
• Site-Specific Conformational Alteration Induced by Sialylation of MUC1 Tandem Repeating Glycopeptides at an Epitope Region for the Anti-KL-6 Monoclonal Antibody

  Takahiko Matsushita, Naoki Ohyabu, Naoki Fujitani, Kentaro Naruchi, Hiroki Shimizu, Hiroshi Hinou, Shin-Ichiro Nishimura

  BIOCHEMISTRY 52 2 402 - 414 2013年01月 [査読有り][通常論文]
   

  Protein O-glycosylation is an essential step for controlling structure and biological functions of glycoproteins involving differentiation, cell adhesion, immune response, inflammation, and tumorigenesis and metastasis. This study provides evidence of site-specific structural alteration induced during multiple sialylation at Ser/Thr residues of the tandem repeats in human MUC1 glycoprotein. Systematic nuclear magnetic resonance (NMR) study revealed that sialylation of the MUC1 tandem repeating glycopeptide, Pro-Pro-Ala-His-Gly-Val-Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr-Ala with core 2-type O-glycans at five potential glycosylation sites, afforded a specific conformational change at one of the most important cancer-relevant epitopes (Pro-Asp-Thr-Arg). This result indicates that disease-relevant epitope structures of human epithelial cell surface mucins can be altered both by the introduction of an inner GalNAc residue and by the distal sialylation in a peptide sequence-dependent manner. These data demonstrate the feasibility of NMR-based structural characterization of glycopeptides synthesized in a chemical and enzymatic manner in examining the conformational impact of the distal glycosylation at multiple O-glycosylation sites of mucin-like domains.
• Novel thiosialosides tethered to metal nanoparticles as potent influenza A virus haemagglutinin blockers

  Fei Feng, Yoshihiro Sakoda, Tatsuya Ohyanagi, Noriko Nagahori, Hitomi Shibuya, Masatoshi Okamastu, Nobuaki Miura, Hiroshi Kida, Shin-Ichiro Nishimura

  Antiviral Chemistry and Chemotherapy 23 2 59 - 65 2013年 [査読有り][通常論文]
   

  Background: The purpose of this study was to develop a new class of influenza A virus haemagglutinin (HA) blockers by tethering thiosialoside molecules to metal nanoparticles and producing glycoclusters that enhance the affinity of HA binding by N-acetylneuraminic acid. Methods: Oxygen of the glycoside bond of sialoside was replaced with sulfur to prevent hydrolytic digestion of the N-acetylneuraminic acid residue by viral neuraminidase. Two novel thiosialosides, α-2-S-[p-(N-levulinyl) aminophenyl]-5-N-acetylneuraminic acid (Neu5Ac-S-Lev) and α-2-S-[m-(N-levulinyl)aminobenzyl]-5-Nacetylneuraminic acid (Neu5Ac-S-CH 2-Lev), were tethered onto the surface of metal nanoparticles via an aminooxy functionalized thiol linker in a glycoblotting reaction. Gold (Au) and silver (Ag) nanoparticles were coated simultaneously with 11-mercaptoundecyl phosphorylcholine to reduce non-specific adsorption of proteins. Phosphorylcholine self-assembled monolayercoated metals displaying clustered Neu5Ac (Neu5Ac-PCSAM-Au and Neu5Ac-PCSAM-Ag) were subjected to haemagglutination inhibition (HI) assays using the influenza A virus strain A/PR/8/1934 (H1N1). Results: Glyconanoparticles with thiosialosides had potent HI activities. In particular, Neu5Ac-PCSAM-Au with a diameter of 20 nm corresponding to 9.8 μM monosaccharide Neu5Ac was the most potent HA inhibitor. The versatility of this strategy was demonstrated by similar submicromolar HI activities of Neu5Ac-PCSAM--Ag with diameters of 50 nm and 150 nm. Conclusions: Glycosylated metal nanoparticles were designed and synthesized as potent influenza A virus HA blockers. This study may contribute to the acceleration of the discovery of a new class of nanoparticle antiinfluenza drugs. © 2013 International Medical Press.
• N- and O-glycome analysis of serum and urine from bladder cancer patients using a high-throughput glycoblotting method

  Motoi Takeuchi, Maho Amano, Hiroshi Kitamura, Taiji Tsukamoto, Naoya Masumori, Kazuko Hirose, Tetsu Ohashi, Shin-Ichiro Nishimura

  Journal of Glycomics and Lipidomics 3 1 108  2013年 [査読有り][通常論文]
  
• Serum N-glycan profiling predicts prognosis in patients undergoing hemodialysis.

  Hatakeyama S, Amano M, Tobisawa Y, Yoneyama T, Tsushima M, Hirose K, Yoneyama T, Hashimoto Y, Koie T, Saitoh H, Yamaya K, Funyu T, Nishimura S, Ohyama C

  TheScientificWorldJournal 2013 268407  2013年 [査読有り][通常論文]
  
• Further examination of polysaccharides causing membrane fouling in membrane bioreactors (MBRs): Application of lectin affinity chromatography and MALDI-TOF/MS

  Katsuki Kimura, Ippei Tanaka, Shin-Ichiro Nishimura, Risho Miyoshi, Taro Miyoshi, Yoshimasa Watanabe

  WATER RESEARCH 46 17 5725 - 5734 2012年11月 [査読有り][通常論文]
   

  Membrane fouling remains a major obstacle for wider application of membrane bioreactors (MBRs) to wastewater treatment. Polysaccharides in mixed liquor suspensions in the reactors are thought to be mainly responsible for the evolution of membrane fouling in MBRs. However, details of polysaccharides causing membrane fouling in MBRs are still unknown. In this study, polysaccharides in a mixed liquor suspension of a pilot-scale MBR treating municipal wastewater were fractionated by using lectins, special proteins that bind to specific polysaccharides depending on their properties. Fouling potentials of the fractionated polysaccharides were assessed by bench-scale dead-end filtration tests. It was clearly shown that the degrees of fouling caused by fractionated polysaccharides were significantly different. The amounts of polysaccharides in each fraction could not explain the variations in the fouling, indicating the presence of polysaccharides with high specific fouling potentials. To investigate structures and origins of the polysaccharides with high fouling potentials, matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)/mass spectrometry (MS) analysis was applied to the fractionated polysaccharides after partial hydrolysis. Several mass peaks obtained could be assigned to fragments of structures of polysaccharides (i.e., oligosaccharides) reported in a database/literature. This is the first report showing the plausible structures of polysaccharides in MBRs based on MS. A deeper understanding and effective control of membrane fouling in MBRs could be achieved with information obtained by the approach used in this study. (C) 2012 Elsevier Ltd. All rights reserved.
• Tumour suppressor p16INK4a-anoikis-favouring decrease in N/O-glycan/cell surface sialylation by down-regulation of enzymes in sialic acid biosynthesis in tandem in a pancreatic carcinoma model

  Maho Amano, Hanna Eriksson, Joachim C. Manning, Katharina M. Detjen, Sabine Andre, Shin-Ichiro Nishimura, Janne Lehtio, Hans-Joachim Gabius

  FEBS JOURNAL 279 21 4062 - 4080 2012年11月 [査読有り][通常論文]
   

  Tumour suppressor p16INK4a is known to exert cell-cycle control via cyclin-dependent kinases. An emerging aspect of its functionality is the orchestrated modulation of N/O-glycosylation and galectin expression to induce anoikis in human Capan-1 pancreatic carcinoma cells. Using chemoselective N/O-glycan enrichment technology (glycoblotting) and product characterization, we first verified a substantial decrease in sialylation. Tests combining genetic (i.e. transfection with a2,6-sialyltransferase-specific cDNA) or metabolic (i.e. medium supplementation with N-acetylmannosamine to track down a bottleneck in sialic acid biosynthesis) engineering with cytofluorometric analysis of lectin binding indicated a role of limited substrate availability, especially for a2,6-sialylation, which switches off reactivity for anoikis-triggering homodimeric galectin-1. Quantitative MS analysis of protein level changes confirmed an enhanced galectin-1 presence along with an influence on glycosyltransferases (beta 1,4-galactosyltransferase-IV, a2,3-sialyltransferase-I) and detected p16INK4a-dependent down-regulation of two enzymes in the biosynthesis pathway for sialic acid [i.e. the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) and N-acetylneuraminic acid 9-phosphate synthase] (P < 0.001). By contrast, quantitative assessment for the presence of nuclear CMP-N-acetylneuraminic acid synthase (which is responsible for providing the donor for enzymatic sialylation that also acts as feedback inhibitor of the epimerase activity of GNE) revealed a trend for an increase. Partial restoration of sialylation in GNE-transfected cells supports the implied role of sialic acid availability for the glycophenotype. Fittingly, the extent of anoikis was reduced in double-transfected (p16INK4a/GNE) cells. Thus, a second means of modulating cell reactivity to the growth effector galectin-1 is established in addition to the common route of altering a2,6-sialyltransferase expression: regulating enzymes of the pathway for sialic acid biosynthesis.
• Molecular shuttle between extracellular and cytoplasmic space allows for monitoring of GAG biosynthesis in human articular chondrocytes.

  Hoshi H, Shimawaki K, Takegawa Y, Ohyanagi T, Amano M, Hinou H, Nishimura S

  Biochimica et biophysica acta 1820 1391 - 1398 9 2012年09月 [査読有り][通常論文]
  
• Determination of O-Glycosylation Heterogeneity Using a Mass-Spectrometric Method Retaining Sugar Modifications

  Naomi Manri, Yasuhiro Takegawa, Naoki Fujitani, Akihito Kaneko, Atsumu Hirabayashi, Shin-Ichiro Nishimura, Takeshi Sakamoto

  ANALYTICAL SCIENCES 28 7 723 - 727 2012年07月 [査読有り][通常論文]
   

  A mass-spectrometric method for a de novo determination of O-glycosylation heterogeneity was developed. We used a mild fragmentation technique, electron capture dissociation (ECD), which enables the determination of glycosylation sites as well as peptide sequencing. To demonstrate the correct identification of glycopeptides, we prepared a series of glycopeptides with the same peptide sequence and 6 different glycan modifications. ECD spectra were obtained at various electron energies, and were analyzed with the Mascot database-search engine. The obtained candidate glycopeptides were further validated by confirming the spectral overlap of ECD fragment peaks with the theoretical peaks. The results indicate that all glycopeptides were unambiguously identified, including glycosylation sites by combining ECD results with different electron energies for each glycopeptide.
• Aglycone-focused randomization of 2-difluoromethylphenyl-type sialoside suicide substrates for neuraminidases

  Hirokazu Kai, Hiroshi Hinou, Shin-Ichiro Nishimura

  BIOORGANIC & MEDICINAL CHEMISTRY 20 8 2739 - 2746 2012年04月 [査読有り][通常論文]
   

  A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycone-focused library of 2-difluoromethylphenyl alpha-sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compound library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic analysis of the selected inhibitors demonstrated that the modification of the aglycone moiety improved the K-I value with little change in the t(1/2) value of the enzyme activity relative to the basic skeleton (2). (C) 2012 Elsevier Ltd. All rights reserved.
• Strict stereocontrol by 2,4-O-di-tert-butylsilylene group on β-glucuronylations.

  Furukawa T, Hinou H, Nishimura S

  Organic letters 14 8 2102 - 2105 8 2012年04月 [査読有り][通常論文]
   

  Strict beta-controlled glucuronylations without classical neighboring-group participation were achieved by the assistance of a 2,4-O-di-tert-butylsilylene group. Comparison of activation conditions and conformational analysis indicated that the strict beta-selectivity was achieved by steric hindrance of the 2,4-O-di-tert-butylsilylene group and not by complex glycosyl intermediates.
• Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates

  Yayoi Yoshimura, Aaron S. Nudelman, Steven B. Levery, Hans H. Wandall, Eric P. Bennett, Ole Hindsgaul, Henrik Clausen, Shin-Ichiro Nishimura

  GLYCOBIOLOGY 22 3 429 - 438 2012年03月 [査読有り][通常論文]
   

  Mucin-type glycosylation [alpha-N-acetyl-d-galactosamine (alpha-GalNAc)-O-Ser/Thr] on proteins is initiated biosynthetically by 16 homologous isoforms of GalNAc-Ts (uridine diphosphate-GalNAc:polypeptide N-acetylgalactosaminyltransferases). All the GalNAc-Ts consist of a catalytic domain and a lectin domain. Previous reports of GalNAc-T assays toward peptides and alpha-GalNAc glycopeptides showed that the lectin domain recognized the sugar on the substrates and affected the reaction; however, the details are not clear. Here, we report a new strategy to give insight on the sugar recognition ability and the function of the GalNAc-T3 lectin domain using chemically synthesized natural-type (alpha-GalNAc-O-Thr) and unnatural-type [beta-GalNAc-O-Thr, alpha-Fuc-O-Thr and beta-GlcNAc-O-Thr] MUC5AC glycopeptides. GalNAc-T3 is one of isoforms expressed in various organs, its substrate specificity extensively characterized and its anomalous expression has been identified in several types of cancer (e.g. pancreas and stomach). The glycopeptides used in this study were designed based on a preliminary peptide assay with a sequence derived from the MUC5AC tandem repeat. Through GalNAc-T3 and lectin-inactivated GalNAc-T3, competition assays between the glycopeptide substrates and product analyses (MALDI-TOF MS, RP-HPLC and ETD-MS/MS), we show that the lectin domain strictly recognized GalNAc on the substrate and this specificity controlled the glycosylation pathway.
• Phosphorylcholine Self-Assembled Monolayer-Coated Quantum Dots: Real-Time Imaging of Live Animals by Cell Surface Mimetic Glyco-Nanoparticles

  Shin-Ichiro Nishimura

  CLINICS IN LABORATORY MEDICINE 32 1 73 - + 2012年03月 [査読有り][通常論文]
   

  Multifunctional phosphorylcholine self-assembled monolayer-coated quantum dots (PCSAM-QDs) displaying glycoconjugates with excellent solubility and long-term stability in aqueous solution without loss of quantum yields were developed. Live animal imaging by PCSAM-QDs displaying various carbohydrates (glyco-PC-QDs) uncovered the evidence of an essential role of the terminal sialic acid residues for achieving prolonged in vivo lifetime and biodistribution of this new class of nanoparticles.
• A cross-linked polymer possessing a high density of hydrazide groups: high-throughput glycan purification and labeling for high-performance liquid chromatography analysis

  Midori Abe, Hideyuki Shimaoka, Masao Fukushima, Shin-Ichiro Nishimura

  POLYMER JOURNAL 44 3 269 - 277 2012年03月 [査読有り][通常論文]
   

  Previously, we communicated that rapid and efficient purification of carbohydrates can be achieved by employing sugar-specific chemical ligation with hydrazide-functionalized polymer beads; we termed this process 'glycoblotting'. The polymer beads are designed to recover carbohydrates by an imine exchange reaction in which hydrazone bonds between the beads and carbohydrates are transferred to oxime bonds between the aminooxy probe and the carbohydrates. To apply our concept to reductive amination with common fluorescent dyes, such as 2-aminobenzamide, the method for releasing the carbohydrates from the beads was examined, and we found that heating the beads with several percentages of acetic acid was efficient. Additionally, we obtained fundamental data on our novel method, such as the recovery ratio, the quantitative capability and the reproducibility. From the results, we concluded that rapid and accurate glycan analysis can be achieved with this novel method. Overall, these novel technologies represent a significant advance toward more efficient glycan analyses, especially by using high-performance liquid chromatography. Polymer Journal (2012) 44, 269-277; doi:10.1038/pj.2011.125; published online 30 November 2011
• Effects of Single Genetic Damage in Carbohydrate-Recognizing Proteins in Mouse Serum N-Glycan Profile Revealed by Simple Glycotyping Analysis

  Maho Amano, Ryo Hashimoto, Shin-Ichiro Nishimura

  CHEMBIOCHEM 13 3 451 - 464 2012年02月 [査読有り][通常論文]
   

  Gene knock-out of C-type lectin receptors expressed in dendritic cells induced significant alteration of serum N-glycans compared with that of gender-matched controls. Glycotyping analysis suggested that putative-core fucosylation is strongly influenced by differences in the dominant mechanisms after carbohydrate recognition by pattern-recognition receptors, endocytosis of ligands, or induction of cytokines/chemokines. However, the loss of galectin-9, a ligand for T-helper type 1-specific cell-surface molecule, did not affect most N-glycan profiles. Interestingly, lack of the Chst3 gene (chondroitin 6-sulfotransferase) appeared to influence markedly the expression of most N-glycans, especially highly modified glycoforms bearing multiple Neu5Gc, Fuc, and LacNAc units. In contrast, genetic mutations in B4galnt1 and B4galnt2 (GalNAc transferase, responsible for the synthesis of many gangliosides) induced no discernable alteration. These results indicate that the biosynthesis of N-glycans of serum glycoproteins can be affected not only by direct genetic mutations in the glycosyltransferases but also by changes in metabolite availability in sugar nucleotide synthesis and Golgi N-glycosylation pathways caused concertedly in whole cells, tissues, and organs by milder deficiencies in immune cell-surface lectins. Many common chronic conditions, such as autoimmunity, metabolic syndrome, and aging/dementia result.
• IFN-γ elevates airway hyperresponsiveness via upregulation of neurokinin A/neurokinin-2 receptor signaling in a severe asthma model

  Kobayashi M, Ashino S, Shiohama Y, Wakita D, Kitamura H, Nishimura T

  Eur J Immunol 42 2 393 - 402 2012年02月 [査読有り][通常論文]
   

  The adoptive transfer of OVA-specific Th1 cells into WT mice followed by OVA inhalation induces a significant elevation of airway hyper-responsiveness (AHR) with neutrophilia but not mucus hypersecretion. Here, we demonstrate that the airway inflammation model, pathogenically characterized as severe asthma, was partly mimicked by i.n. administration of IFN-gamma. The administration of IFN-gamma instead of Th1 cells caused AHR elevation but not neutrophilia, and remarkably induced neurokinin-2 receptor (NK2R) expression along with neurokinin A (NKA) production in the lung. To evaluate whether NKA/NK2R was involved in airway inflammation, we first investigated the role of NKA/NK2R-signaling in airway smooth muscle cells (ASMCs) in vitro. NK2R mRNA expression was significantly augmented in tracheal tube-derived ASMCs of WT mice but not STAT-1-/- mice after stimulation with IFN-gamma. In addition, methacholine-mediated Ca2+ influx into the ASMCs was significantly reduced in the presence of NK2R antagonist. Moreover, the NK2R antagonist strongly inhibited IFN-gamma-dependent AHR elevation in vivo. Thus, these results demonstrated that IFN-gamma directly acts on ASMCs to elevate AHR via the NKA/NK2R-signaling cascade. Our present findings suggested that NK2R-mediated neuro-immuno crosstalk would be a promising target for developing novel drugs in Th1-cell-mediated airway inflammation, including severe asthma.
• An efficient protocol for the solid-phase synthesis of glycopeptides under microwave irradiation

  Fayna Garcia-Martin, Hiroshi Hinou, Takahiko Matsushita, Shun Hayakawa, Shin-Ichiro Nishimura

  ORGANIC & BIOMOLECULAR CHEMISTRY 10 8 1612 - 1617 2012年 [査読有り][通常論文]
   

  A standardized and smooth protocol for solid-phase glycopeptides synthesis under microwave irradiation was developed. Double activation system was proved to allow for highly efficient coupling of Tn-Ser/Thr and bulky core 2-Ser/Thr derivatives. Versatility and robustness of the present strategy was demonstrated by constructing a Mucine-1 (MUC1) fragment and glycosylated fragments of tau protein. The success of this approach relies on the combination of microwave energy, a resin consisting totally of polyethylene glycol, a low excess of sugar amino acid and the "double activation" method.
• Glycomics for Drug Discovery: Metabolic Perturbation in Androgen-Independent Prostate Cancer Cells Induced by Unnatural Hexosamine Mimics

  Shin-Ichiro Nishimura, Megumi Hato, Satoshi Hyugaji, Fei Feng, Maho Amano

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 51 14 3386 - 3390 2012年 [査読有り][通常論文]
  
• H-bonding promotion of peptide solubility and cyclization by fluorinated alcohols

  Hiroshi Hinou, Kei Hyugaji, Fayna Garcia-Martin, Shin-Ichiro Nishimura, Fernando Albericio

  RSC ADVANCES 2 7 2729 - 2731 2012年 [査読有り][通常論文]
   

  A promotion effect for peptide cyclization by strong H-bonding of fluorinated alcohols was revealed via a synthetic study of a cyclic AFGP skeleton. Combination of fluorinated alcohol-DCM solvent system and DIC-additive system afforded the cyclic hexapeptide skeleton in more than 80% yield. The ratio of intra- vs. inter-peptide condensation depended upon the H-bonding donor strength. This effect was quenched by H-bond acceptor solvents.
• Alterations of High-Mannose Type N-Glycosylation in Human and Mouse Osteoarthritis Cartilage

  Atsushi Urita, Tomoya Matsuhashi, Tomohiro Onodera, Hiroaki Nakagawa, Megumi Hato, Maho Amano, Naoki Seito, Akio Minami, Shin-Ichiro Nishimura, Norimasa Iwasaki

  ARTHRITIS AND RHEUMATISM 63 11 3428 - 3438 2011年11月 [査読有り][通常論文]
   

  Objective. The process of N-glycosylation is involved in the pathogenesis of various diseases. However, little is known about the contribution of changes in N-glycans in osteoarthritis (OA). The aim of this study was to identify the alterations in N-glycans in human OA cartilage, to characterize the messenger RNA (mRNA) expression of N-glycan biosynthesis enzyme genes (N-glycogenes) in mouse articular chondrocytes during cartilage degradation, and to analyze the relationship between altered N-glycan patterns and mechanisms of cartilage degradation. Methods. Alterations in N-glycans were analyzed in human OA cartilage and degraded mouse cartilage by high-performance liquid chromatography and mass spectrometry. N-glycogene mRNA expression in mouse chondrocytes was measured using reverse transcription-polymerase chain reaction. To assess the relationship between the altered N-glycans and degradation of mouse cartilage, experiments involving either knockdown or overexpression of N-glycogenes were performed in mouse articular chondrocytes. Results. Alterations in high-mannose type N-glycans were observed in both human OA cartilage and degraded mouse cartilage. The expression of beta 1,2N-acetylglucosaminyltransferase I (GlcNAc-TI) mRNA, which converts high-mannose type N-glycans, was significantly increased in degraded mouse cartilage. Mouse chondrocytes with suppressed GlcNAc-TI expression had reduced levels of matrix metalloproteinase 13 (MMP-13) and ADAMTS-5 (aggrecanase 2) mRNA following stimulation with interleukin-1 alpha (IL-1 alpha). In contrast, mouse chondrocytes overexpressing GlcNAc-TI had increased levels of MMP-13 and ADAMTS-5 mRNA following stimulation with IL-1 alpha. Conclusion. These findings indicate that alterations in high-mannose type N-glycans and N-glycogenes in chondrocytes correlate with the release of MMP-13 and ADAMTS-5 during cartilage degradation. These findings suggest that N-glycans play a crucial role in the initiation and progression of OA.
• Alteration of the N-glycome of bovine milk glycoproteins during early lactation

  Shota Takimori, Hideyuki Shimaoka, Jun-Ichi Furukawa, Tadashi Yamashita, Maho Amano, Naoki Fujitani, Yasuhiro Takegawa, Lennart Hammarstrom, Imre Kacskovics, Yasuro Shinohara, Shin-Ichiro Nishimura

  FEBS JOURNAL 278 19 3769 - 3781 2011年10月 [査読有り][通常論文]
   

  Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc)/N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG(2) better than IgG(1), strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG(2) from the udder to blood, rather than to secrete IgG(1) into colostrum.
• A potential glucuronate glycosyl donor with 2-O-acyl-6,3-lactone structure: efficient synthesis of glycosaminoglycan disaccharides

  Takayuki Furukawa, Hiroshi Hinou, Ken Shimawaki, Shin-Ichiro Nishimura

  Tetrahedron Letters 52 43 5567 - 5570 2011年10月 [査読有り][通常論文]
  
• Development of quantitative plasma N-glycoproteomics using label-free 2-D LC-MALDI MS and its applicability for biomarker discovery in hepatocellular carcinoma

  Takeshi Ishihara, Isao Fukuda, Atsushi Morita, Yoshihiko Takinami, Hiroyuki Okamoto, Shin-Ichiro Nishimura, Yoshito Numata

  JOURNAL OF PROTEOMICS 74 10 2159 - 2168 2011年09月 [査読有り][通常論文]
   

  There has been rapid progress in the development of clinical proteomic methodologies with improvements in mass spectrometric technologies and bioinformatics, leading to many new methodologies for biomarker discovery from human plasma. However, it is not easy to find new biomarkers because of the wide dynamic range of plasma proteins and the need for their quantification. Here, we report a new methodology for relative quantitative proteomic analysis combining large-scale glycoproteomics with label-free 2-D LC-MALDI MS. In this method, enrichment of glycopeptides using hydrazide resin enables focusing on plasma proteins with lower abundance corresponding to the tissue leakage region. On quantitative analysis, signal intensities by 2-D LC-MALDI MS were normalized using a peptide internal control, and the values linked to LC data were treated with DeView (TM) software. Our proteomic method revealed that the quantitative dynamic ranged from 10(2) to 10(6) pg/mL of plasma proteins with good reproducibility, and the limit of detection was of the order of a few ng/mL of proteins in biological samples. To evaluate the applicability of our method for biomarker discovery, we performed a feasibility study using plasma samples from patients with hepatocellular carcinoma, and identified biomarker candidates, including ceruloplasmin, alpha-1 antichymotrypsin, and multimerin-1. (C) 2011 Elsevier B.V. All rights reserved.
• 前立腺癌患者血清に含まれるAcute Phase Proteinの糖鎖構造解析

  藤村 務, 數野 彩子, 古川 潤一, 篠原 康郎, 西村 紳一郎, 藤目 真, 村山 季美枝

  日本生化学会大会プログラム・講演要旨集 84回 4P - 0600 (公社)日本生化学会 2011年09月
• Toward Green and Sustainable Chemical Glycosylation: Enhanced Lewis Acidity of Recyclable Solid Super Acid Catalyst, SO4/ZrO2 by CaCl2 Doping

  Hiroshi Hinou, Naohiro Saito, Takahiro Maeda, Masao Matsuda, Yuichi Kamiya, Shin-Ichiro Nishimura

  Journal of Carbohydrate Chemistry 30 7-9 575 - 586 2011年09月01日 [査読有り][通常論文]
  
• Importance of Sialic Acid Residues Illuminated by Live Animal Imaging Using Phosphorylcholine Self-Assembled Monolayer-Coated Quantum Dots

  Tatsuya Ohyanagi, Noriko Nagahori, Ken Shimawaki, Hiroshi Hinou, Tadashi Yamashita, Akira Sasaki, Takashi Jin, Toshihiko Iwanaga, Masataka Kinjo, Shin-Ichiro Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 133 32 12507 - 12517 2011年08月 [査読有り][通常論文]
   

  Glycans are expected to be one of the potential signal molecules for controlling drug targeting/delivery or long-term circulation of biopharmaceuticals. However, the effect of the carbohydrates of artificially glycosylated derivatives on in vivo dynamic distribution profiles after intravenous injection of model animals remains unclear due to the lack of standardized methodology and a suitable platform. We report herein an efficient and versatile method for the preparation of multifunctional quantum dots (QDs) displaying common synthetic glycosides with excellent solubility and long-term stability in aqueous solution without loss of quantum yields. Combined use of an aminooxy-terminated thiol derivative, 11,11'-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino hexa(ethyleneglycol)], and a phosphorylcholine derivative, 11-mercaptoundecylphosphorylcholine, provided QDs with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific protein adsorption concurrently. In vivo near-infrared (NIR) fluorescence imaging of phosphorylcholine self-assembled monolayer (SAM)-coated QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse revealed that distinct long-term delocalization over 2 h can be achieved in cases of QDs modified with alpha-sialic acid residue (Neu5Ac-PC-QDs) and control PC-QDs, while QDs bearing other common sugars, such as alpha-glucose (Glc-PC-QDs), alpha-mannose (Man-PC-QDs), alpha-fucose (Fuc-PC-QDs), lactose (Lac-PC-QDs), beta-glucuronic acid (GlcA-PC-QDs), N-acetyl-beta-D-glucosamine (GlcNAc-PC-QDs), and N-acetyl-beta-D-galactosamine (GalNAc-PC-QDs) residues, accumulated rapidly (5-10 min) in the liver. Sequential enzymatic modifications of GlcNAc-PC-QDs gave Gal beta 1,4GlcNAc-PC-QDs (LacNAc-PC-QDs), Gal beta 1,4(Fuc alpha 1,3)GlcNAc-PC-QDs (Le(x)-PC-QDs), Neu5Ac alpha 2,3Gal beta 1,4GlcNAc-PC-QDs (sialyl LacNAc-PC-QDs), and Neu5Ac alpha 2,3Gal beta 1,4(Fuc alpha 1,3)GlcNAc-PC-QDs (sialyl Le(x)-PC-QDs) in quantitative yield as monitored by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses. Live animal imaging uncovered for the first time that Le(x)-PC-QDs also distributed rapidly in the liver after intravenous injection and almost quenched over 1 h in similar profiles to those of LacNAc-PC-QDs and Lac-PC-QDs. On the other hand, sialyl LacNAc-PC-QDs and sialyl Le(x)-PC-QDs were still retained stably in the whole body after 2 h, while they showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution. The present results clearly visualize the evidence of an essential role of the terminal sialic acid residue(s) for achieving prolonged in vivo lifetime and biodistribution of various glyco-PC-QDs as a novel class of functional platforms for nanomaterial-based drug targeting/delivery. A standardized protocol using multifunctional PC-QDs should facilitate live animal imaging of ligand-displayed QDs using versatile NIR fluorescence photometry without influence of size-dependent accumulation/excretion pathway for nanoparticles (e.g., viruses) >10 nm in hydrodynamic diameter by the liver.
• Biomechanical and histologic evaluation of tissue engineered ligaments using chitosan and hyaluronan hybrid polymer fibers: A rabbit medial collateral ligament reconstruction model

  Toru Irie, Tokifumi Majima, Naohiro Sawaguchi, Tadanao Funakoshi, Shin-ichiro Nishimura, Akio Minami

  JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A 97A 2 111 - 117 2011年05月 [査読有り][通常論文]
   

  In this study, we used a rabbit medial collateral ligament reconstruction model to evaluate a novel chitosan-based hyaluronan hybrid polymer fiber scaffold for ligament tissue engineering and to examine whether mechanical forces exerted in an in vivo model increased extracellular matrix production by seeded fibroblasts. Scaffolds were used 2 weeks after incubation with fibroblasts obtained from the same rabbit in a cell-seeded scaffold (CSS) group and without cells in a noncell-seeded scaffold (NCSS) group. At 3, 6, and 12 weeks after surgery, the failure loads of the engineered ligaments in the CSS groups were significantly greater than those in the NCSS groups. At 6 weeks after surgery, the reconstructed tissue of the CSS group was positive for type I collagen, whereas that in the NCSS group was negative for type I collagen. At 12 weeks after surgery, the reconstructed tissue stained positive for type I collagen in the CSS group, but negative in the NCSS group. Our results indicate that the scaffold material enhanced the production of type I collagen and led to improved mechanical strength in the engineered ligament in vivo. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 97A: 111-117, 2011.
• Insight into Glycan Diversity and Evolutionary Lineage Based on Comparative Avio-N-glycomics and Sialic Acid Analysis of 88 Egg Whites of Galloanserae

  Kazuko Hirose, Maho Amano, Ryo Hashimoto, Yuan Chuan Lee, Shin-Ichiro Nishimura

  BIOCHEMISTRY 50 21 4757 - 4774 2011年05月 [査読有り][通常論文]
   

  A large set of glycome information was obtained from egg white proteins of 88 samples from Galloanserae (63 Anseriformes and 25 Galliformes). The data were obtained on whole N-glycan structures and types of sialic acids of these egg whites by glycoblotting-based high-throughput and quantitative glycomics. The results revealed clear trends and complexity patterns as well as diversity among taxonomic groups. It is well-known that chicken, a representative domesticated poultry involved in Galliformes, can become an influenza host. However, our data demonstrate that duck, wild goose, and swan of Anseriformes are representative migratory birds that are known as natural hosts of the influenza virus. Hierarchical clustering analysis of the expression pattern of N-glycome (total of 61 N-glycan peaks) revealed that the members of Galloanserae can be classified into two major groups and five submajor clusters (clusters 1-5) on the basis of simple m/z values obtained by MALDI-TOF MS. It is dear that expression patterns of N-glycomes in the five dusters are influenced significantly by the features such as the body size of the birds, rather than by the difference of the family. On the other hand, quantitative analysis showed that the total amounts of sialic acids in egg whites of Galliformes were distinctly larger than those of Anseriformes. However, it was also revealed in Anseriformes that Neu5Gc and KDN, in addition to common Neu5Ac, were expressed significantly in both N- and O-glycans of glycoproteins and glycosphingolipids, suggesting the influence of their lifestyles and diet. This is the first report that KDN exists in egg white. These results and the environmental factors are discussed preliminarily with respect to their evolutionary lineage.
• A Strategy for Neuraminidase Inhibitors Using Mechanism-Based Labeling Information

  Hiroshi Hinou, Risho Miyoshi, Yasuaki Takasu, Hirokazu Kai, Masaki Kurogochi, Shingo Arioka, Xiao-Dong Gao, Nobuaki Miura, Naoki Fujitani, Shinya Omoto, Tomokazu Yoshinaga, Tamio Fujiwara, Takeshi Noshi, Hiroko Togame, Hiroshi Takemoto, Shin-Ichiro Nishimura

  CHEMISTRY-AN ASIAN JOURNAL 6 4 1048 - 1056 2011年04月 [査読有り][通常論文]
   

  A potent inhibitor for Vibrio cholerae neuraminidase (VCNA) was developed by using a novel two-step strategy, a target amino acid validation using mechanism-based labeling information, and a potent inhibitor search using a focused library. The labeling information suggested the hidden dynamics of a loop structure of VCNA, which can be a potential target of the novel inhibitor. A focused library composed of 187 compounds was prepared from a 9-azide derivative of 2,3-dehydro-N-acetylneuraminic acid (DANA) to interrupt the function of the loop of the labeled residues. Inhibitor 3c showed potent inhibition properties and was the strongest inhibitor with FANA, a N-trifluoroacetyl derivative of DANA. Validation studies of the inhibitor with a detergent and a Lineweaver-Burk plot suggested that the 9-substitution group would interact hydrophobically with the target loop moiety, adding a noncompetitive inhibition property to the DANA skeleton. This information enabled us to design compound 4 having the combined structure of 3c and FANA. Compound 4 showed the most potent inhibition (K-i = 73 nm, mixed inhibition) of VCNA with high selectivity among the tested viral, bacterial, and mammal neuraminidases.
• An Efficient Approach for the Characterization of Mucin-Type Glycopeptides: The Effect of O-Glycosylation on the Conformation of Synthetic Mucin Peptides

  Ryo Hashimoto, Naoki Fujitani, Yasuhiro Takegawa, Masaki Kurogochi, Takahiko Matsushita, Kentaro Naruchi, Naoki Ohyabu, Hiroshi Hinou, Xiao Dong Gao, Naomi Manri, Hiroyuki Satake, Akihito Kaneko, Takeshi Sakamoto, Shin-Ichiro Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 17 8 2393 - 2404 2011年02月 [査読有り][通常論文]
   

  Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of alpha-GalNAc residues by UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by alpha-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of alpha-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six alpha-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four alpha-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of alpha-GalNAc at Thr10 of MUC4 stabilizes specifically a beta-like extended backbone structure at this area, whereas other synthetic models with a single alpha-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.
• Membrane-Bound Stable Glycosyltransferases: Highly Oriented Protein Immobilization by a C-Terminal Cationic Amphipathic Peptide

  Kentaro Naruchi, Shin-Ichiro Nishimura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 50 6 1328 - 1331 2011年 [査読有り][通常論文]
  
• TOWARD AUTOMATED GLYCAN ANALYSIS

  Shin-Ichiro Nishimura

  ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL 65 65 219 - 271 2011年 [査読有り][通常論文]
  
• The extract of Japanese soybean, Kurosengoku activates the production of IL-12 and IFN-γ by DC or NK1.1(+) cells in a TLR4- and TLR2-dependent manner

  Tanaka S, Koizumi S, Makiuchi N, Aoyagi Y, Quivy E, Mitamura R, Kano T, Wakita D, Chamoto K, Kitamura H, Nishimura T

  Cell Immunol 266 2 135 - 42 2011年 [査読有り][通常論文]
   

  During the search for immuno-improving foods, we found that a variety of the Japanese soybean, Glycine max cv. Kurosengoku (Kurosengoku), which activated Type-1 immunity in a Toll-like receptor (TLR)4-and TLR2-dependent manner. Namely, the extract of Kurosengoku first caused production of IL-12 from DC and sequentially induced IFN-gamma production by NK1.1(+) NK cells and NKT cells. The IFN-gamma production was significantly blocked by neutralizing mAb against IL-12 or TLR4- and TLR2-deficient condition, indicating that TLR4- and TLR2-dependent activation of DC to produce IL-12 was essential for the production of IFN-gamma from spleen cells by Kurosengoku. Moreover, the extract of Kurosengoku also enhanced production of IFN-gamma from human PBMC by co-stimulation with anti-CD3 mAb in a TLR2- and TLR4-dependent manner. Thus, our findings strongly suggest that Kurosengoku might a novel immuno-improving food, which would be a useful tool for preventing the tip of immune balance in developed countries. (C) 2010 Elsevier Inc. All rights reserved.
• Glycoblotting-Assisted O-Glycomics: Ammonium Carbamate Allows for Highly Efficient O-Glycan Release from Glycoproteins

  Yoshiaki Miura, Kentaro Kato, Yasuhiro Takegawa, Masaki Kurogochi, Jun-ichi Furukawa, Yasuro Shinohara, Noriko Nagahori, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura

  ANALYTICAL CHEMISTRY 82 24 10021 - 10029 2010年12月 [査読有り][通常論文]
   

  Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hyclrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers.
• Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay

  Masaki Kurogochi, Takahiko Matsushista, Maho Amano, Jun-ichi Furukawa, Yasuro Shinohara, Masato Aoshima, Shin-Ichiro Nishimura

  MOLECULAR & CELLULAR PROTEOMICS 9 11 2354 - 2368 2010年11月 [査読有り][通常論文]
   

  Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model. Molecular & Cellular Proteomics 9:2354-2368, 2010.
• Artificial Golgi Apparatus: Globular Protein-like Dendrimer Facilitates Fully Automated Enzymatic Glycan Synthesis

  Takahiko Matsushita, Izuru Nagashima, Masataka Fumoto, Takashi Ohta, Kuriko Yamada, Hiroki Shimizu, Hiroshi Hinou, Kentaro Naruchi, Takaomi Ito, Hirosato Kondo, Shin-Ichiro Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 132 46 16651 - 16656 2010年11月 [査読有り][通常論文]
   

  Despite the growing importance of synthetic glycans as tools for biological studies and drug discovery, a lack of common methods for the routine synthesis remains a major obstacle. We have developed a new method for automated glycan synthesis that employs the enzymatic approach and a dendrimer as an ideal support within the chemical process. Recovery tests using a hollow fiber ultrafiltration module have revealed that monodisperse G6 (MW = 58 kDa) and G7 (MW = 116 kDa) poly(amidoamine) dendrimers exhibit a similar profile to BSA (MW = 66 kDa). Characteristics of the globular protein-like G7 dendrimer with high solubility and low viscosity in water greatly enhanced throughput and efficiency in automated synthesis while random polyacrylamide-based supports entail significant loss during the repetitive reaction/separation step. The present protocol allowed for the fully automated enzymatic synthesis of sialyl Lewis X tetrasaccharide derivatives over a period of 4 days in 16% overall yield from a simple N-acetyl-D-glucosamine linked to an aminooxy-functionalized G7 dendrimer.
• Chemical Synthesis, Folding, and Structural Insights into O-Fucosylated Epidermal Growth Factor-like Repeat 12 of Mouse Notch-1 Receptor

  Kazumi Hiruma-Shimizu, Kensaku Hosoguchi, Yan Liu, Naoki Fujitani, Takashi Ohta, Hiroshi Hinou, Takahiko Matsushita, Hiroki Shimizu, Ten Feizo, Shin-Ichiro Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 132 42 14857 - 14865 2010年10月 [査読有り][通常論文]
   

  Notch receptors are cell surface glycoproteins that play key roles in a number of developmental cascades in metazoa. The extracellular domains of Notch-1 receptors are composed of 36 tandem epidermal growth factor (EGF)-like repeats, many of which are modified at highly conserved consensus sites by an unusual form of O-glycan, with O-fucose. The O-fucose residues on certain EGF repeats may be elongated. In mammalian cells this can be a tetrasaccharide, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Fuc alpha 1 ->. This elongation process is initiated by the action of O-fucose-specific beta 1,3 N-acetylglucosaminyltransferases of the Fringe family. There is evidence that the addition of GlcNAc by Fringe serves as an essential modulator of the interaction of Notch with its ligands and the triggering of activation. Here we describe the efficient synthesis, folding, and structural characterization of EGF repeat 12 (EGF 12) of a mouse Notch-1 receptor bearing different O-fucose glycan chains. We demonstrate that the three disulfide bonds, Cys(456)-Cys(467) (C1-C3), Cys(461)-Cys(476) (C2-C4), and Cys(478)-Cys(487) (C5-C6) were correctly formed in the nonglycosylated as well as the O-fucosylated forms of EGF 12. Three-dimensional structural studies by NMR reveal that the methyl group of fucose is in close contact with ILe(475), Met(477), Pro(478) residues and this stabilizes the conformation of the antiparallel beta-sheet of EGF 12. The addition of the GlcNAc residue on O-fucosylated EGF 12 induces a significant conformational change in the adjacent tripeptide sequence, Gln(462)Asn(463)Asp(464), which is a motif involved in the natural, enzymatic O-fucosylation at the conserved site (Cys(461)X(4)Ser/ThrCys(467)).
• A new class of mechanism-based inhibitors for Trypanosoma cruzi trans-sialidase and their influence on parasite virulence

  Sebastiao T. Carvalho, Mauro Sola-Penna, Isadora A. Oliveira, Samuel Pita, Arlan S. Goncalves, Bianca C. Neves, Francisco R. Sousa, Leonardo Freire-de-Lima, Masaki Kurogochi, Hiroshi Hinou, Shin-Ichiro Nishimura, Lucia Mendonca-Previato, Jose O. Previato, Adriane R. Todeschini

  GLYCOBIOLOGY 20 8 1034 - 1045 2010年08月 [査読有り][通常論文]
   

  One of the most interesting aspects of Trypanosoma cruzi is its adaptation to obtain sialic acid from its host, fulfilling this need exclusively through the reaction catalyzed by enzymatically active trans-sialidase (aTS), thought to play an important role in the pathogenesis of Chagas' disease. Herein, we report that 2-difluoromethyl-4-nitrophenyl-3,5-dideoxy-d-glycero-alpha-d-galacto-2-nonulopyranosid acid (NeuNAcFNP) inactivates aTS time- and dose-dependently, and this inhibition was not relieved removing the inhibitor. Also, NeuNAcFNP causes a decrease in infection of mammalian cells. Characterization of labeled aTS by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that inactivation of the enzyme occurs through formation of a covalent bond between Arg245 and Asp247 and the inhibitor aglycone. Participation of Asp247 in the catalytic mechanism was proved by constructing a TSD247A mutant, which presents only residual activity. Molecular dynamic simulations indicate that the D247A mutation results in a more open catalytic cleft. In summary, NeuNAcFNP is the first reported mechanism-based inhibitor of aTS, representing a new template for drug design and opening new possibilities for chemotherapy of Chagas' disease, as well as for the elucidation of aTS function in T. cruzi pathogenesis and biology.
• Identification of glycosylated exendin-4 analogue with prolonged blood glucose-lowering activity through glycosylation scanning substitution

  Taichi Ueda, Takaomi Ito, Kazuyoshi Tomita, Hiroko Togame, Masataka Fumoto, Kenji Asakura, Takeo Oshima, Shin-Ichiro Nishimura, Kohji Hanasaki

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 20 15 4631 - 4634 2010年08月 [査読有り][通常論文]
   

  Exendin-4, a glucagon-like peptide 1 receptor agonist, is a potent therapeutic xenopeptide hormone for the treatment of type 2 diabetes. In order to further improve in vivo activity, we examined the introduction of sialyl N-acetyllactosamine (sialyl LacNAc) to exendin-4. The glycosylated analogue having sialyl LacNAc at position 28 was found to have improved in vivo activity with prolonged glucose-lowering activity. (C) 2010 Elsevier Ltd. All rights reserved.
• An Efficient Approach to the Discovery of Potent Inhibitors against Glycosyltransferases

  Kensaku Hosoguchi, Takahiro Maeda, Jun-ichi Furukawa, Yasuro Shinohara, Hiroshi Hinou, Mitsuaki Sekiguchi, Hiroko Togame, Hiroshi Takemoto, Hirosato Kondo, Shin-Ichiro Nishimura

  JOURNAL OF MEDICINAL CHEMISTRY 53 15 5607 - 5619 2010年08月 [査読有り][通常論文]
   

  We describe a standardized approach for searching potent and selective inhibitors of glycosyltransferases by high throughput quantitative MALDI-TOFMS-based screening of focused compound libraries constructed by 1,3-dipolar cycloaddition of the desired azidosugar nucleotides with various alkynes. An aminooxy-functionalized reagent with a stable isotope was conjugated with oligosaccharides to afford glycopeptides as acceptor substrates with improved ion sensitivity. Enhanced ionization potency of new substrates allowed for MALDI-TOFMS-based facile and quantitative analysis of enzymatic glycosylation in the presence of glycosyl donor substrates. A non-natural synthetic sugar nucleotide was identified to be the first highly specific inhibitor for rat recombinant alpha 2,3-(N)-sialyltransferase (alpha 2,3ST, IC(50) = 8.2 mu M), while this compound was proved to become a favorable substrate for rat recombinant alpha 2,6-(N)-sialyltransferase (alpha 2,6ST, K(m) = 125 mu M). Versatility of this strategy was demonstrated by identification of two selective inhibitors for human recombinant alpha 1,3-fucosyltransferase V (alpha 1,3-FucT, K(i) = 293 nM) and alpha 1,6-fucosyltransferase VIII (alpha 1,6-FucT, K(i) = 13.8 mu M).
• Unexpected Tolerance of Glycosylation by UDP-GalNAc:Polypeptide alpha-N-Acetylgalactosaminyltransferase Revealed by Electron Capture Dissociation Mass Spectrometry: Carbohydrate as Potential Protective Groups

  Yayoi Yoshimura, Takahiko Matsushita, Naoki Fujitani, Yasuhiro Takegawa, Haruhiko Fujihira, Kentarou Naruchi, Xiao-Dong Gao, Naomi Manri, Takeshi Sakamoto, Kentaro Kato, Hiroshi Hinou, Shin-Ichiro Nishimura

  BIOCHEMISTRY 49 28 5929 - 5941 2010年07月 [査読有り][通常論文]
   

  UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs, EC 2.4.1.41), a family of key enzymes that initiate posttranslational modification with O-glycans in mucin synthesis by introduction of alpha-GalNAc residues, are structurally composed of a catalytic domain and a lectin domain. It has been known that multiple Ser/Thr residues are assigned in common mucin glycoproteins as potential O-glycosylation sites and more than 20 distinct isoforms of this enzyme family contribute to produce densely O-glycosylated mucin glycoproteins. However, it seems that the functional role of the lectin domain of ppGalNAcTs remains unclear. We considered that electron capture dissociation mass spectrometry (ECD-MS), a promising method for highly selective fragmentation at peptide linkages of glycopeptides to generate unique c and z series of ions, should allow for precise structural characterization to uncover the mechanism in O-glycosylation of mucin peptides by ppGalNAcTs. In the present study, it was demonstrated that a system composed of an electrospray source, a linear RFQ ion trap that isolates precursor ions, the ECD device, and a TOF mass spectrometer is a nice tool to identify the preferential O-glycosylation sites without any decomposition of the carbohydrate moiety. It should be noted that electrons used for ECD are accelerated within a range from 1.75 to 9.75 eV depending on the structures of glycopeptides of interest. We revealed for the first time that additional installation of a alpha-GalNAc residue at potential glycosylation sites by ppGalNAcT2 proceeds smoothly in various unnatural glycopeptides having alpha-Man, alpha-Fuc, and beta-Gal residues as well as alpha-GalNAc residues. The results may suggest that ppGalNAcT2 did not differentiate totally presubstituted sugar residues in terms of configuration of functional groups, D-, L-configuration, and even alpha-, beta-stereochemistry at an anomeric carbon atom when relatively short synthetic peptides were employed for the acceptor substrates. Unexpected characteristics of ppGalNAcT2 motivated us to challenge site-directed installation of alpha-GalNAc residues at desired position(s) by protecting some hydroxyl groups of Thr/Ser residues with selectively removable sugars, notably a novel concept as "carbohydrate as protective groups", toward a goal of the systematic chemical and enzymatic synthesis of biologically important mucin glycopeptides.
• Effect of cyclic three-dimensional strain on cell proliferation and collagen synthesis of fibroblast-seeded chitosan-hyaluronan hybrid polymer fiber

  Naohiro Sawaguchi, Tokifumi Majima, Tadanao Funakoshi, Kazumi Shimode, Kazuo Harada, Akio Minami, Shin-Ichiro Nishimura

  JOURNAL OF ORTHOPAEDIC SCIENCE 15 4 569 - 577 2010年07月 [査読有り][通常論文]
   

  Tissue engineering techniques using biodegradable three-dimensional (3D) scaffolds with cultured cells offer more potential alternatives for the treatment of severe ligament and tendon injuries. In tissue engineering, one of the crucial roles of 3D scaffolds is to provide a temporary template with the biomechanical characteristics of the native extracellular matrix (ECM) until the regenerated tissue matures. The purpose of the present study was to assess the effect of various cyclic mechanical stresses on cell proliferation and ECM production in a 3D scaffold made from chitosan and hyaluronan for ligament and tendon tissue engineering. Three-dimensional scaffolds seeded with rabbit patella tendon fibroblasts were attached to a bioreactor under various conditions: static group, no strain; stretch group, tensile strain; rotational group, rotational strain; combined group, rotational and tensile strain. In the Static group, 3 weeks of stationary culture was performed. In the remaining three groups, a loading regimen of 0.5 Hz for 18 h and then 6 h rest was carried out for 2 weeks after 1 week of static culture. The DNA content was determined to quantify cell proliferation. Real-time reverse transcription polymerase chain reaction analysis was performed to assess the mRNA levels of the ECM products. DNA content of the combined group was significantly higher than that of the static and stretch groups, and that of the rotational group was significant higher than that of the static and stretch groups at 21 days after cultivation. The mRNA level of types I and III collagen and fibromodulin in the combined group was significantly higher than that in the other three groups. The amount of collagen synthesis in the combined group was higher than that in the static group, but the difference was not significant. Multidimensional cyclic mechanical strain to mimic the physiological condition in vivo has the potential to improve or accelerate tissue regeneration in ligament and tendon tissue engineering using 3D scaffolds in vitro.
• Novel trivalent anti-influenza reagent

  Fei Feng, Nobuaki Miura, Norikazu Isoda, Yoshihiro Sakoda, Masatoshi Okamatsu, Hiroshi Kida, Shin-Ichiro Nishimura

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 20 12 3772 - 3776 2010年06月 [査読有り][通常論文]
   

  We designed and synthesized novel trivalent anti-influenza reagents. Sialyllactose was located at the terminal of each valence which aimed to block each receptor-binding site of the hemagglutinin (HA) trimer on the surface of the virus. Structural analyses were carried out with a model which was constructed with a computer simulation. A previously reported cyclic glycopeptide blocker [Ohta, T.; Miura, N.; Fujitani, N.; Nakajima, F.; Niikura, K.; Sadamoto, R.; Guo, C.-T.; Suzuki, T.; Suzuki, Y.; Monde, K.; Nishimura, S.-I. Angew. Chem. Int. Ed., 2003, 42, 5186] bound to the HA in the model. The analyses suggest that the glutamine residue in the cyclic peptide bearing Neu5A alpha 2,3Gal beta 1,4Glc trisaccharide via a linker interacts with the Gln189 in HA through hydrogen bonding. The present anti-influenza reagents likely interact with a glutamine residue included in the vicinity of Gln189. A plague reduction assay of the influenza virus, A/PR/8/1934 (H1N1),was performed in MDCK cells to evaluate for the synthesized compounds to inhibit viral replication. One of the compounds showed approximately 85% inhibition at the concentration of 400 mu M at 4 degrees C. (C) 2010 Elsevier Ltd. All rights reserved.
• Functional network of glycan-related molecules: Glyco-Net in Glycoconjugate Data Bank

  Ryo Hashimoto, Kazuko Hirose, Taku Sato, Nobuhiro Fukushima, Nobuaki Miura, Shin-Ichiro Nishimura

  BMC SYSTEMS BIOLOGY 4 91  2010年06月 [査読有り][通常論文]
   

  Background: Glycans are involved in a wide range of biological process, and they play an essential role in functions such as cell differentiation, cell adhesion, pathogen-host recognition, toxin-receptor interactions, signal transduction, cancer metastasis, and immune responses. Elucidating pathways related to post-translational modifications (PTMs) such as glycosylation are of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids and various biological events are considered extremely valuable and convenient tools for the systematic investigation of PTMs. However, there is no database which dynamically draws functional networks related to glycans. Description: We have created a database called Glyco-Net http://www.glycoconjugate.jp/functions/, with many binary relationships among glycan-related molecules. Using search results, we can dynamically draw figures of the functional relationships among these components with nodes and arrows. A certain molecule or event corresponds to a node in the network figures, and the relationship between the molecule and the event are indicated by arrows. Since all components are treated equally, an arrow is also a node. Conclusions: In this paper, we describe our new database, Glyco-Net, which is the first database to dynamically show networks of the functional profiles of glycan related molecules. The graphical networks will assist in the understanding of the role of the PTMs. In addition, since various kinds of bio-objects such as genes, proteins, and inhibitors are equally treated in Glyco-Net, we can obtain a large amount of information on the PTMs.
• Threshold in Stage-specific Embryonic Glycotypes Uncovered by a Full Portrait of Dynamic N-Glycan Expression during Cell Differentiation

  Maho Amano, Misa Yamaguchi, Yasuhiro Takegawa, Tadashi Yamashita, Michiyo Terashima, Jun-ichi Furukawa, Yoshiaki Miura, Yasuro Shinohara, Norimasa Iwasaki, Akio Minami, Shin-ichiro Nishimura

  MOLECULAR & CELLULAR PROTEOMICS 9 3 523 - 537 2010年03月 [査読有り][通常論文]
   

  Although various glycoforms appear to participate independently in multiple molecular interactions in cellular adhesion that contribute to embryogenesis and organogenesis, a full portrait of the glycome diversity and the effect of the structural variations of cellular glycoforms on individual cell stages in proliferation and differentiation remain unclear. Here we describe a novel concept for the characterization of dynamic glycoform alteration during cell differentiation by means of "glycoblotting-based cellular glycomics," the only method allowing for rapid and quantitative glycan analysis. We demonstrated that processes of dynamic cellular differentiation of mouse embryonic carcinoma cells, P19CL6 and P19C6, and mouse embryonic stem cells into cardiomyocytes or neural cells can be monitored and characterized quantitatively by profiling entire N-glycan structures of total cell glycoproteins. Whole N-glycans enriched and identified by the glycoblotting method (67 glycans for P19CL6, 75 glycans for P19C6, and 72 glycans for embryonic stem cells) were profiled and bar-coded quantitatively with respect to the ratio of subgroups composed of characteristic glycoforms, namely glycotypes. Molecular & Cellular Proteomics 9: 523-537, 2010.
• Carbohydrate Moieties Contribute Significantly to the Physicochemical Properties of French Bean 7S Globulin Phaseolin

  Aiko Kimura, Mary Rose G. Tandang-Silvas, Takako Fukuda, Cerrone Cabanos, Yasuhiro Takegawa, Maho Amano, Shin-Ichiro Nishimura, Yasuki Matsumura, Shigeru Utsumi, Nobuyuki Maruyama

  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 58 5 2923 - 2930 2010年03月 [査読有り][通常論文]
   

  We have previously reported that the solubility of French bean 7S globulin (phaseolin) at low ionic strength and its emulsifying stability are remarkably high compared with those of 7S globulins prepared from other plant species, including soybean (Kimura et al. J. Agric. Food Chem. 2008, 56, 10273-10279). In this study, we examined the role of carbohydrate moieties in the properties of phaseolin. Three preparations of phaseolin were analyzed: (i) N7S, prepared from defatted seed meal and having intact carbohydrate moieties: (ii) R7S, expressed in E coli and lacking N-linked glycans; and (iii) EN7S, having partial N-linked glycans after treatment with Endo H. The solubilities of N7S and EN7S were much higher than that of R7S at a low ionic strength (mu = 0.08). N7S exhibited good emulsifying ability under the conditions examined, but R7S did not. In terms of emulsion stability, an emulsion of R7S separated into two phases after 1 h at mu = 0.01, 0.08, and 0.5, whereas the emulsion of N7S was stable for 5 days at mu = 0.01 and for at least 10 days at mu = 0.08 and 0.5. The emulsion stability of EN7S was comparable to that of N7S under most conditions examined. These results indicate the carbohydrate modifications are necessary for the good solubility, emulsifying ability, and emulsion stability of phaseolin. Further, a structural analysis of the carbohydrate moieties indicates that truncated carbohydrate moieties are sufficient for conferring these physicochemical properties to phaseolin.
• Highly Oriented Recombinant Glycosyltransferases: Site-Specific Immobilization of Unstable Membrane Proteins by Using Staphylococcus aureus Sortase A

  Takaomi Ito, Reiko Sadamoto, Kentaro Naruchi, Hiroko Togame, Hiroshi Takemoto, Hirosato Kondo, Shin-Ichiro Nishimura

  BIOCHEMISTRY 49 11 2604 - 2614 2010年03月 [査読有り][通常論文]
   

  Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides. and various art let artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both ill the basic Studies of their functional roles in Various biological processes and ill the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that file Immobilization of extremely unstable membrane-bound glycosyltransferases Oil some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation Of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate it versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the First time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human beta 1,4-galactosyltranseferase or recombinant Helicobacter pylori alpha 1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically Immobilized enzymes exhibited the desired sugar transfer activity, all improved stability, and it practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, its well its glycosyltransferases are unstable and highly oriented membrane proteins. the merit of four strategy based oil "site-specific" transpeptidation is evident because the reaction proceeds only ill all engineered C-terminus without any conformational influence around the active sites of both enzymes its well its heptad repeats of rH FucT required to maintain native secondary and quaternary structures during the dimerization on cell Surfaces.
• Potent inhibitor scaffold against Trypanosoma cruzi trans-sialidase

  Shingo Arioka, Masahiro Sakagami, Rie Uematsu, Hiroto Yamaguchi, Hiroko Togame, Hiroshi Takemoto, Hiroshi Hinou, Shin-ichiro Nishimura

  BIOORGANIC & MEDICINAL CHEMISTRY 18 4 1633 - 1640 2010年02月 [査読有り][通常論文]
   

  The protozoan Trypanosoma cruzi, the causative agent of Chagas' disease, can infect the heart, causing cardiac arrest frequently followed by death. To treat this disease, a potential molecular drug target is T. cruzi trans-sialidase (TcTS). However, inhibitors found to date are not strong enough to serve as a lead scaffold; most inhibitors reported thus far are derivatives of the substrate sialic acid or a transition state analogue known as 2,3-dehydro-3-deoxy-N-acetylneuraminic acid (DANA) with an IC(50) value of more than hundreds of micromolar. Since natural products are highly stereodiversified and often provide highly specific biological activity, we screened a natural product library for inhibitors of TcTS and identified promising flavonoid and anthraquinone derivatives. A structure-activity relationship (SAR) analysis of the flavonoids revealed that apigenin had the minimal and sufficient structure for inhibition. Intriguingly, the compound has been reported to possess trypanocidal activity. An SAR analysis of anthraquinones showed that 6-chloro-9,10-dihydro-4,5,7-trihydroxy-9,10-dioxo-2-anthracenecarboxylic acid had the strongest inhibitory activity ever found against TcTS. Moreover, its inhibitory activity appeared to be specific to TcTS. These compounds may serve as potent lead chemotherapeutic scaffolds against Chagas' disease. (C) 2010 Elsevier Ltd. All rights reserved.
• An Essential Epitope of Anti-MUC1 Monoclonal Antibody KL-6 Revealed by Focused Glycopeptide Library

  Naoki Ohyabu, Hiroshi Hinou, Takahiko Matsushita, Ryukou Izumi, Hiroki Shimizu, Keiko Kawamoto, Yoshito Numata, Hiroko Togame, Hiroshi Takemoto, Hirosato Kondo, Shin-Ichiro Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 131 47 17102 - 17109 2009年12月 [査読有り][通常論文]
   

  Human serum Krebs von den Lungen-6 (KL-6) antigen, a high-molecular-weight glycoprotein classified as a polymorphic epithelial mucin (MUC1), is a biomarker of diseases such as interstitial pneumonia, lung adenocarcinoma, breast cancer, colorectal adenocarcinoma, and hepatocellular carcinoma. Anti-KL-6 monoclonal antibody (anti-KL-6 MAb) is therefore a potential diagnostic and therapeutic reagent. Although glycosylation at Thr/Ser residues of the tandem-repeating MUC1 peptides appears to determine the disease-associated antigenic structures of KL-6, an essential epitope structure recognized by anti-KL-6 MAb remains unclear. In the present study, a novel compound library of synthetic MUC1 glycopeptides allowed the first rapid and precise evaluation of the specific epitope structure of anti-KL-6 MAb by combined use of a tailored glycopeptides library and common ELISA protocol. We demonstrated that the minimal antigenic structure, an essential epitope, recognized by anti-KL-6 MAb is a heptapeptide sequence Pro-Asp-Thr-Arg-Pro-Ala-Pro (PDTRPAP), in which the Thr residue is modified by Neu5Ac alpha 2,3Gal beta 1,3GalNAc alpha (2,3-sialyl T antigen, core 1-type O-glycan). Anti-KL-6 MAb did not bind with other tumor-relevant antigens, such as GalNA alpha(Tn), Neu5Ac alpha 2,6GalNAc alpha(STn), and Gal beta 1,3GalNAc alpha (T), except for Neu5Ac alpha 2,3Gal beta 1,3(Neu5Ac alpha 2,6) GalNAc alpha(2,3/2,6-disialyl T). However, anti-KL-6 MAb could not differentiate the above minimal antigenic glycopepticle from some core 2-based glycopeptides involving this crucial epitope structure and showed a similar binding affinity toward these compounds, indicating that branching at the O-6 position of GalNAc residue does not influence the interaction of anti-KL-6 MAb with some MUC1 glycoproteins involving an essential epitope. Actually, anti-KL-6 MAb reacts with 2,3/2,6-disialyl T having a 2,3-sialyl T component. This is why anti-KL-6 MAb often reacts with various kinds of tumor-derived MUC1 glycoproteins as well as a clinically important MUC1 glycoprotein biomarker of interstitial pneumonia, namely KL-6, originally discovered as a circulating pulmonary adenocarcinoma-associated antigen. In other words, combined use of anti-KL-6 MAb and some probes that can differentiate the sugars substituted at the O-6 position of the GalNAc residue in MUC1 glycopeptides including the PDTRPAP sequence might be a promising diagnostic protocol for individual disease-specific biomarkers. It was also revealed that glycosylation at neighboring Thr/Ser residues outside the immunodominant PDTRPAP motif strongly influences the interaction between anti-KL-6 MAb and MUC1 glycopeptides involving the identified epitope. Our novel strategy will greatly facilitate the processes for the identification of the tumor-specific and strong epitopes of various known anti-MUC1 MAbs and allow for their practical application in the generation of improved antibody immunotherapeutics, diagnostics, and MUC1-based cancer vaccines.
• Microwave Effect for Glycosylation Promoted by Solid Super Acid in Supercritical Carbon Dioxide

  Hiroshi Hinou, Naohiro Saito, Masato Ogawa, Takahiko Maeda, Shin-Ichiro Nishimura

  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 10 12 5285 - 5295 2009年12月 [査読有り][通常論文]
   

  The effects of microwave irradiation (2.45 GHz, 200 W) on glycosylation promoted by a solid super acid in supercritical carbon dioxide was investigated with particular attention paid to the structure of the acceptor substrate. Because of the symmetrical structure and high diffusive property of supercritical carbon dioxide, microwave irradiation did not alter the temperature of the reaction solution, but enhanced reaction yield when aliphatic acceptors are employed. Interestingly, the use of a phenolic acceptor under the same reaction conditions did not show these promoting effects due to microwave irradiation. In the case of aliphatic diol acceptors, the yield seemed to be dependent on the symmetrical properties of the acceptors. The results suggest that microwave irradiation do not affect the reactivity of the donor nor promoter independently. We conclude that the effect of acceptor structure on glycosylation yield is due to electric delocalization of hydroxyl group and dielectrically symmetric structure of whole molecule.
• Functional Neoglycopeptides: Synthesis and Characterization of a New Class of MUC1 Glycoprotein Models Having Core 2-Based O-Glycan and Complex-Type N-Glycan Chains

  Takahiko Matsushita, Reiko Sadamoto, Naoki Ohyabu, Hideki Nakata, Masataka Fumoto, Naoki Fujitani, Yasuhiro Takegawa, Takeshi Sakamoto, Masaki Kurogochi, Hiroshi Hinou, Hiroki Shimizu, Takaomi Ito, Kentarou Naruchi, Hiroko Togame, Hiroshi Takemoto, Hirosato Kondo, Shin-Ichiro Nishimura

  BIOCHEMISTRY 48 46 11117 - 11133 2009年11月 [査読有り][通常論文]
   

  An efficient protocol for the construction of MUC1-related glycopeptide analogues having complex O-glycan and N-glycan chains was established by integrating chemical and enzymatic approaches on the functional polymer platforms. We demonstrated the feasibility of sortase A-mediated ligation between two glycopeptide segments by tagging with signal peptides, LPKTGLR and GG, at each C- or N-terminal position. Structural analysis of the macromolecular N,O-glycopeptides was performed by means of ESI-TOFMS (MS/MS) equipped with an electron-captured dissociation device. Immunological assay using MUC1 glycopeptides synthesized in this study revealed that N-glycosylation near the antigenic O-glycosylated PDTR motif did not disturb the interaction between the anti-MUC1 monoclonal antibody and this crucial O-glycopeptide moiety. NMR study indicated that the N-terminal immunodominant region [Ala-ProAsp-Thr(O-glycan)-Arg] forms an inverse gamma-turn-like structure, while the C-terminal region composed of N-glycopeptide and linker SrtA-peptide was proved to be an independently random structure. These results indicate that the bulky O- and N-glycan chains can function independently as disease-relevant epitopes and ligands for carbohydrate-binding proteins, when both are combined by an artificial intervening peptide having a possible effect of separating N- and C-terminal regions. The present strategy will greatly facilitate rapid synthesis of multiply functionalized complex neoglycopeptides as new types of convenient tools or models for the investigation of the structure-function relationship of various glycoproteins and development of novel class glycopeptide-based biopharmaceuticals, drug delivery systems, and biomedical materials.
• Chemoenzymatic Synthesis of Glycosylated Glucagon-like Peptide 1: Effect of Glycosylation on Proteolytic Resistance and in Vivo Blood Glucose-Lowering Activity

  Taichi Ueda, Kazuyoshi Tomita, Yoshihide Notsu, Takaomi Ito, Masataka Fumoto, Tomoaki Takakura, Hirofumi Nagatome, Akio Takimoto, Shin-Ichi Mihara, Hiroko Togame, Keiko Kawamoto, Takanori Iwasaki, Kenji Asakura, Takeo Oshima, Kohji Hanasaki, Shin-Ichiro Nishimura, Hirosato Kondo

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 131 17 6237 - 6245 2009年05月 [査読有り][通常論文]
   

  Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogues with N-acetylglucosamine (GIcNAc), N-acetyllactosamine (LacNAc), and alpha 2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepared by chemoenzymatic approaches. We assessed the receptor binding affinity and CAMP production activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addition of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic analysis of the degradation reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addition, the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogues with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogues of other bioactive peptides as desirable pharmaceuticals.
• Identification of novel helper epitopes of MAGE-A4 tumour antigen: useful tool for the propagation of Th1 cells

  T. Ohkuri, D. Wakita, K. Chamoto, Y. Togashi, H. Kitamura, T. Nishimura

  BRITISH JOURNAL OF CANCER 100 7 1135 - 1143 2009年03月 [査読有り][通常論文]
   

  MAGE-A4 has been considered as an attractive cancer-testis (CT) antigen for tumour immunotherapy. It has been well accepted that T-helper type 1 (Th1) cell-dominant immunity is critical for the successful induction of antitumour immunity in a tumour-bearing host. The adoptive Th1 cell therapy has been shown to be an attractive strategy for inducing tumour eradication in mouse systems. However, Th1-cell therapy using human tumour-specific Th1 cells, which were expanded from peripheral blood mononuclear cells (PBMCs) in a clinically useful protocol, has never been performed. Here, we first identified MAGE-A4-derived promiscuous helper epitope, peptide (MAGE-A4 280-299), bound to both HLA-DPB1*0501 and DRB1*1403. Using the peptide, we established a suitable protocol for the propagation of MAGE-A4-specific Th1 cells in vitro. Culture of CD4(+) T cells with IFN-gamma-treated PBMC-derived adherent cells in the presence of helper epitope peptide resulted in a great expansion of MAGE-A4-reactive Th cells producing IFN-gamma, but not IL-4. Moreover, it was shown that ligation of MAGE-A4-reactive Th1 cells with the cognate peptide caused the production of IFN-gamma and IL-2. Thus, our identified MAGE-A4 helper epitope peptide will become a good tool for the propagation of tumour-specific Th1 cells applicable to adoptive immunotherapy of human cancer.
• Glycosylation Specific for Adhesion Molecules in Epidermis and Its Receptor Revealed by Glycoform-focused Reverse Genomics

  Rie Uematsu, Yasuro Shinohara, Hiroaki Nakagawa, Masaki Kurogochi, Jun-ichi Furukawa, Yoshiaki Miura, Masashi Akiyama, Hiroshi Shimizu, Shin-Ichiro Nishimura

  MOLECULAR & CELLULAR PROTEOMICS 8 2 232 - 244 2009年02月 [査読有り][通常論文]
   

  Glycosylation of proteins greatly affects their structure and function, but traditional genomics and transcriptomics are not able to precisely capture tissue- or species-specific glycosylation patterns. We describe here a novel approach to link different "omics" data based on exhaustive quantitative glycomics of murine dermis and epidermis. We first examined the dermal and epidermal N-glycome of mouse by a recently established glycoblotting technique. We found that the Gal alpha 1-3Gal epitope was solely expressed in epidermis tissue and was preferentially attached to adhesion molecules in a glycosylation site-specific manner. Clarified glycomic and protemic information combined with publicly available microarray data sets allowed us to identify galectin-3 as a receptor of Gal alpha 1-3Gal epitope. These findings provide mechanistic insight into the causal connection between the genotype and the phenotype seen in alpha 3GalT-1-deficient mice and transgenic mice expressing endo-beta-galactosidase C. Because humans do not possess the Gal alpha 1-3Gal structure on their tissues, we further examined the human dermal and epidermal N-glycome. Comparative glycomics revealed that the GalNAc beta 1-4GlcNAc ( N, N'-diacetyllactosediamine) epitope, instead of the Gal alpha 1-3Gal epitope, was highly expressed in human epidermis. Molecular & Cellular Proteomics 8:232-244, 2009.
• MALDI-TOF Mass-Spectrometry-Based Versatile Method for the Characterization of Protein Kinases

  Noriyasu Kondo, Shin-Ichiro Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 15 6 1413 - 1421 2009年 [査読有り][通常論文]
   

  We describe a MALDI-TOF mass-spectrometry-based method that is rapid and versatile for the characterization of protein kinases and their inhibitors. We have designed new kinase substrates by the modification of common synthetic peptides, such as kemptide (LRRALSG), CaMKII substrate (KRQQSFDLF), erktide (ATGPLSPGPFGRR), abltide (EAIYAAPFAKKK), srctide (AEEEIYGEFEAKKKK), neurogranin (AAAKIQASFRGHMARKK), and casein kinase I (CKI) substrate (RRKDLHDDEEDEAMSITA). There are two fundamental points on which the proposed method is based to improve the mass-spectrometric response: 1) mass tag technology by N-derivatization through stable isotope labeling and 2) C-terminal conjugation with tryptophanylarginine (WR). It was suggested that C-terminal conjugation with the WR moiety enhances the ionization potency of these new substrates 1.5-13.7 times as much as those of the original peptides. We demonstrated, by using modified abltide (Ac-EAIYAAPFAKKKWR-NH(2)), that WR conjugation at the C-terminus in combination with stable-isotope labeling at the N-terminus allowed the quantitative assay of recombinant c-Abl kinase in the presence of adenosine 5'-triphosphate (ATP; K(M,ATP)= 48.6 mu m and V(max)=642 pmol min(-1) mu g(-1)). The present protocol made a simple and reliable inhibition assay of recombinant c-Abl kinase by imatinib possible (IC(50(recombinant))=291 nM; ST1571, Gleevec; Novartis Pharma). Moreover, it was also demonstrated that this ATP noncompetitive inhibitor differentiates between two conformers of c-Abl kinases: the phosphorylated active and dephosphorylated inactive forms (IC(50(active form))=1049 nM and IC(50(inactive form))=54 nM). The merit of this approach is evident because the present protocol can be applied to the direct monitoring of the activities of living cell kinases by using cancer-cell lines, such as mouse B16 melanoma cells and human lung cancer K562 cells. A multiple-kinase assay that uses K562 cell lysate in the presence of seven new synthetic substrates made high-throughput inhibitor profiling possible. It should be emphasized that this radioactive isotope-free quantitative kinase assay will greatly accelerate the discovery of a new generation of potential kinase inhibitors that exhibit highly selective or unique inhibitory profiles.
• Structural and Functional Glycosphingolipidomics by Glycoblotting with an Aminooxy-Functionalized Gold Nanoparticle

  Noriko Nagahori, Midori Abe, Shin-Ichiro Nishimura

  BIOCHEMISTRY 48 3 583 - 594 2009年01月 [査読有り][通常論文]
   

  Glycosphingolipids (GSLs) synthesized in Golgi apparatus by sequential transfer of sugar residues to a ceramide lipid anchor are ubiquitously distributing on vertebrate plasma membranes. A standardized method allowing for high-throughput structural profiling and functional characterization of living cell surface GSLs is of growing importance because they function as crucial signal transduction molecules in various processes of dynamic cellular recognitions. However, methods are not available for amplification of GSLs, while the genomic scale PCR amplification permits large-scale mammalian proteomic analysis. Here we communicate such an approach to a novel "omics", namely, glycosphin-golipidomics based on the "glycoblotting" method. The method, which involves selective ozonolysis of the C-C double bond in the ceramide moiety and subsequent enrichment of generated GSL aldehydes by chemical ligation using an aminooxy-functionalized gold nanoparticle (aoGNP) should be of widespread utility for identifying and characterizing whole GSLs present in the living cell surfaces. The present protocol using glycoblotting permitted MALDI-TOFMS-based high-throughput structural profiling of mouse brain gangliosides such as GM1, GD1a/GD1b, and GT1b for adult or GD3 in the case for the embryonic mouse. When mouse melanoma B16 cells were subjected to this protocol, it was demonstrated that gangliosides enriched from the plasma membranes are the only GM3 bearing microheteogeneity in the structure of the N-acyl chain. Surface plasmon resonance analysis revealed that aoGNP displaying whole GSLs blotted from mouse B 16 melanoma cell surfaces can be used directly for monitoring the specific interaction with the self-assembled monolayer (SAM) of Gg3Cer (gangliotriaosylceramide). Our results indicate that GSL-selective enrichment onto aoGNP from living cell surfaces allows for rapid reconstruction of plasma membrane models mimicking the intact GSL microdomain feasible for further structural and functional characterization.
• One-pot synthesis of cyclic antifreeze glycopeptides

  Masakazu Hachisu, Hiroshi Hinou, Manabu Takamichi, Sakae Tsuda, Shuhei Koshidaa, Shin-Ichiro Nishimura

  CHEMICAL COMMUNICATIONS 13 1641 - 1643 2009年 [査読有り][通常論文]
   

  The first cyclic glycopeptides exhibiting significant antifreeze activity by forming hexagonal-bipyramidal ice crystals, denoted cyclic antifreeze glycopeptides (cyclic AFGPs), were constructed by a one-pot synthesis based on the controlled cyclization reaction of pre-formed small linear glycopeptides.
• Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

  Xiao-Dong Gao, Satoru Moriyama, Nobuaki Miura, Neta Dean, Shin-Ichiro Nishimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 283 47 32534 - 32541 2008年11月 [査読有り][通常論文]
   

  The second step of eukaryotic N-linked glycosylation in endoplasmic reticulum is catalyzed by an UDP-N-acetylglucosamine transferase that is comprised of two subunits, Alg13 and Alg14. The interaction between Alg13 and 14 is crucial for UDP-Glc-NAc transferase activity, so formation of the Alg13/14 complex is likely to play a key role in the regulation of N-glycosylation. Using a combination of bioinformatics and molecular biological methods, we have undertaken a functional analysis of yeast Alg13 and Alg14 proteins to elucidate the mechanism of their interaction. Our mutational studies demonstrated that a short C-terminal alpha-helix of Alg13 is required for interaction with Alg14 and for enzyme activity. Electrostatic surface views of the modeled Alg13/14 complex suggest the presence of a hydrophobic cleft in Alg14 that provides a pocket for the Alg13 C-terminal alpha-helix. Co-immunoprecipitation assays confirmed the C-terminal three amino acids of Alg14 are required for maintaining the integrity of Alg13/Alg14 complex, and this depends on their hydrophobicity. Modeling studies place these three Alg14 residues at the entrance of the hydrophobic-binding pocket, suggesting their role in the stabilization of the interaction between the C termini of Alg13 and Alg14. Together, these results demonstrate that formation of this hetero-oligomeric complex is mediated by a short C-terminal alpha-helix of Alg13 in cooperation with the last three amino acids of Alg14. In addition, deletion of the N-terminal beta-strand of Alg13 caused the destruction of protein, indicating the structural importance of this region in protein stability.
• Crucial Role of Aspartic Acid at Position 265 in the CH2 Domain for Murine IgG2a and IgG2b Fc-Associated Effector Functions

  Lucie Baudino, Yasuro Shinohara, Falk Nimmerjahn, Jun-Ichi Furukawa, Munehiro Nakata, Eduardo Martinez-Soria, Franz Petry, Jeffery V. Ravetch, Shin-Ichiro Nishimura, Shozo Izui

  JOURNAL OF IMMUNOLOGY 181 9 6664 - 6669 2008年11月 [査読有り][通常論文]
   

  Replacement of aspartic acid by alanine at position 265 (D265A) in mouse IgG1 results in a complete loss of interaction between this isotype and low-affinity IgG Fc receptors (Fc gamma RIIB and Fc gamma RIII). However, it has not yet been defined whether the D265A substitution could exhibit similar effects on the interaction with two other Fc gamma R (Fc gamma RI and Fc gamma RIV) and on the activation of complement. To address this question, 34-3C anti-RBC IgG2a and IgG2b switch variants bearing the D265A mutation were generated, and their effector functions and in vivo pathogenicity were compared with those of the respective wild-type Abs. The introduction of the D265A mutation almost completely abolished the binding of 34-3C IgG2a and IgG2b to all four classes of Fc gamma R and the activation of complement. Consequently, these mutants were hardly pathogenic. Although oligosaccharide side chains of these mutants were found to contain higher levels of sialic acids than those of wild-type Abs, the analysis of enzymatically desialylated D265A variants ruled out the possibility that very poor Fc-associated effector functions of the D265A mutants were due to an increased level of the mutant Fc sialylation. Thus, our results demonstrate that aspartic acid at position 265 is a residue critically implicated in triggering the Fc-associated effector functions of IgG, probably by defining a crucial three-dimensional structure of the Fc region. The Journal of Immunology, 2008, 181: 6664-6669.
• Alterations in the glycoform of cisplatin-resistant human carcinoma cells are caused by defects in the endoplasmic reticulum-associated degradation system

  Hiroaki Nakagawa, Miki Ohira, Shunji Hayashi, Shigeaki Abe, Shin Saito, Noriko Nagahori, Kenji Monde, Yasuro Shinohara, Naoki Fujitani, Hirosato Kondo, Shin-Ichi Akiyama, Akira Nakagawara, Shin-Ichiro Nishimura

  Cancer Letters 270 2 295 - 301 2008年11月 [査読有り][通常論文]
  
• A novel beta-defensin structure: A potential strategy of big defensin for overcoming resistance by gram-positive bacteria

  Takahide Kouno, Naoki Fujitani, Mineyuki Mizuguchi, Tsukasa Osaki, Shin-ichiro Nishimura, Shun-ichiro Kawabata, Tomoyasu Aizawa, Makoto Demura, Katsutoshi Nitta, Keiichi Kawan

  BIOCHEMISTRY 47 40 10611 - 10619 2008年10月 [査読有り][通常論文]
   

  Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. It has antimicrobial activities against Gram-positive and -negative bacteria. The amino acid sequence of big defensin can be divided into an N-terminal hydrophobic half and a C-terminal cationic half. Interestingly, the trypsin cleaves big defensin into two fragments, the N-terminal and C-terminal fragments, which are responsible for antimicrobial activity against Gram-positive and -negative bacteria, respectively. To explore the antimicrobial mechanism of big defensin, we determined the solution structure of mature big defensin and performed a titration experiment with DPC micelles. Big defensin has a novel defensin structure; the C-terminal domain adopts a P-defensin structure, and the N-terminal domain forms a unique globular conformation. It is noteworthy that the hydrophobic N-terminal domain undergoes a conformational change in micelle solution, while the C-terminal domain remains unchanged. Here, we propose that the N-terminal domain achieves its antimicrobial activity in a novel fashion and explain that big defensin has developed a strategy different from those of other beta-defensins to suppress the growth of Gram-positive bacteria.
• Magnetic nanoparticles for improving cell invasion in tissue engineering

  Takuro Sasaki, Norimasa Iwasaki, Kenji Kohno, Mikio Kishimoto, Tokifumi Majima, Shin-Ichiro Nishimura, Akio Minami

  Journal of Biomedical Materials Research Part A 86A 4 969 - 978 2008年09月15日 [査読有り][通常論文]
  
• Impact of a three amino acid deletion in the CH2 domain of murine IgG1 on Fc-associated effector functions

  Lucie Baudino, Falk Nimmerjahn, Yasuro Shinohara, Jun-Ichi Furukawa, Franz Petry, J. Sjef Verbeek, Shin-Ichiro Nishimura, Jeffery V. Ravetch, Shozo Izui

  JOURNAL OF IMMUNOLOGY 181 6 4107 - 4112 2008年09月 [査読有り][通常論文]
   

  Four murine IgG subclasses display markedly different Fc-associated effector functions because of their differential binding to three activating IgG Fc receptors (Fc gamma RI, Fc gamma RIII, and Fc gamma RIV) and C1q. Previous analysis of IgG subclass switch variants of 34-3C anti-RBC monoclonal autoantibodies revealed that the IgG1 subclass, which binds only to Fc gamma RIII and fails to activate complement, displayed the poorest pathogenic potential. This could be related to the presence of a three amino acid deletion at positions 233-235 in the CH2 domain uniquely found in this subclass. To address this question, IgG1 insertion and IgG2b deletion mutants at positions 233-235 of 34-3C anti-RBC Abs were generated, and their ability to initiate effector functions and their pathogenicity were compared with those of the respective wild-type Abs. The insertion of amino acid residues at positions 233-235 enabled the IgG1 subclass to bind Fc gamma RIV but did not improve the binding to C1q. Accordingly, its pathogenicity was enhanced but still inferior to that of IgG2b. In contrast, the IgG2b deletion mutant lost its ability to bind to Fc gamma RIV and activate complement. Consequently, its pathogenicity was markedly diminished to a level comparable to that of lgG1. Our results demonstrated that the initiation of Fc gamma R- and complement-mediated effector functions of IgG2b was profoundly affected by the three amino acid deletion at positions 233-235, but that this natural three amino acid deletion could only partially explain the poor binding of IgG1 to Fc gamma RIV and C1q. This indicates the lack in the IgG1 subclass of as yet unknown motifs promoting efficient interaction with Fc gamma RIV and C1q.
• Detection of Carcinoembryonic Antigens Using a Surface Plasmon Resonance Biosensor

  Fengyu Su, Chunye Xu, Minoru Taya, Kimie Murayama, Yasuro Shinohara, Shin-Ichiro Nishimura

  Sensors 8 7 4282 - 4295 2008年07月18日 [査読有り][通常論文]
  
• Development of mature cartilage constructs using novel three-dimensional porous scaffolds for enhanced repair of osteochondral defects

  Yasuhiko Kasahara, Norimasa Iwasaki, Shintaro Yamane, Tatsuya Igarashi, Tokifumi Majima, Sachiko Nonaka, Kazuo Harada, Shin-Ichiro Nishimura, Akio Minami

  Journal of Biomedical Materials Research Part A 86A 1 127 - 136 2008年07月 [査読有り][通常論文]
  
• Alteration of N-glycans related to articular cartilage deterioration after anterior cruciate ligament transection in rabbits.

  Matsuhashi T, Iwasaki N, Nakagawa H, Hato M, Kurogochi M, Majima T, Minami A, Nishimura SI

  Osteoarthritis Cartilage. 16 7 772 - 778 2008年07月 [査読有り][通常論文]
  
• Novel long-acting incretin mimetics glycosylated glucagon-like peptide 1 exert improved dipeptidyl peptidase IV resistance and potent anti-diabetic efficacy

  Kenji Asakura, Kazuyoshi Tomita, Taichi Ueda, Takeo Oshima, Takanori Iwasaki, Takaomi Ito, Hirofumi Nagatome, Akio Takimoto, Shin-Ichi Mihara, Hiroko Togame, Kohji Hanasaki, Shin-Ichiro Nishimura, Hirosato Kondo

  DIABETES 57 A4 - A4 2008年06月 [査読有り][通常論文]
  
• Comprehensive analysis of N-glycans in serum glycoproteins of subjects with high fasting glucose levels

  Hiroshi Ohira, Shinji Sakaue, Naofumi Itoh, Jun-Ichi Furukawa, Yoshiaki Miura, Ichizo Tsujino, Shin-Ichiro Nishimura, Masaharu Nishimura

  DIABETES 57 A704 - A704 2008年06月 [査読有り][通常論文]
  
• Two-dimensional hydrophilic interaction chromatography coupling anion-exchange and hydrophilic interaction columns for separation of 2-pyridylamino derivatives of neutral and sialylated N-glycans

  Kisaburo Deguchi, Takuro Keira, Kuriko Yamada, Hiroki Ito, Yasuhiro Takegawa, Hiroaki Nakagawa, Shin-Ichiro Nishimura

  JOURNAL OF CHROMATOGRAPHY A 1189 1-2 169 - 174 2008年05月 [査読有り][通常論文]
   

  A novel chromatographic approach coupling anion-exchange (diethylaminoethylene) and hydrophilic-interaction (amide or zwitterionic type) columns was developed for the separating of 2-pyridylamino derivatives of N-glycans (PA-N-glycans). This is a kind of on-line, two-dimensional (2D) separation approach in hydrophilic-interaction chromatography (called the 2D-HILIC method), analogous to that of coupling cation- (or anion-, or mixed ion-) exchange and reversed-phase columns in hydrophobic interaction (reversed-phase) chromatography. The efficiency of the 2D-HILIC method was tested with bianternnary neutral and sialylated PA-N-glycan standards by properly combining linear gradient elutions of water-acetonitrile and spiked-salt (ammonium acetate) elutions. The retention time RSDs of all the peaks in three sequential runs of a 100 min cycle are less than 0.52%, which indicates a reasonably good repeatability of the 2D-HILIC method. Then, the method was applied to a complex mixture of PA-N-glycans from human serum proteins. It was demonstrated that the neutral PA-N-glycans and mono-, di-, tri-, and tetrasialylated PA-N-glycans are able to be eluted in turn according to the number of sialic acids in an automated (programmed) single run. (C) 2007 Elsevier B.V. All rights reserved.
• Alg13p, the catalytic subunit of the endoplasmic reticulum UDP-GlcNAc glycosyltransferase, is a target for proteasomal degradation

  Nicole Averbeck, Xiao-Dong Gao, Shin-Ichiro Nishimura, Neta Dean

  MOLECULAR BIOLOGY OF THE CELL 19 5 2169 - 2178 2008年05月 [査読有り][通常論文]
   

  The second step of dolichol-linked oligosaccharide synthesis in the N-linked glycosylation pathway at the endoplasmic reticulum ( ER) membrane is catalyzed by an unusual hetero-oligomeric UDP-N-acetylglucosamine transferase that in most eukaryotes is comprised of at least two subunits, Alg13p and Alg14p. Alg13p is the cytosolic and catalytic subunit that is recruited to the ER by the membrane protein Alg14p. We show that in Saccharomyces cerevisiae, cytosolic Alg13p is very short-lived, whereas membrane-associated Alg13 is relatively stable. Cytosolic Alg13p is a target for proteasomal degradation, and the failure to degrade excess Alg13p leads to glycosylation defects. Alg13p degradation does not require ubiquitin but instead, requires a C-terminal domain whose deletion results in Alg13p stability. Conversely, appending this sequence onto normally long-lived beta-galactosidase causes it to undergo rapid degradation, demonstrating that this C-terminal domain represents a novel and autonomous degradation motif. These data lead to the model that proteasomal degradation of excess unassembled Alg13p is an important quality control mechanism that ensures proper protein complex assembly and correct N-linked glycosylation.
• Chemical and enzymatic synthesis of neoglycolipids in the presence of cyclodextrins

  Izuru Nagashima, Hiroki Shimizu, Takahiko Matsushita, Shin-Ichiro Nishimura

  Tetrahedron Letters 49 21 3413 - 3418 2008年05月 [査読有り][通常論文]
  
• Profiling ofN- andO-glycopeptides of erythropoietin by capillary zwitterionic type of hydrophilic interaction chromatography/electrospray ionization mass spectrometry

  Yasuhiro Takegawa, Hiroki Ito, Takuro Keira, Kisaburo Deguchi, Hiroaki Nakagawa, Shin-Ichiro Nishimura

  Journal of Separation Science 31 9 1585 - 1593 2008年05月 [査読有り][通常論文]
  
• Chromatographic deuterium isotope effects of derivatizedN-glycans andN-glycopeptides in a zwitterionic type of hydrophilic interaction chromatography

  Yasuhiro Takegawa, Megumi Hato, Kisaburo Deguchi, Hiroaki Nakagawa, Shin-Ichiro Nishimura

  Journal of Separation Science 31 9 1594 - 1597 2008年05月 [査読有り][通常論文]
  
• 臨床検体情報をどう治療へむすびつけるか? 血清中糖鎖の網羅的解析による肝細胞癌新規バイオマーカーの開発

  横尾 英樹, 三浦 嘉晃, 神山 俊哉, 中西 一彰, 濱口 純, 工藤 岳秋, 古川 潤一, 中川 隆公, 田原 宗徳, 中馬 誠, 髭 修平, 篠原 康郎, 尾崎 倫孝, 多田 光宏, 松下 通明, 浅香 正博, 西村 紳一郎, 藤堂 省

  日本外科学会雑誌 109 臨増2 144 - 144 (一社)日本外科学会 2008年04月
• Comprehensive Approach to Structural and Functional Glycomics Based on Chemoselective Glycoblotting and Sequential Tag Conversion

  Jun-ichi Furukawa, Yasuro Shinohara, Hiromitsu Kuramoto, Yoshiaki Miura, Hideyuki Shimaoka, Masaki Kurogochi, Mika Nakano, Shin-Ichiro Nishimura

  Analytical Chemistry 80 4 1094 - 1101 2008年02月 [査読有り][通常論文]
  
• BlotGlycoABC™, an Integrated Glycoblotting Technique for Rapid and Large Scale Clinical Glycomics

  Yoshiaki Miura, Megumi Hato, Yasuro Shinohara, Hiromitsu Kuramoto, Jun-ichi Furukawa, Masaki Kurogochi, Hideyuki Shimaoka, Mitsuhiro Tada, Kazuaki Nakanishi, Michitaka Ozaki, Satoru Todo, Shin-Ichiro Nishimura

  Molecular & Cellular Proteomics 7 2 370 - 377 2008年02月 [査読有り][通常論文]
  
• Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjects

  Tsutomu Fujimura, Yasuro Shinohara, Berangere Tissot, Poh-Choo Pang, Masaki Kurogochi, Seiichi Saito, Yoichi Arai, Martin Sadilek, Kimie Murayama, Anne Dell, Shin-Ichiro Nishimura, Sen-Itiroh Hakomori

  INTERNATIONAL JOURNAL OF CANCER 122 1 39 - 49 2008年01月 [査読有り][通常論文]
   

  We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewis(x/a) antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the P-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the beta-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GaINAc beta 4(NeuAc alpha 3)GaI beta 3(NeuAc alpha 6)GlcNAc beta Gal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O-glycosylation were identified in prostate cancer haptoglobin for the first time. Mono-and disialyl core Type 1 O-linked structures were identified after reductive beta-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this O-glycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N-glycans attached to a defined peptide region of its beta-chain are characterized by enhanced branching as well as antenna fucosylation. (C) 2007 Wiley-Liss, Inc.
• FRET-based direct and continuous monitoring of human fucosyltransferases activity: An efficient synthesis of versatile GDP-L-fucose derivatives from abundant D-galactose

  Takahiro Maeda, Shin-Ichiro Nishimuira

  CHEMISTRY-A EUROPEAN JOURNAL 14 2 478 - 487 2008年 [査読有り][通常論文]
   

  We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6-deoxy-6-N-(2-naphalene-2-yl-acetamide)-beta-L-galactopyranos-1-yl-guanosine 5'-diphosphate disodium salt (1), was efficiently synthesized from naturally abundant D-galactopyranose via a key intermediate, 6-azide-1,2,3,4-tetra-O-benzoyl-6-deoxy-beta-L-galactopyranose (10). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl-alpha-2,3-LacNAc derivative (2) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis X (SLe(x)), which is catalyzed by human alpha-1,3-fucosyltransferase VI (FUT-VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate (K-M=0.94 mu M and V-max = 0.14 mu m min(-1)) for detecting human FUT-VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT-VI among all of the known GDP-Fuc analogues, including the parent GDP-Fuc. When a dansylated asparagine-linked glycopeptide 20, which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET-based assay with compound 1 could be used to directly monitor the alpha 1,6-fucosylation at the reducing terminal GlcNAc residue by human FUT-VIII (K-M= 175 mu m and V-max=0.06 mu m/ min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23, which was obtained by a 1,3-dipolar cycloaddition between the disodiurn salt 16 and 1-ethynylnaphthalene exhibits highly potent inhibitory effects against the FUT-VI-mediated sialyl Lewis X synthesis (IC50 = 5.4 mu m).
• Glycoconjugate Data Bank : Structures - an annotated glycan structure database and N-glycan primary structure verification service

  Taku Nakahara, Ryo Hashimoto, Hiroaki Nakagawa, Kenji Monde, Nobuaki Miura, Shin-Ichiro Nishimura

  NUCLEIC ACIDS RESEARCH 36 D368 - D371 2008年01月 [査読有り][通常論文]
   

  Glycobiology has been brought to public attention as a frontier in the post-genomic era. Structural information about glycans has been accumulating in the Protein Data Bank (PDB) for years. It has been recognized, however, that there are many questionable glycan models in the PDB. A tool for verifying the primary structures of glycan 3D structures is evidently required, yet there have been no such publicly available tools. The Glycoconjugate Data Bank: Structures (GDB:Structures, http://www.glycostructures.jp) is an annotated glycan structure database, which also provides an N-glycan primary structure (or glycoform) verification service. All the glycan 3D structures are detected and annotated by an in-house program named 'getCARBO'. When an N-glycan is detected in a query coordinate by getCARBO, the primary structure of the glycan is compared with the most similar entry in the glycan primary structure database (KEGG GLYCAN), and unmatched substructure(s) are indicated if observed. The results of getCARBO are stored and presented in GDB: Structures.
• Bacterial Surface Engineering Utilizing Gluosamine Phosphate Derivatives as Cell Wall Precursor Surrogates

  Reiko Sadamoto, Takeshi Matsubayashi, Masataka Shimizu, Taichi Ueda, Shuhei Koshida, Toshiaki Koda, Shin-Ichiro Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 14 33 10192 - 10195 2008年 [査読有り][通常論文]
  
• Positive expressions of N-acetylglucosaminyltransferase-V (GnT-V) and β1-6 branching N-linked oligosaccharides in human testicular germ cells diminish during malignant transformation and progression

  Atsushi Kyan, Noritaka Kamimura, Shigeru Hagisawa, Shingo Hatakeyama, Takuya Koie, Takahiro Yoneyama, Yoichi Arai, Hiroaki Nakagawa, Shin-Ichiro Nishimura, Eiji Miyoshi, Yasuhiro Hashimoto, Chikara Ohyama

  International Journal of Oncology 2008年01月01日 [査読有り][通常論文]
  
• Analysis of N-glycan in serum glycoproteins from db/db mice and humans with type 2 diabetes

  Naofumi Itoh, Shinji Sakaue, Hiroaki Nakagawa, Masaki Kurogochi, Hiroshi Ohira, Kisaburo Deguchi, Shin-Ichiro Nishimura, Masaharu Nishimura

  AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 293 4 E1069 - E1077 2007年10月 [査読有り][通常論文]
   

  Glycosylation has an important role in regulating properties of proteins and is associated with many diseases. To examine the alteration of serum N-glycans in type 2 diabetes, we used the db/db mouse model. Serum N-glycans were fluorescence labeled and applied to HPLC. There were reproducible differences in N-glycan profiles between the db/db mouse model and the db/+ control. The structures of the oligosaccharides, which had changed in their amounts, were analyzed by a two-dimensional mapping method, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and exoglycosidase digestion. Those analyses revealed an increase in the N-glycans possessing alpha 1,6-fucose in the serum of db/db mice. The level of alpha 1,6-fucosyltransferase mRNA was increased in the liver of the db/db mice. The ratio of a biantennary N-glycan with alpha 1,6-fucose to that without alpha 1,6-fucose in the liver tissue of the db/db mouse was increased relative to the db/+ control. Next, we analyzed the serum N-glycan profile in human subjects with type 2 diabetes and found an increased amount of a biantennary N-glycan that had an alpha 1,6-fucose with a bisecting N-acetylglucosamine. In conclusion, the increase in alpha 1,6-fucosylation is a striking change in the serum N-glycans of the db/db mice, whereas the change in the fucosylation in humans with type 2 diabetes was small, albeit statistically significant. It is likely that the change is caused, at least partially, by the increase in the alpha 1,6- fucosyltransferase mRNA level in the liver. The increased alpha 1,6-fucosylation may affect protein properties associated with the pathophysiology of type 2 diabetes.
• Swainsonine reduces 5-fluorouracil tolerance in the multistage resistance of colorectal cancer cell lines.

  Jun Hamaguchi, Hiroaki Nakagawa, Masato Takahashi, Takeaki Kudo, Naoya Kamiyama, Bailong Sun, Takahiro Oshima, Yuji Sato, Kisaburo Deguchi, Satoru Todo, Shin-Ichiro Nishimura

  Molecular cancer 6 58 - 58 2007年09月21日 

  BACKGROUND: Drug resistance is a major problem in cancer chemotherapy. Acquisition of chemo-resistance not only reduces the effectiveness of drugs, but also promotes side effects and markedly reduces the patient's quality of life. However, a number of resistance mechanisms have been reported and are thought to be the reason for the difficulties in solving drug-resistance problems. RESULT: To investigate the mechanisms of drug resistance, a set of cell lines with different levels of sensitivity and possessing different mechanisms of resistance to 5-fluorouracil (5-FU) was established from a colorectal cancer cell line. The expression of thymidylate synthase, orotic acid phosphoribosyltransferase and dihydropyrimidine dehydrogenase, which are well known to be related to drug resistance, differed among these cell lines, indicating that these cell lines acquired different resistance mechanisms. However, swainsonine, an inhibitor of N-glycan biosynthesis, reduced 5-FU-tolerance in all resistant cells, whereas the sensitivity of the parental cells was unchanged. Further analysis of the N-glycan profiles of all cell lines showed partial inhibition of biosynthesis and no cytotoxicity at the swainsonine dosage tested. CONCLUSION: These observations suggest that N-linked oligosaccharides affect 5-FU resistance more widely than do drug-resistance related enzymes in colorectal cancer cells, and that the N-glycan could be a universal target for chemotherapy. Further, swainsonine may enhance the performance of chemotherapy by reducing tolerance.
• Structural transition of a 15 amino acid residue peptide induced by GM1

  Naoki Fujitani, Hiroki Shimizu, Teruhiko Matsubara, Takashi Ohta, Yuuki Komata, Nobuaki Miura, Toshinori Sato, Shin-Ichiro Nishimura

  CARBOHYDRATE RESEARCH 342 12-13 1895 - 1903 2007年09月 [査読有り][通常論文]
   

  The ganglioside GM1-binding peptide, p3, with a sequence of VWRLLAPPFSNRLLP, displayed a clear structural alteration depending on the presence or absence of GM1 micelles. The three-dimensional structures of the p3 peptide in the free and GM1 bound states were analyzed using two-dimensional NMR spectroscopic experiments with distance-rest rained simulated annealing calculations. The NMR experiments for the p3 peptide alone indicated that the peptide has two conformers derived from the exchange of cis and trans forms at Pro(7)-Pro(8). Further study with theoretical modeling revealed that the p3 peptide has a curb conformation without regular secondary structure. On the other band, the NMR studies for the p3 peptide with the GM1 micelles elucidated a trans conformer and gave a structure stabilized by hydrophobic interactions of beta- and helical turns. Based on these structural investigations, tryptophan, a core residue of the hydrophobic cluster, might be an essential residue for the recognition of the GM1 saccharides. The dynamic transition of the p3 peptide may play an important role in the function of GM1 as a multiple receptor as in the traditional pathway of the infection by cholera toxin. (c) 2007 Elsevier Ltd. All rights reserved.
• Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N-glycans

  Yoko Kita, Yoshiaki Miura, Jun-ichi Furukawa, Mika Nakano, Yasuro Shinohara, Masahiro Ohno, Akio Takimoto, Shin-lchiro Nishimura

  MOLECULAR & CELLULAR PROTEOMICS 6 8 1437 - 1445 2007年08月 [査読有り][通常論文]
   

  Global glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases. Despite the fact that quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol for which deglycosylation efficiency is proven to be quantitative. To establish a standard procedure to evaluate N-glycan release from whole human serum glycoproteins by peptide-N-glycosidase F ( PNGase F) treatment, we determined the efficiencies of major N-glycan liberation from serum glycoproteins in the presence of reducing agents, surfactants, protease treatment, or combinations of pretreatments prior to PNGase F digestion. We show that de-N-glycosylation efficiency differed significantly depending on the condition used, indicative of the importance of a standardized protocol for the accumulation and comparison of glycomics data. Maximal de-N-glycosylation was achieved when serum was subjected to reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant used for solubilizing proteins, or related analogues, followed by tryptic digestion prior to PNGase F treatment. An optimized de-N-glycosylation protocol permitted relative and absolute quantitation of up to 34 major N-glycans present in serum glycoproteins of normal subjects for the first time. Moreover PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, allowing accurate simulation of PNGase F-catalyzed de-N-glycosylation required for clinical glycomics using human serum samples. The results of the current study may provide a firm basis to identify new diagnostic markers based on serum glycomics analysis.
• Tumor suppressor p16(INK4a) - modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells

  Sabine Andre, Hugo Sanchez-Ruderisch, Hiroaki Nakagawa, Malte Buchholz, Jurgen Kopitz, Pia Forberich, Wolfgang Kemmner, Corina Boeck, Kisaburo Deguchi, Katharia M. Detjen, Bertram Wiedenmann, Magnus von Knebel Doeberitz, Thomas M. Gress, Shin-Ichiro Nishimura, Stefan Rosewicz, Hans-Joachim Gabius

  FEBS JOURNAL 274 13 3233 - 3256 2007年07月 [査読有り][通常論文]
   

  Expression of the tumor suppressor p16(INK4a) after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis. This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor. Considering its glycan chains as pivotal signals, we assumed an effect of p16(INK4a) on glycosylation. To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding. Major differences between p16-positive and control cells were detected. They concerned expression of beta 1,4-galactosyltransferases (down-regulation of beta 1,4-galactosyltransferases-I/V and up-regulation of beta 1,4-galactosyltransferase-IV) as well as decreased alpha 2,3-sialylation of O-glycans and alpha 2,6-sialylation of N-glycans. The changes are compatible with increased beta(1)-integrin maturation, subunit assembly and binding activity of the alpha(5)beta(1)-integrin. Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1. As a result of reduced sialylation, the cells' capacity to bind galectin-1 was enhanced. In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16(INK4a)-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis. The cells therefore gain optimal responsiveness. The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance. To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated. We conclude that p16(INK4a) orchestrates distinct aspects of glycosylation that are relevant for integrin maturation and reactivity to an endogenous effector as well as the effector's expression. This mechanism establishes a new aspect of p16(INK4a) functionality.
• Effect of pore size on in vitro cartilage formation using chitosan-based hyaluronic acid hybrid polymer fibers

  Shintaro Yamane, Norimasa Iwasaki, Yasuhiko Kasahara, Kazuo Harada, Tokifumi Majima, Kenji Monde, Shin-ichiro Nishimura, Akio Minami

  JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A 81A 3 586 - 593 2007年06月 [査読有り][通常論文]
   

  In this study, we successfully developed three-dimensional scaffolds fabricated from the chitosan-based hyaluronic acid hybrid polymer fibers, which can control the porous structure. To determine the adequate pore size for enhancing the chondrogenesis of cultured cells, we compared the behaviors of rabbit chondrocytes in scaffolds comprising different pore sizes (100, 200, and 400 mu m pore size). Regarding the cell proliferation, there was no significant difference among the three groups. On the other hand, glycosaminoglycan contents in the 400 pm group significantly increased during the culture period, compared with those in the other groups. The ratio of type II to type I collagen mRNA level was also significantly higher in the 400 mu m group than in the other groups. These results indicate that our scaffold with 400 mu m pore size significantly enhances the extracellular matrix synthesis by chondrocytes. Additionally, the current scaffolds showed high mechanical properties, compared with liquid and gel materials. The data derived from this study suggest great promise for the future of a novel fabricated material with relatively large pore size as a scaffold for cartilage regeneration. The biological and mechanical advantages presented here will make it possible to apply our scaffold to relatively wide cartilaginous lesions. (C) 2006 Wiley Periodicals, Inc. J Biomed Mater Res 81A: 586-593, 2007.
• Cell growth arrest by sialic acid clusters in ganglioside GM3 mimetic polymers

  Satoshi Uemura, Fei Feng, Maya Kume, Kuriko Yamada, Kazuya Kabayama, Shin-Ichiro Nishimura, Yasuyuki Igarashi, Jin-Ichi Inokuchi

  GLYCOBIOLOGY 17 6 568 - 577 2007年06月 [査読有り][通常論文]
   

  Ganglioside GM3, one of the sialic acid containing glycosphingolipids, is known to form clusters in lipid microdomains, which serve as platforms for effective signal transduction. In an attempt to clarify the GM3 cluster effect, we enzymatically synthesized GM3 mimetic polymer (GM3-p), with an acrylamide backbone from LacCer mimetic polymer (LacCer-p). Interestingly, GM3-p, but not LacCer-p, reversibly inhibited proliferation of NIH3T3 cells, which are normally resistant to exogenously added GM3. Moreover, we found that the introduction of carbonic acid into the acrylamide chain aided well-oriented cluster formation and enhanced the inhibitory effect of GM3-p. Since sialyllactosyl polymer and GM4 mimetic polymer, but not GM2 mimetic polymer, also inhibited cell proliferation, sialic acid-galactose units must be essential for the biological activity of GM3-p. These results suggest that the formation of sialic acid-galactose clusters is necessary for the suppressive effect of GM3-p. GM3-p treatment did not affect the serum-dependent activation of ERK1/2 or c-fos expression, but caused a reduction in the gene and/or protein expression of cyclin D1, cyclin E, cyclin-dependent kinase (cdk)4, and cdk2, which are involved in the cell cycle. Therefore, GM3-p inhibits cell proliferation by reducing cyclin D1-cdk4 and cyclin E-cdk2 complexes without affecting growth factor signaling from serum to c-fos.
• Detection of altered N-glycan profiles in whole serum from rheumatoid arthritis patients

  Hiroaki Nakagawa, Megumi Hato, Yasuhiro Takegawa, Kisaburo Deguchi, Hiroki Ito, Masahiko Takahata, Norimasa Iwasaki, Akio Minami, Shin-Ichiro Nishimura

  JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 853 1-2 133 - 137 2007年06月 [査読有り][通常論文]
   

  Altered N-glycosylation occurs in many diseases. In rheumatoid arthritis (RA), for example, reduction in galactose residues in IgG and an increase in fucose residues in alpha 1-acid glycoprotein have been observed. To further analyse N-glycans in disease, we show N-glycan profiling from whole serum employing reversed phase high performance liquid chromatography/negative-ion mode by sonic spray ionization ion trap mass spectrometry with pyridylamination. Profiles from female 15 RA patients and 18 aged-matched healthy women were compared. The most significant change seen in RA was decreased levels of mono-galactosyl bi-antennary N-glycans, in agreement with the previous reports regarding IgG. We also show previously unreported differences between isomers and increased tri-antennary oligosaccharides. These results indicate that LC-MS analysis of whole serum N-glycans can identify N-glycan alterations in RA and that this is a promising method both for studies of RA mechanisms and diagnosis. (c) 2007 Elsevier B.V. All rights reserved.
• N-glycan alterations are associated with drug resistance in human hepatocellular carcinoma

  Takeaki Kudo, Hiroaki Nakagawa, Masato Takahashi, Jun Hamaguchi, Naoya Kamiyama, Hideki Yokoo, Kazuaki Nakanishi, Takahito Nakagawa, Toshiya Kamiyama, Kisaburo Deguchi, Shin-Ichiro Nishimura, Satoru Todo

  MOLECULAR CANCER 6 32  2007年05月 [査読有り][通常論文]
   

  Background: Correlations of disease phenotypes with glycosylation changes have been analysed intensively in the tumor biology field. Glycoforms potentially associated with carcinogenesis, tumor progression and cancer metastasis have been identified. In cancer therapy, drug resistance is a severe problem, reducing therapeutic effect of drugs and adding to patient suffering. Although multiple mechanisms likely underlie resistance of cancer cells to anticancer drugs, including overexpression of transporters, the relationship of glycans to drug resistance is not well understood. Results: We established epirubicin (EPI)- and mitoxantrone (MIT)-resistant cell lines (HLE-EPI and HLE-MIT) from the human hepatocellular carcinoma cell line (HLE). HLE-EPI and HLE-MIT overexpressed transporters MDR1/ ABCB1 and BCRP/ ABCG2, respectively. Here we compared the glycomics of HLEEP1 and HLE-MIT cells with the parental HLE line. Core fucosylated triantennary oligosaccharides were increased in the two resistant lines. We investigated mRNA levels of glycosyltransferases synthesizing this oligosaccharide, namely, N-acetylglucosaminyltransferase (GnT)-IVa, GnT-IVb and alpha 1,6-fucosyltransferase (a1,6-FucT), and found that a1,6-FucT was particularly overexpressed in HLE-MIT cells. In HLE-EPI cells, GnT-IVa expression was decreased, while GnT-IVb was increased. Both GnT-IVs were downregulated in HLE-MIT cells. HLE-MIT cells also showed decreases in fucosylated tetraantennary oligosaccharide, the product of GnT-V. GnT-V expression was decreased in both lines, but particularly so in HLE-MIT cells. Thus both N-glycan and glycosyltransferase expression was altered as cells acquired tolerance, suggesting novel mechanisms of drug resistance. Conclusion: N-glycan and glycosyltransferase expression in HLE-EPI and HLE-MIT were analysed and presented that glycans altered according with acquired tolerance. These results suggested novel mechanisms of drug resistance.
• Natural killer T cells from interleukin-4-deficient mice are defective in early interferon-γ production in response to α-galactosylceramide

  Togashi Y, Chamoto K, Wakita D, Tsutsumi N, Iwakura Y, Matsubara N, Kitamura H, Nishimura T

  Cancer Science 98 5 721 - 725 2007年05月 [査読有り][通常論文]
   

  Discovery of the natural killer (NK) T cell-specific ligand, alpha-galactosylceramide (alpha-GalCer) has enabled us to investigate the functional regulation of NKT cells. However, the detailed mechanism of cytokine production by NKT cells remains to be elucidated. Here we evaluated the role of interleukin (IL)-4 in the production of interferon (IFN)-gamma from NKT cells using IL-4-deficient C57BL/6 mice (IL-4(-/-) mice). Administration of alpha-GalCer into wild-type C57BL/6 mice caused the production of both IFN-gamma and IL-4 in serum or cytoplasm within 4 h of the injection. Unexpectedly, however, IL-4(-/-) mice-derived NKT cells did not produce any IFN-gamma at early phase after primary stimulation with alpha-GalCer. Because NKT cells from IL-4(-/-) mice produced IFN-gamma when they were stimulated secondarily with alpha-GalCer in vitro for 72 h, NKT cells from IL-4(-/-) mice were not completely genetically deficient for IFN-gamma production. To elucidate which cells, NKT cells or dendritic cells (DC), were responsible for the deficiency in IFN-gamma production in IL-4(-/-) mice, we carried out an add-back experiment using purified NKT cells and DC, which were prepared from either wild-type mice or IL-4(-/-) mice. NKT cells from wild-type mice produced IFN-gamma when they were cocultured with DC prepared from either wild-type or IL-4(-/-) mice, whereas NKT cells from IL-4(-/-) mice did not produce IFN-gamma by coculturing with DC from either wild-type or IL-4(-/-) mice. These results indicate that NKT cells, not DC, were responsible for the deficiency in IFN-gamma production in IL-4(-/-) mice. Thus, IL-4 is required for the activation of NKT cells to produce IFN-gamma in response to alpha-GalCer.
• Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

  Chihiro Kitatsuji, Masaki Kurogochi, Shin-Ichiro Nishimura, Koichiro Ishimori, Keisuke Wakasugi

  JOURNAL OF MOLECULAR BIOLOGY 368 1 150 - 160 2007年04月 [査読有り][通常論文]
   

  Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (G(xi). Here, to elucidate the molecular mechanism underlying the GD1 activity of Ngb, we used an glutathione-S-transferase pun-down assay to confirm that Ngb competes with G-protein beta gamma-subunits (G beta gamma) for binding to G alpha(i), and identified the G alpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-G alpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Gai peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (G alpha(i)), and between Glu53 (Ngb) and Ser44 (G alpha(i)). Because Ser206 of G alpha(i) is located in the region that contacts G beta gamma, binding of Ngb could facilitate the release of G beta gamma from G alpha(i). Binding of Ngb to Gai would also inhibit the exchange of GDP for GTP, because Ser44 (G alpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (G alpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Gai, enabling us to discuss the functional significance of this binding on the GDI activity of Ngb. (c) 2007 Elsevier Ltd. All rights reserved.
• The solution structure of horseshoe crab antimicrobial peptide tachystatin B with an inhibitory cystine-knot motif

  Naoki Fujitani, Takahide Kouno, Taku Nakahara, Kenji Takaya, Tsukasa Osaki, Shun-Ichiro Kawabata, Mineyuki Mizuguchi, Tomoyasu Aizawa, Makoto Demura, Shin-Ichiro Nishimura, Keiichi Kawano

  JOURNAL OF PEPTIDE SCIENCE 13 4 269 - 279 2007年04月 [査読有り][通常論文]
   

  Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 angstrom, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.
• AsialoGM1(+)CD8(+) central memory-type T cells in unimmunized mice as novel immunomodulator of IFN-gamma-dependent type 1 immunity

  Akemi Kosaka, Daiko Wakita, Naoki Matsubara, Yuji Togashi, Shin-Ichiro Nishimura, Hidemitsu Kitamura, Takashi Nishimura

  INTERNATIONAL IMMUNOLOGY 19 3 249 - 256 2007年03月 [査読有り][通常論文]
   

  In unimmunized specific pathogen-free mice, there are unique memory-type CD8(+) T cell populations expressing asialoGM1 (ASGM1). These cells were classified into central memory-type T cells (T-CMT) judging from their expression profile of CD44, IL-2R beta, CD62L and CCR7 cell-surface molecules. Among CD44(high)CD8(+) so-called memory CD8(+) T cell population, ASGM1(+)CD44(high)CD8(+) T-CMT, but not ASGM1(-)CD44(high)CD8(+) memory T cells, produced IFN-gamma by stimulation with anti-CD3 mAb. The physiological significance of ASGM1(+)CD8(+) T-CMT as early source of IFN-gamma was also demonstrated in vivo. Namely, intravenous injection of anti-CD3 mAb (2 mu g) resulted in early activation of IFN-gamma-producing ASGM1(+)CD8(+) T-CMT cells as well as NKT and NK cells. Unexpectedly, however, few IFN-gamma-producing CD4(+) T cells were detected until 4 h after anti-CD3 mAb administration. Thus, ASGM1(+)CD8(+) T-CMT were demonstrated to be early IFN-gamma producer, which may be crucial for T(h)1-dependent cellular immunity. Indeed, co-culture of naive CD4(+) T cells with ASGM1(+)CD8(+) T-CMT but not ASGM1(-)CD8(+) T cells caused a great acceleration of IFN-gamma-producing T(h)1 cells in vitro. Finally, we found that T(h)1-prone C57BL/6 mice possessed higher percentage (10%) of ASGM1(+)CD8(+) T-CMT in CD8(+) T cells compared with that (3%) of T(h)2-prone BALB/c mice. Moreover, ASGM1(+)CD8(+) T-CMT derived from C57BL/6 mice produced higher levels of IFN-gamma compared with those from BALB/c mice. Thus, ASGM1(+)CD8(+) T-CMT, whose differentiation in vivo is genetically controlled, appear to play a critical role in the control of type 1 immunity, which is essential for therapy of tumors and infectious diseases.
• A new glycosylation method. Part 2: Study of carbohydrate elongation onto the gold nanoparticles in a colloidal phase

  Hiroki Shimizu, Masahiro Sakamoto, Noriko Nagahori, Shin-Ichiro Nishimura

  Tetrahedron 63 11 2418 - 2425 2007年03月 [査読有り][通常論文]
  
• N-Linked neutral oligosaccharides in the stratum corneum of normal and ichthyotic skin

  Hiroko Ito, Masashi Akiyama, Hiroaki Nakagawa, Rie Uematsu, Kisaburo Deguchi, James R. McMillan, Shin-Ichiro Nishimura, Hiroshi Shimizu

  ARCHIVES OF DERMATOLOGICAL RESEARCH 298 8 403 - 407 2007年01月 [査読有り][通常論文]
   

  N-Glycan oligosaccharides are thought to play multiple, important roles in a variety of biological events. However, N-glycan profiles in the stratum corneum of human skin have not yet been studied in detail. To clarify the N-glycan profiles in the stratum corneum of normal and ichthyotic epidermis, N-glycan profiles were studied by high-performance liquid chromatography using normal human epidermal samples and scales from hyperkeratotic skin of ichthyosis patients. Chromatograms of patient scale samples showed unique alterations in three peaks eluted at 15.8, 18.8 and 26.9 min. The N-glycan profiles were significantly altered in ichthyotic hyperkeratotic skin compared with normal non-hyperkeratotic controls. These findings indicate the reduction of N-acetylglucosaminyltransferase II and fucosyltransferase 8 activities. Alteration of N-glycan structures in hyperkeratotic skin suggests the biological role of N-glycans in keratinization.
• Structural assignment of disialylated biantennary N-glycan isomers derivatized with 2-aminopyridine using negative-ion multistage tandem mass spectral matching

  Hiroki Ito, Kuriko Yamada, Kisaburo Deguchi, Hiroaki Nakagawa, Shin-Ichiro Nishimura

  RAPID COMMUNICATIONS IN MASS SPECTROMETRY 21 2 212 - 218 2007年 [査読有り][通常論文]
   

  To investigate the possibility of structural assignment based on negative-ion multistage tandem mass (MSn) spectral matching, four isomers of disialylated biantennary N-glycans (alpha 2-6 and/or alpha 2-3 linked sialic acid on alpha 1-6 and alpha 1-3 antennae) derivatized with 2-aminopyridine (PA) were analyzed by employing high-performance liquid chromatography/electrospray ionization linear ion trap time-of-flight mass spectrometry (HPLC/ESI-LIT-TOMS), which uses helium gas for ion trapping and collision-induced dissociation (CID). It is shown that the MS2 spectra derived from each precursor ion [M-2H](2-) are reproducible and useful for distinguishing the four isomers. Thus, they can be assigned by negative-ion MS2 spectral matching based on correlation coefficients. In addition, MS3 spectra derived from D-type fragment ions clearly differentiate the alpha 2-3- or alpha 2-6-linked sialic acid on the alpha 1-6 antenna due to their characteristic spectral patterns. The C-4-type fragment ions, which are produced from both the alpha 1-6 and alpha 1-3 antennae, show the characteristic MS3 spectra reflecting alpha 2-3- or alpha 2-6- linkage type or a mixture of both types. Thus, the differentiation and assignment of these disialylated biantennary N-glycan isomers can also be supported with the MS3 spectra of C-4- and D-type ions. Copyright (c) 2006 John Wiley & Sons, Ltd.
• One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics

  Hideyuki Shimaoka, Hiromitsu Kuramoto, Jun-ichi Furukawa, Yoshiaki Miura, Masaki Kurogochi, Yoko Kita, Hiroshi Hinou, Yasuro Shinohara, Shin-Ichiro Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 13 6 1664 - 1673 2007年 [査読有り][通常論文]
   

  The development of rapid and efficient methods for high-throughput protein glycomics is of growing importance because the glycoform-focused reverse proteomics/genomics strategy will greatly contribute to the discovery of novel biomarkers closely related to cellular development, differentiation, growth, and aging as well as a variety of diseases such as cancers and vital infection. Recently, we communicated that rapid and efficient purification of carbohydrates can be achieved by employing sugar-specific chemical ligation with aminooxy-functionalized polymers, which we termed "glycoblotting" (see SA. Nishimura et al., Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 9196). The chemoselective blotting of oligosaccharides present in crude biological materials onto synthetic polymers relies on the unique oxime-bond formation between aminooxy group displayed on the supporting materials and aldehyde/ketone group at the reducing terminal of all oligosaccharides, thus enabling highly selective and rapid oligoosaccharide purification. Aiming to improve the detection sensitivity of the released oligosaccharides, we introduce here a novel strategy for one-pot solid-phase glycoblotting and probing by transoximization. We found that oligosaccharides captured by the polymer supports via the oxime bond can be released in the presence of excess O-substituted aminooxy derivatives in a weakly acidic condition. The released oligosaccharides could be recovered as newly formed oxime derivatives of the O-substituted aminooxy compound added, thus demonstrating the simultaneous releasing and probing. In addition, we synthesized a novel aminooxy-functionalized monomer, N-[2-[2-(2tert-butoxycarbonylaminooxyacetylami-no-ethoxy)ethoxy]ethyl]-2-methacrylamide, which allows for the large-scale preparation of a versatile polymer characterized by its high stability, high blotting capacity, and easy use. The one-pot protocol allowed to profile 23 kinds of N-glycan chains of human serum glycoproteins. This concept was further applied for the glycopeptides analysis in a crude mixture followed by galactose oxidase treatment to generate free aldehyde group at the non-reducing terminal of oligosaccharide moiety of glycopeptides. Our technique may be implemented in existing bio-chemistry and molecular diagnostics laboratories because enriched oligosaccharides and glycopeptides by solid-phase transoximization with high-sensitive labeling reagents are widely applicable in a variety of common analytical methods using two-dimensional HPLC, LC/MS, and capillary electrophoresis as well as modern mass spectrometry.
• Structural analysis of O-glycopeptides employing negative- and positive-ion multi-stage mass spectra obtained by collision-induced and electron-capture dissociations in linear ion trap time-of-flight mass spectrometry

  Kisaburo Deguchi, Hiroki Ito, Takashi Baba, Atsumu Hirabayashi, Hiroaki Nakagawa, Masataka Fumoto, Hiroshi Hinou, Shin-Ichiro Nishimura

  RAPID COMMUNICATIONS IN MASS SPECTROMETRY 21 5 691 - 698 2007年 [査読有り][通常論文]
   

  Structural analyses of various glycans attached to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on mass spectrometry (MS) combining both collision-induced dissociation (CID) and electron-capture dissociation (ECD) in the positive- and negative-ion modes has been proposed as a simple and direct method of assigning an O-glycan without releasing it from the peptide and of determining the amino acid sequence of the peptide and glycosylation site. The instrument used is an electrospray ionization (ESI) linear ion trap (LIT) time-of-flight (TOF) mass spectrometer with tandem LITs for CID by He gas and ECD. The proposed approach was tested with two synthetic O-glycopeptides binding a sialyl Lewis x (sLe(x)) oligosaccharide and a 3'-sialyl N-acetyllactosamine (3'-SLN) on a serine (S) residue. In the negative-ion mode, the CID MS2 spectra of O-glycopeptides showed a relatively abundant glycoside-bond cleavage between the core N-acetylglucosamine (GlcNAc) and serine (S) that yields deprotonated C-3-type fragment ions of O-glycan and deprotonated Z(0)-type peptide ions. The structure of the sLe(x) (3'-SLN) oligosaccharide was simply assigned by comparing the CID MS3 spectrum derived from the C-3-type fragment ion with the CID MS2 spectra of the sLe(x) and sLe(a) (3'- and 6'-SLN) standards (i.e., negative-ion MSn spectral matching). The amino acid sequence of the peptide including the glycosylation site was determined from the ECD MS2 spectrum in the positive-ion mode. Copyright (c) 2007 John Wiley & Sons, Ltd.
• Highly efficient and versatile synthesis of proteoglycan core structures from 1,6-anhydro-beta-lactose as a key starting material

  Ken Shimawaki, Yoshinori Fujisawa, Fumihiro Sato, Naoki Fujitani, Masaki Kurogochi, Hiroko Hoshi, Hiroshi Hinou, Shin-Ichiro Nishiinura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 46 17 3074 - 3079 2007年 [査読有り][通常論文]
  
• Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry

  Yoshiaki Miura, Yasuro Shinohara, Jun-ichi Furukawa, Noriko Nagahori, Shin-ichiro Nishimurara

  CHEMISTRY-A EUROPEAN JOURNAL 13 17 4797 - 4804 2007年 [査読有り][通常論文]
   

  A rapid and quantitative method for solid-phase methyl esterification of carboxy groups of various sialylated oligosaccharides has been established. The method employed a triazene derivative, 3-methyl-1-p-tolyltriazene, for facile derivatization of oligosaccharides immobilized onto general solid supports such as Affi-Gel Hz and gold colloidal nanoparticles in a multi-well plate. The workflow protocol was optimized for the solid-phase processing of captured sialylated/unsialylated oligosaccharides separated from crude sample mixtures by chemical ligation. From tryptic and/or PNGase F-digest mixtures of glycoproteins, purification by chemoselective immobilization, esterification and recovery were achieved in the same well of the filter plate within three hours when used in conjunction with "glycoblotting technology" (SA. Nishimura, K. Niikura, M. Kurogochi, T Matsushita, M. Fumoto, H. Hinou, R. Kamitani, H. Nakagawa, K. Deguchi, N. Miura, K. Monde, H. Kondo, High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry: Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 9196). The recovered materials were directly applicable to subsequent characterization by mass spectrometric techniques such as MALDI-TOF for large-scale glycomics of both neutral and sialylated oligosaccharides. On-bead/on-gold nanoparticle derivatization of glycans containing sialic acids allowed rapid and quantitative glycoform profiling by MALDI-TOF MS with reflector and positive ion mode. In addition to its simplicity and speed, the method eliminates the use of unfavorable halogenated solvents such as chloroform and dichloromethane or volatile solvents such as diethyl ether and hexane, resulting in a practical and green chemical method for automated robotic adaptation.
• Reverse glycoblotting allows rapid-enrichment glycoproteomics of biopharmaceuticals and disease-related biomarkers

  Masaki Kurogochi, Maho Amano, Masataka Fumoto, Akio Takimoto, Hirosato Kondo, Shin-ichiro Nishimura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 46 46 8808 - 8813 2007年 [査読有り][通常論文]
  
• Glycopolymers

  S. I. Nishimura, N. Nagahori

  Comprehensive Glycoscience: From Chemistry to Systems Biology 3-4 453 - 476 2007年01月01日
• Computational design and experimental evaluation of glycosyltransferase mutants: engineering of a blood type B galactosyltransferase with enhanced glucosyltransferase activity

  Taku Nakahara, Ole Hindsgaul, Monica M. Palcic, Shin-Ichiro Nishimura

  PROTEIN ENGINEERING DESIGN & SELECTION 19 12 571 - 578 2006年12月 [査読有り][通常論文]
   

  Glycosyltransferases are an enormous and diverse class of enzyme encompassing 1% of all sequenced genomes. They catalyze the transfer of a monosaccharide from an activated donor such as a sugar-nucleotide to an acceptor molecule. Though the primary sequences of glycosyltransferases have little homology, X-ray structural studies on glycosyltransferases have revealed that there are two main folds and that the orientation of the sugar donors with respect to the folds is highly conserved. It seems that glycosyltransferases have evolved diversified specificities toward donor sugars by changing the amino acids around the monosaccharide moiety without altering the orientation of the nucleotide moiety. In this study, we designed new glycosyltransferases with altered donor specificities by use of a novel empirical model called the Epimer Propensity Index (EPI). The EPI was constructed using 221 carbohydrate-protein complex structures in the Protein Data Bank with either galactose or glucose in the complex. The blood type B synthesizing glycosyltransferase GTB, a galactosyltransferase was our target enzyme. Two GTB mutants designed to exhibit enhanced glucosyltransferase activity were cloned, expressed and characterized experimentally. The predicted GTB mutants, Ser185Asn and Ser185Cys, exhibited 4.3- and 4.8-fold elevations in k(cat)/K-m for UDP-Glc relative to that of wild-type enzyme.
• Construction and structural characterization of versatile lactosaminoglycan-related compound library for the synthesis of complex glycopeptides and glycosphingolipids

  Kentarou Naruchi, Tomoki Hamamoto, Masaki Kurogochi, Hiroshi Hinou, Hiroki Shimizu, Takahiko Matsushita, Naoki Fujitani, Hirosato Kondo, Shin-Ichiro Nishimura

  JOURNAL OF ORGANIC CHEMISTRY 71 26 9609 - 9621 2006年12月 [査読有り][通常論文]
   

  We have established a facile and efficient protocol for the preparative-scale synthesis of various compound libraries related to lactosaminoglycans: cell surface oligosaccharides composed of N-acetyllactosamine as a repeating disaccharide unit, based on chemical and enzymatic approaches. Substrate specificity and feasibility of a bacterial glycosyltransferase, Neisseria meningitidis beta 1,3-N-acetylglucosaminyltransferase (LgtA), were investigated in order to synthesize various key intermediates suited for the construction of mammalian O-glycopeptides and glycosphingolipids containing poly-N-acetyllactosamine structures. Recombinant LgtA exhibited the highest glycosyltransferase activity with strongly basic conditions (pH = 10, glycine-NaOH buffer) and a broad range of optimal temperatures from 20 to 30 C. Interestingly, it was found that LgtA discriminates L-serine and L-threonine and functions both as a core-1 beta 1,3-N-acetylglucosaminyltransferase and core-2 beta 1,3-N-acetylglucosaminyltransferase toward Fmoc-Ser derivatives, while LgtA showed only core-2 beta 1,3-N-acetylglucosaminyltransferase activity in the presence of Fmoc-Thr derivatives. Combined use of LgtA with human beta 1,4-galactosyltransferase allowed for controlled sugar extension reactions from synthetic sugar amino acids and gave synthetic lactosaminoglycans, such as a decasaccharide derivative, Gal,(1 -> 4) GlcNAc beta(1 -> 3) Gal beta(1 -> 4) GlcNAc beta( 1 -> 3) Gal beta(1 -> 4) GlcNAc beta(1 -> 3) Ga beta(1 -> 4) GlcNAc beta(1 -> 6)[Gal beta(1 -> 3)] GalNAc alpha 1 f Fmoc-Ser-OH (6), and a dodecasaccharide derivative, Gal beta( 1 f 4) GlcNAc beta(1 -> 3) Gal beta( 1 -> 4) GlcNAc beta(1 -> 3) Gal beta(1 -> 4)GlcNAc beta(1 -> 6)[Gal beta(1 -> 4) GlcNAc beta(1 -> 3) Gal beta(1 -> 4) GlcNAc beta(1 -> 3) Gal beta(1 -> 3)] GalNAc alpha 1 f Fmoc-Ser-OH(9). A partially protected pentasaccharide intermediate, GlcNAc beta(1 -> 3) Gal beta(1 -> 4)GlcNAc,(1 -> 6)[Gal beta(1 -> 3)] GalNAc alpha 1 -> Fmoc-Thr-OH (11), was applied for the microwave-assisted solid-phase synthesis of a MUC1-related glycopeptide 19 (MW = 2610.1). The findings suggest that this sugar extension strategy can be employed for the modification of lactosyl ceramide mimetic polymers to afford convenient precursors for the synthesis of various glycosphingolipids.
• Rewinding of Helical Systems by Use of the Cr(III) Complex as a Photoresponsive Chiral Dopant

  Tohru Taniguchi, Kenji Monde, Shin-Ichiro Nishimura, Jun Yoshida, Hisako Sato, Akihiko Yamagishi

  Molecular Crystals and Liquid Crystals 460 1 107 - 116 2006年12月01日
• Unusual N-glycan structures in alpha-mannosidase II/IIx double null embryos identified by a systematic glycomics approach based on two-dimensional LC mapping and matrix-dependent selective fragmentation method in MALDI-TOF/TOF mass spectrometry

  Megumi Hato, Hiroaki Nakagawa, Masaki Kurogochi, Tomoya O. Akama, Jamey D. Marth, Michiko N. Fukuda, Shin-Ichiro Nishimura

  MOLECULAR & CELLULAR PROTEOMICS 5 11 2146 - 2157 2006年11月 [査読有り][通常論文]
   

  alpha-Mannosidase IIx (MX) is an enzyme closely related to alpha-mannosidase II (MII), a key enzyme in N-glycan biosynthesis that catalyzes the first step in conversion of hybrid-to complex-type N-glycans in Golgi apparatus. Recently we generated MII/MX double knock-out mice and found that double nulls completely lack the complex-type N-glycans (Akama, T.O., Nakagawa, H., Wong, N.K., Sutton-Smith, M., Dell, A., Morris, H. R., Nakayama, J., Nishimura, S.-I., Pai, A., Moremen, K. W., Marth, J. D., and Fukuda, M.N. ( 2006) Essential and mutually compensatory roles of alpha-mannosidase II and alpha-mannosidase IIx in N-glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 8983-8988). In the present study, we determined minor but unusual N-glycan structures found in MII/ MX double knock-out mice. We identified such N-glycans by a systematic glycomics approach applying a two-dimensional LC mapping database and matrix-dependent selective fragmentation technique in MALDI-TOF/TOF MS, a highly sensitive and reliable technique that provides specific fragmentations enabling the determination of precise oligosaccharide structures including regioisomers (Kurogochi, M., and Nishimura, S.-I. ( 2004) Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097-6101). Quantitative profiling of all N-glycan structures including minor components from MII/ MX nulls, MII nulls, MX nulls, and wildtype mice at embryonic day 15.5 yielded a total of 37 species when structural heterogeneity was reduced by the removal of the sialic acids. Among six unusual N-glycan structures, two glycoforms were novel and were found only in MII/ MX double nulls. We characterize such structure as pseudocomplex-type N-glycans. The present study demonstrated that use of the versatile matrix-dependent selective fragmentation method in MALDI-TOF/TOF MS greatly accelerates detailed structural analysis of a trace amount of N-glycans.
• Interleukin-12-responding asialoGM1(+)CD8(+) central memory-type T cells as precursor cells for interferon-gamma-producing killer T cells

  Akemi Kosaka, Ushaku Lee, Daiko Wakita, Naoki Matsubara, Yuji Togashi, Shin-Ichiro Nishimura, Hidemitsu Kitamura, Takashi Nishimura

  CANCER SCIENCE 97 11 1236 - 1241 2006年11月 [査読有り][通常論文]
   

  While investigating CD8(+) memory T cells in unimmunized C57BL/6 mice, we found that there were unique memory-type CD8(+) T cells expressing asialoGM1 (ASGM1), CD62L and CCR7 cell surface molecules, which occupied approximately 10% of CD8(+) T cells and 35% of CD44(+) memory CD8(+) T cells. Culture of freshly isolated ASGM1(+)CD8(+) T cells with interleukin (IL)-12 plus IL-2 caused the proliferation and generation of killer T cells. Moreover, ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, produced high levels of interferon (IFN)-gamma in response to IL-12 plus IL-2. Although ASGM1(+)CD8(+) T cells showed no significant responses to IL-12 alone or IL-2 alone, pulse incubation of ASGM1(+)CD8(+) T cells with IL-12 at an earlier time (0-12 h), and subsequently with IL-2 at a later time (12-24 h), caused the same levels of proliferation, killer cell generation and IFN-gamma production as when they were incubated simultaneously with IL-12 plus IL-2 for 24 h. Thus, ASGM1(+)CD8(+) T cells appeared to respond to IL-12 directly to acquire IL-2 responsiveness and differentiate into IFN-gamma-producing killer T cells. Indeed, freshly isolated ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, expressed higher levels of IL-12R beta 2 mRNA. The fact that IL-12 administration in vivo caused the generation of ASGM1(+)CD8(+) killer T cells in an IFN-gamma-dependent manner further indicated a physiological significance of ASGM1(+)CD8(+) central memory-type T cells in IL-12-induced immunoregulation for the therapy of tumors and infectious diseases.
• Specific cell behavior of human fibroblast onto carbohydrate surface detected by glycoblotting films

  Tomohiro Onodera, Kenichi Niikura, Norimasa Iwasaki, Noriko Nagahori, Hideyuki Shimaoka, Ryusuke Kamitani, Tokifumi Majima, Akio Minami, Shin-Ichiro Nishimura

  BIOMACROMOLECULES 7 11 2949 - 2955 2006年11月 [査読有り][通常論文]
   

  We synthesized an aminooxyl polymer that is reactive with the reduced end of carbohydrates using our sugar-displaying approach. The carbohydrates were easily immobilized on the polymer film (glycoblotting film) by simple immersion in a in sugar solution through stable oxime bond. The in vitro behaviors of human fibroblasts on the carbohydrate-coated surface were investigated. The adhesion of human fibroblasts on the cellobiose- and cellotriose-coated surfaces was much greater than on the other coated surfaces and the noncoated surface. This result indicated that simple structural differences in carbohydrates induced biological changes in human cells, especially cell adhesion. Our approach provides a high-throughput assay system for carbohydrate-related cell adhesion and proliferation.
• Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography

  Yasuhiro Takegawa, Kisaburo Deguchi, Hiroki Ito, Takuro Keira, Hiroaki Nakagawa, Shin-Ichiro Nishimura

  JOURNAL OF SEPARATION SCIENCE 29 16 2533 - 2540 2006年11月 [査読有り][通常論文]
   

  Asparagine-linked oligosaccharides (N-glycans) usually show structural heterogeneity, especially in proteins with sialylated N-glycans and, therefore, their structural analysis is still very difficult. A zwitterionic type of hydrophilic interaction chromatography column with sulfobetaine functional groups (called a ZIC-HILIC column) was applied to the separation of tryptic peptides of alpha-l-acid glycoprotein. It was demonstrated that the ZIC-HILIC separation column has a selectivity for sialylated N-glycopeptides and a high capability for separation based on the structural recognition of sialylated N-glycan isomers as well as for the previously reported neutral N-glycans and N-glycopeptides. The retention characteristics of neutral and sialylated N-glycans derivatized with 2-aminopyridine (PA N-glycans) demonstrate that the retentions of the N-glycans are based primarily on hydrophilic interaction with the water-rich liquid layer generated on the surface of the ZIC-HILIC column. In addition, the electrostatic repulsion interaction shielded with counter ions effectively tunes the separation and recognition of sialylated N-glycan isomers.
• Construction of MUC1 related compound library

  Ohyabu Naoki, Matsushita Takahiko, Hinou Hiroshi, Izumi Ryuko, Shimizu Hiroki, Kondo Hirosato, Nishimura Shin-Ichiro

  GLYCOBIOLOGY 16 11 1152 - 1152 2006年11月 [査読有り][通常論文]
  
• Structural analysis of O-glycopeptides employing negative- and positive-ion MSn spectra acquired by nanoHPLC/ESI-linear ion trap time-of-flight mass spectrometer

  Ito Hiroki, Deguchi Kisaburo, Yamada Kuriko, Nagai Shinji, Fumoto Masataka, Hinou Hiroshi, Nakagawa Hiroaki, Shinohara Yasuro, Nishimura Shin-Ichiro

  GLYCOBIOLOGY 16 11 1136  2006年11月 [査読有り][通常論文]
  
• 抗癌剤耐性ヒト肝癌細胞株におけるN-結合型糖鎖の構造変化

  工藤 岳秋, 中川 裕章, 高橋 將人, 神山 直也, 濱口 純, 横尾 英樹, 中西 一彰, 中川 隆公, 尾崎 倫孝, 神山 俊哉, 出口 喜三郎, 西村 紳一郎, 藤堂 省

  日本癌学会総会記事 65回 100 - 100 日本癌学会 2006年09月
• Direct and efficient monitoring of glycosyltransferase reactions on gold colloidal nanoparticles by using mass spectrometry

  Noriko Nagahori, Shin-Ichiro Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 12 25 6478 - 6485 2006年08月 [査読有り][通常論文]
   

  A simple and efficient assay for glycosyltransferase activity on gold colloidal nanoparticles (GCNPs) by using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) is demonstrated by the enzymatic synthesis of the Lewis X trisaccharide on GCNPs containing GlcNAc residues. GCNPs containing multivalent sugars were well dispersed in aqueous solution and proved to be excellent acceptor substrates for the glycosyltransferase reaction. Direct LDI-TOF MS analysis of these GCNPs provided the ion peaks of the sugar derivatives, chemisorbed through S-Au linkages onto the GCNPs, even in the presence of contaminants such as proteins and salts. Thus, it enabled the rapid and direct detection of the enzymatic reaction on the GCNPs by subjecting a small amount (0.15 mu L) of the reaction mixture to MS analysis without purification. Subsequent MS/MS analyses (LDI-LIFT-TOF/TOF method) of the product-carrying GCNPs enabled the structures of the sugar derivatives that had been constructed on the GCNPs by enzymatic glycosylation to be determined. A quantitative inhibition assay for glycosyltransferase by using LDI-TOF MS analysis on the GCNPs was demonstrated by using uridine 5'-diphosphate (UDP) as the inhibitor. This simple assay was then applied to the detection of the enzymatic activity of a crude cell extract of Escherichia coli, which produces Neisseria meningitidis beta-1,4-galactosyltransferase (beta-1,4-GalT). In this case, the GCNPs were roughly purified by means of ultrafiltration to remove the buffer and detergents before MS analysis. That the GCNPs are dissolved in solution in the reaction medium but are solid in the purification process is greatly advantageous for the simple and efficient detection of enzymatic activity in crude biological samples. Thus, GCNPs containing a variety of biomolecules may become a versatile and efficient tool for the rapid and direct monitoring of metabolism (metabolomics) in living cells when combined with LDI-TOF MS analysis.
• Essential and mutually compensatory roles of alpha-mannosidase II and alpha-mannosidase IIx in N-glycan processing in vivo in mice

  TO Akama, H Nakagawa, NK Wong, M Sutton-Smith, A Dell, HR Morris, J Nakayama, SI Nishimura, A Pai, KW Moremen, JD Marth, MN Fukuda

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 24 8983 - 8988 2006年06月 [査読有り][通常論文]
   

  Many proteins synthesized through the secretory pathway receive posttranslational modifications, including N-glycosylation. a-Mannosidase II (MII) is a key enzyme converting precursor high-mannose-type N-glycans to matured complex-type structures. Previous studies showed that Mll-null mice synthesize complextype N-glycans, indicating the presence of an alternative pathway. Because alpha-mannosiclase IN (MX) is a candidate enzyme for this pathway, we asked whether MX functions in N-glycan processing by generating MII/MX double-null mice. Some double-nulls died between embryonic days 15.5 and 18.5, but most survived until shortly after birth and died of respiratory failure, which represents a more severe phenotype than that seen in single-nulls for either gene. Structural analysis of N-glycans revealed that double-nulls completely lack complex-type N-glycans, demonstrating a critical role for at least one of these enzymes for effective N-glycan processing. Recombinant mouse MX and MII showed identical substrate specificities toward N-glycan substrates, suggesting that MX is an isozyme of MII. Thus, either MII or MX can biochemically compensate for the deficiency of the other in vivo, and either of two is required for late embryonic and early postnatal development.
• Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography

  Y Takegawa, K Deguchi, T Keira, H Ito, H Nakagawa, S Nishimura

  JOURNAL OF CHROMATOGRAPHY A 1113 1-2 177 - 181 2006年04月 [査読有り][通常論文]
   

  Isomeric oligosaccharides and isomeric glycopeptides are sometimes difficult to separate on normal-phase (NP) and reversed-phase (RP) columns. A zwitterionic type of hydrophilic-interaction chromatography column with sulfobetaine groups (called ZIC-HILIC column) was first applied to the separation of 2-aminopyridine derivatized (PA) N-glycans and tryptic peptides of human serum immunoglobulin G (IgG). It is shown that the ZIC-HILIC column has high capability for structural recognition of isomeric N-glycans as well as high selectivity for glycopeptides. The former feature (i.e., structural recognition) was proven by sufficient separation of neutral PA N-glycan isomers, which are usually difficult to separate on NP and RP columns. In addition, it is noteworthy that IgG glycopeptides consisting of isomeric N-glycans and the same peptide sequences can be sufficiently separated on a ZIC-HILIC column. The latter feature (i.e., selectivity) was also demonstrated by easily separating two peptide groups with/without N-glycans. Thus, we note that the ZIC-HILIC column is highly promising for a simple analysis of N-glycans and N-glycopeptide samples. (c) 2006 Elsevier B.V. All rights reserved.
• N-acetylglucosaminyltransferase V and beta 1-6 branching N-linked oligosaccharides are associated with good prognosis of patients with bladder cancer

  H Ishimura, T Takahashi, H Nakagawa, SI Nishimura, Y Arai, Y Horikawa, T Habuchi, E Miyoshi, A Kyan, S Hagisawa, C Ohyama

  CLINICAL CANCER RESEARCH 12 8 2506 - 2511 2006年04月 [査読有り][通常論文]
   

  Purpose: N-acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes beta 1-6 branching of N-acetylglucosamine on asparagine (N)-linked oligosaccharides (N-glycan) of cell proteins. We examined the relationship between GnT-V expression and clinicopathologic features of the patients with bladder cancer. Experimental Design: We immunohistochemically examined GnT-V expression in paraffin-embedded bladder cancer specimen using anti-GnT-V monoclonal antibody. We compared GnT-V expression with cause-specific survival of the patients with bladder cancer treated by radical cystectomy. Kaplan-Meier survival curves were generated to show the cause-specific survival. Univariate and multivariate analyses were carried out to compare GnT-V expression with other clinical and pathologic variables. We also evaluated mRNA expression of GnT-V and N-linked oligosaccharide structure in bladder cancer specimens. Results: Immunohistochemistry revealed that GnT-V expression inversely correlated with tumor grade and stage. The incidence of positive GnT-V expression in bladder cancer was significantly higher in low-grade/superficial cancer than in high-grade/invasive cancer. The patients whose tumor was positive for GnT-V survived significantly longer than those whose tumor was negative for GnT-V. Univariate and multivariate analyses revealed that GnT-V expression was an independent predictor of prognosis of the patient. The expression of GnT-V mRNA determined by reverse transcription-PCR was consistent with the results with immunohistochemistry for tumor samples. Carbohydrate structural analysis revealed that superficial bladder cancer is rich in branched N-linked oligosaccharides, for which biosynthesis GnT-V is responsible. Conclusions: GnT-V and its resultant beta 1-6 branching N-linked oligosaccharides are closely related to low malignant potential and good prognosis of the patients with bladder cancer.
• Construction of Highly Glycosylated Mucin-Type Glycopeptides Based on Microwave-Assisted Solid-Phase Syntheses and Enzymatic Modifications

  Takahiko Matsushita, Hiroshi Hinou, Masataka Fumoto, Masaki Kurogochi, Naoki Fujitani, Hiroki Shimizu, Shin-Ichiro Nishimura

  The Journal of Organic Chemistry 71 8 3051 - 3063 2006年04月 [査読有り]
  
• Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography

  Yasuhiro Takegawa, Kisaburo Deguchi, Takuro Keira, Hiroki Ito, Hiroaki Nakagawa, Shin-Ichiro Nishimura

  Journal of Chromatography A 1113 1-2 177 - 181 2006年04月 [査読有り][通常論文]
  
• Effects of synthetic antifreeze glycoprotein analogue on islet cell survival and function during cryopreservation

  S Matsumoto, M Matsusita, T Morita, H Kamachi, S Tsukiyama, Y Furukawa, S Koshida, Y Tachibana, S Nishimura, S Todo

  CRYOBIOLOGY 52 1 90 - 98 2006年02月 [査読有り][通常論文]
   

  The antifreeze glycoprotein (AFGP), found in the blood of polar fish, is known to prevent ice crystal growth and to depress the freezing temperature, which may in turn protect tissues from freezing injury. The chemical synthesis of AFGP is an attractive alternative to its difficult isolation from natural sources, and this would permit quality control and mass production. In spite of recent success in islet transplantation for the treatment of type I diabetes mellitus, existing methods for the long-term preservation of islets are considered to be suboptimal and inadequate, which indicates the need for the development of improved methods. Rat islets were isolated from male Wistar rats, using intraductal collagenase distention, mechanical dissociation, and Ficoll-Conray gradient purification. Islets were cultured overnight and then cryopreserved in RPMI1640 in the presence of dimethyl sulfoxide (Me,SO) and 10% FCS with various concentrations of syAFGP, followed by slow cooling (0.3 degrees C/min) and rapid thawing (200 degrees C/min) as described by Rajotte. The freezing process was observed by cryomicroscopy. Islet recovery post-cryo preservation was 85.0 +/- 6.2% with syAFGP and 63.3 +/- 14.2% without syAFGP, both compared with the pre-cryopreservation counts (P < 0.05). The in vitro islet function measured by insulin release was equivalent to a static stimulation index of 3.86 +/- 0.43 for the islets that were frozen-and-thawed with syAFGP, compared to 2.98 +/- 0.22 without syAFGP (P < 0.05). At a concentration of around 500 mu g/ml syAFGP, a strong attenuation of ice crystal growth and formation was observed by cryomicroscopy and these ice crystals did not cause cryoinjury. In conclusion, the attenuation of ice crystallization by syAFGP improves islet survival and function following cryopreservation and thawing. (C) 2005 Elsevier Inc. All rights reserved.
• A theoretical study of α- and β-d-glucopyranose conformations by the density functional theory

  Nobuaki Miura, Tohru Taniguchi, Kenji Monde, Shin-Ichiro Nishimura

  Chemical Physics Letters 419 4-6 326 - 332 2006年02月 [査読有り][通常論文]
  
• Structural assignment of isomeric 2-aminopyridine-derivatized monosialylated biantennary N-linked oligosaccharides using negative-ion multistage tandem mass spectral matching

  K Deguchi, Y Takegawa, H Ito, N Miura, S Yoshioka, S Nagai, H Nakagawa, SI Nishimura

  RAPID COMMUNICATIONS IN MASS SPECTROMETRY 20 3 412 - 418 2006年 [査読有り][通常論文]
   

  To investigate the possibility of structural assignment based on negative-ion tandem multistage (MSn) mass spectral matching, four isomers of 2-aminopyridine (PA)-derivatized monosialylated oligosaccharides (i.e., complex-type N-glycans with an alpha 2-3- or alpha 2-6-linked sialic acid on alpha 1-6 or alpha 1-3 antennae) were analyzed using high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry (HPLC/ESI-IT-TOFMS). The negative ion [M-2H](2-) is observed predominantly in the MS' spectra without the loss of a sialic acid. The MS2 spectra derived from it are sufficiently reproducible that MS2 spectral matching based on correlation coefficients can be applied to the assignment of these isomers. The isomers containing a sialic acid on alpha 1-6 or alpha 1-3 antennae can be distinguished by MS2 spectral matching, but the alpha 2-3 and alpha 2-6 linkage types of sialic acid cannot be distinguished by their MS2 spectra. However, MS3 spectra derived from fragment ions containing a sialic acid (i.e., C-4- and D-type ions) clearly differentiate the alpha 2-3 and alpha 2-6 linkage types of sialic acid in their MS3 spectral patterns. This difference might be rationalized in terms of a proton transfer from the reducing-end mannose to the negatively charged sialic acid. These two moieties are very close in the structural conformations of the precursor C-4-type fragment ions of alpha 2-6 linkage type, as predicted by molecular mechanics calculations. Thus, negative-ion MSn (n = 2, 3) spectral matching was demonstrated to be useful for the structural assignment of these four monosialylated PA N-glycan isomers. Copyright (c) 2005 John Wiley & Sons, Ltd.
• Complementary structural information of positive- and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycans

  K Deguchi, H Ito, Y Takegawa, N Shinji, H Nakagawa, SI Nishimura

  RAPID COMMUNICATIONS IN MASS SPECTROMETRY 20 5 741 - 746 2006年 [査読有り][通常論文]
   

  Positive- and negative-ion MSn spectra of chicken egg yolk glycopeptides binding a neutral and a sialylated N-glycan were acquired by using electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) and collision-induced dissociation (CID) with helium as collision gas. Several characteristic differences were observed between the positive- and negative-ion CID MSn (n = 2,3) spectra. In the positive-ion MS2 Spectra, the peptide moiety was presumably stable, but the neutral N-glycan moiety caused several B-type fragmentations and the sialylated N-glycan almost lost sialic acid(s). In contrast, in the negative-ion MS2 spectra, the peptide moiety caused several side-chain and N-glycan residue (e.g., N-acetylglucosamine (GlcNAc) residue) fragmentations in addition to backbone cleavages, but the N-glycan moieties were relatively stable. The positive-ion MS3 spectra derived from the protonated peptide ion containing a GlcNAc residue (203.1 Da) provided enough information to determine the peptide amino-acid sequence including the glycosylation site, while the negative-ion MS3 spectra derived from the deprotonated peptide containing a X-0,2(1)-type cross-ring cleavage (83.1 Da) complicated the peptide sequence analysis due to side-chain and X-0,2(1) residue related fragmentations. However, for the structural information of the N-glycan moiety of the glycopeptides, the negative-ion CID MS3 spectra derived from the deprotonated (2,4)A(6)-type cross-ring cleavage ion (neutral N-glycan) or the doubly deprotonated B-6-type fragment ion (sialylated N-glycan) are more informative than are those of the corresponding positive-ion CID MS3 spectra. Thus, the positive-ion mode of CID is useful for the analyses of peptide amino-acid sequences including the glycosylation site. The negative-ion mode of CID is especially useful for sialylated N-glycan structural analysis. Therefore, in the structural analysis of N-glycopeptides, their roles are complementary. Copyright (c) 2006 John Wiley & Sons, Ltd.
• Glycosidation promoted by a reusable solid superacid in supercritical carbon dioxide

  Xue-Bing Li, Masato Ogawa, Toshiki Monden, Takahiro Maeda, Eri Yamashita, Mariko Naka, Masao Matsuda, Hiroshi Hinou, Shin-Ichiro Nishimura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 45 34 5652 - 5655 2006年 [査読有り][通常論文]
  
• Direct structural assignment of neutral and sialylated N-glycans of glycopeptides using collision-induced dissociation MSn spectral matching

  Hiroki Ito, Yasuhiro Takegawa, Kisaburo Deguchi, Shinji Nagai, Hiroaki Nakagawa, Yasuro Shinohara, Shin-Ichiro Nishimura

  RAPID COMMUNICATIONS IN MASS SPECTROMETRY 20 23 3557 - 3565 2006年 [査読有り][通常論文]
   

  Mass spectrometric analyses of various N-glycans binding to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on collision-induced dissociation (CID) MSn spectra acquired by electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) in the positive- and negative-ion modes has been proposed as a direct method of assigning N-glycans without releasing them from N-glycopeptides. In the positive-ion mode of this approach, the MS2 spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAc beta 1-4GlcNAc, GlcNAc: N-acetylglucosamine) attached to asparagine (N), and two charges on the [M+H+Na](2+) precursor ion are shared with both of the resulting fragments. These fragments are sodiated B-n-type fragment ions of oligosaccharide (N-glycan) and a protonated peptide ion retaining one GlcNAc residue on the asparagine (N) residue. The structure of N-glycan was assigned by comparing MS3 spectra derived from both the sodiated Bn-type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MSn spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MSn spectra of deprotonated B-n-type fragment ions of N-glycopeptide and the PA N-glycan standard. The efficacy of this approach was tested with chicken egg yolk glycopeptides with a neutral and a sialylated N-glycan, and human serum IgG glycopeptides with neutral N-glycan isomers. These results suggest that the approach based on MSn spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides. Copyright (c) 2006 John Wiley & Sons, Ltd.
• Mechanism-based inhibitors to probe transitional states of glycoside hydrolases

  Hiroshi Hinou, Masaki Kurogochi, Shin-Ichiro Nishimura

  GLYCOBIOLOGY 415 202 - + 2006年 [査読有り][通常論文]
   

  Recent structural and kinetic studies indicate that glycosidases (glycoside hydrolases) change the peripheral structure of their catalytic sites dynamically to trim glycan structures. Inhibitors that label specific.amino acid residues in the active site of these enzymes based on its mechanism of action are powerful tools to probe such a hidden transitional state. This chapter describes methods of mechanism-based irreversible inhibitors having fluorescence tags, including synthesis, inhibitory assay, rapid separation of the peptides containing labeled residues using antibody column, and proteomic analysis of key amino acid residues using matrix-assisted laser desorption/ionization-time-of-flight (TOF)/TOF mass spectrometry.
• Controlled synthesis of artificial glycoprotein using cell-free system

  Reiko Sadamoto, Kei Oiwa, Naoki Matsubara, Takeshi Matsubayashi, Norio Fukuhara, Hiroaki Suga, Shin-Ichiro Nishimura

  Polymer Preprints, Japan 55 2 5274 - 5275 2006年 

  Carbohydrates are known as biomolecules in cell recognitions. In this research, we use a site-specific introduction of carbohydrates into proteins. As carbohydrate-reactive unnatural amino acids, Tyr or Phe derivatives with an aminooxyl residue, were synthesized. We then prepared aminoacyl-tRNAs on a resin-immobilized ribozyme. The protein containing the unnatural amino acids were synthesized in Escherichia coli- in vitro translation system for glycosylation.
• High density sugar-displaying materials using the glycoblotting methods for cell patterning

  Ryosuke Kamitani, Kenichi Niikura, Tomohiro Onodera, Norimasa Iwasaki, Masataka Fumoto, Hideyuki Shimaoka, Kuniharu Ijiro, Shin-Ichiro Nishimura

  Polymer Preprints, Japan 55 1 1895  2006年 

  Synthetic sugar-based materials have been interested in important tools, because cell surface carbohydrates are involved in numerous biological functions, including cellular recognition, adhesion, cell growth regulation. In this study, we present novel sugar-displaying materials by using the glycoblotting method combined with photo-patterning. The glycoblotting method is based on the chemoselective ligation of carbohydrates with reactive aminooxyl groups attached to the polymer side chains. Cell patternings were created on the sugar-displaying substrate by the specific cell-carbohydrate interactions.
• High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis

  R Uematsu, J Furukawa, H Nakagawa, Y Shinohara, K Deguchi, K Monde, SI Nishimura

  MOLECULAR & CELLULAR PROTEOMICS 4 12 1977 - 1989 2005年12月 [査読有り][通常論文]
   

  Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan derivatization with novel, stable isotope-coded derivatization reagents followed by MALDI-TOF(/TOF) analysis. This analysis revealed distinct features of the N-glycosylation profile of epidermis and dermis for the first time. A high abundance of high mannose type oligosaccharides was found to be characteristic of murine epidermis glycoproteins. Based on this observation, we performed high mannose type glycoform-focused proteomics by direct tryptic digestion of protein mixtures and affinity enrichment. We identified 15 glycoproteins with 19 N-glycosylation sites that carry high mannose type glycans by off-line LC-MALDI-TOF/TOF mass spectrometry. Moreover the relative quantity of microheterogeneity of different glycoforms present at each N-glycan binding site was determined. Glycoproteins identified were often contained in lysosomes (e.g. cathepsin L and gamma-glutamyl hydrolase), lamellar granules (e.g. glucosylceramidase and cathepsin D), and desmosomes (e g. desmocollin 1, desmocollin 3, and desmoglein). Lamellar granules are organelles found in the terminally differentiating cells of keratinizing epithelia, and desmosomes are intercellular junctions in vertebrate epithelial cells, thus indicating that N-glycosylation of tissue-specific glycoproteins may contribute to increase the relative proportion of high mannose glycans. The striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation may also reflect the observed high abundance of high mannose glycans.
• Molecular design of glycoprotein mimetics: Glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue

  N Matsubara, K Oiwa, T Hohsaka, R Sadamoto, K Niikura, N Fukuhara, A Takimoto, H Kondo, S Nishimurar

  CHEMISTRY-A EUROPEAN JOURNAL 11 23 6974 - 6981 2005年11月 [査読有り][通常論文]
   

  The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.
• Chitosan-RGDSGGC conjugate as a scaffold material for musculoskeletal tissue engineering

  T Masuko, N Iwasaki, S Yamane, T Funakoshi, T Majima, A Minami, N Ohsuga, T Ohta, SI Nishimura

  BIOMATERIALS 26 26 5339 - 5347 2005年09月 [査読有り][通常論文]
   

  In the present study, we have developed a novel and versatile method for the preparation of chitosan-peptide complex based on the selective reaction of chitosan with 2-iminothiolane. The new type of SH-chitosan derivative showed an excellent solubility to aqueous solution even in the alkaline conditions. This characteristic greatly facilitated further modification study of chifosan with a variety of bioactive substances. A synthetic peptide, RGDSGGC containing RGDS moiety that is known as one of the most important cell adhesive peptides, was readily coupled by disulfide bonds formation with sulfhydryl groups of SH-chitosan in the presence of dimethyl sulfoxide. Next, the effect of the introduction of RGDSGGC moiety to chitosan on cell adhesion and proliferation activity of chondrocytes and fibroblasts were evaluated. As a result, it was suggested that this polysaccharide-peptide conjugate exhibited excellent capacities for both cell adhesion and cell proliferation of chondrocytes and fibroblasts. Considering the growing importance of the biocompatible scaffolds in the recent tailored tissue engineering technique, these results indicate that the present strategy of 2-iminothiolane-based conjugation of polysaccharides with biologically active peptides will become a key and potential technology to develop desirable scaffold materials for the tissue regenerations. (c) 2005 Elsevier Ltd. All rights reserved.
• Characterization of Vibrio cholerae neuraminidase by a novel mechanism-based fluorescent labeling reagent

  H Hinou, M Kurogochi, H Shimizu, SI Nishimura

  BIOCHEMISTRY 44 35 11669 - 11675 2005年09月 [査読有り][通常論文]
   

  Vibrio cholerae neuraminidase (VCNA) plays a significant role in the pathogenesis of cholera by removing sialic acid residues from higher-order gangliosides to an unmasked GM1, the essential receptor for cholera toxin. Here we report that a novel mechanism-based fluorescent labeling reagent, 5-acetamido2-(4-N-5-dimethylaminonaphthalene-1-sulfonyl-2-difluoroi-nethylphenyl)-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosonic acid (1), becomes a unique irreversible inhibitor of VCNA. Characterization of an inactivated VCNA by MALDI-TOF/TOFMS analysis revealed that the Asp-576 and Arg-577 residues, which are located within the (DRFF571)-D-576 sequence, were specifically labeled with this suicide-type fluorescent substrate. Neither Asp-576 nor Arg-577 has ever been known to contribute to a specific residue in the rigid and highly conserved active site of VCNA investigated by crystallographic analysis, suggesting that a flexible beta-turn structure containing this sequence may have a crucial role in the dynamic nature of substrate recognition and catalytic action by VCNA.
• Structural analysis of an N-glycan with "beta 1-4 bisecting branch" from human serum IgG by negative-ion MSn spectral matching and exoglycosidase digestion

  Y Takegawa, K Deguchi, H Nakagawa, SI Nishimura

  ANALYTICAL CHEMISTRY 77 18 6062 - 6068 2005年09月 [査読有り][通常論文]
   

  A novel N-linked oligosaccharide (N-glycan) with "beta 1-4 bisecting branch (galactose beta 1-4 bisecting N-acetylglucosamine)" was found in human serum IgG. Its structure was efficiently analyzed by using beta-galactosidase digestion, a MSn spectral library database, and negative-ion MS2 spectral matching. For confirmation, the novel N-glycan was synthesized by using an expected standard N-glycan (acceptor), UDP-galactose (donor), and beta 1 -4 galactosyltransferase. This work also demonstrates that the MSn spectral library database, in particular, negative-ion MS2 spectral matching, can efficiently reduce the number of specific, sequential exoglycosidase digestions required and is useful for rapid structural analysis of unknown glycans not in the database.
• Rational design, synthesis, and characterization of novel inhibitors for human beta 1,4-galactosyltransferase

  K Takaya, N Nagahori, M Kurogochi, T Furuike, N Miura, K Monde, YC Lee, SI Nishimura

  JOURNAL OF MEDICINAL CHEMISTRY 48 19 6054 - 6065 2005年09月 [査読有り][通常論文]
   

  An affinity labeling reagent, uridine 5'-(6-amino-{2-[(7-bromomethyl-2-naphthyl)methoxy-carbonylmethoxy]ethoxy}acetyl-6-deoxy-alpha-D-galactopyranosyl) diphosphate (1a), was designed on the basis of 3D docking simulation and synthesized to investigate the functional role of Trp310 residue located in the small loop near the active site of human recombinant galactosyltransferase (beta GalT-1). Mass spectrometric analysis revealed that the Trp310 residue of beta GalT1 can be selectively modified with the naphthylmethyl group of compound la at the C-3 position of the indole ring. This result motivated us to synthesize novel uridine-5'-diphosphogalactose (UDP-Gal) analogues as candidates for mechanism-based inhibitors for beta GalT-1. We found that uridine 5'-(6-O-[10-(2-naphthyl)-3,6,9-trioxadecanyl]-alpha-D-galactopyranosyl) diphosphate (2) is the strongest inhibitor (K-i = 1.86 mu M) against UDP-Gal (K-m = 4.91 mu M) among compounds reported previously. A cold spray ionization time-of-flight mass spectrometry study demonstrated that the complex of this inhibitor and beta GalT-1 cannot interact with an acceptor substrate in the presence of Mn2+.
• Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering.

  Tadanao Funakoshi, Tokifumi Majima, Norimasa Iwasaki, Shintaro Yamane, Tatsuya Masuko, Akio Minami, Kazuo Harada, Hiroshi Tamura, Seiichi Tokura, Shin-Ichiro Nishimura

  Journal of biomedical materials research. Part A 74 3 338 - 46 2005年09月01日 

  To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts.
• Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering

  Tadanao Funakoshi, Tokifumi Majima, Norimasa Iwasaki, Shintaro Yamane, Tatsuya Masuko, Akio Minami, Kazuo Harada, Hiroshi Tamura, Seiichi Tokura, Shin-Ichiro Nishimura

  Journal of Biomedical Materials Research - Part A 74 3 338 - 346 2005年09月01日 [査読有り][通常論文]
   

  To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts. © 2005 Wiley Periodicals, Inc.
• Application of tissue engineering techniques for rotator cuff regeneration using a chitosan-based hyaluronan hybrid fiber scaffold

  T Funakoshi, T Majima, N Iwasaki, N Suenaga, N Sawaguchi, K Shimode, A Minami, K Harada, S Nishimura

  AMERICAN JOURNAL OF SPORTS MEDICINE 33 8 1193 - 1201 2005年08月 [査読有り][通常論文]
   

  Background: The current surgical procedures for irreparable rotator cuff tears have considerable limitations. Tissue engineering techniques using novel scaffold materials offer potential alternatives for managing these conditions. Hypothesis: A chitosan-based hyaluronan hybrid scaffold could enhance type I collagen products with seeded fibroblasts and thereby increase the mechanical strength of regenerated tendon in vivo. Study Design: Controlled laboratory study. Methods: The scaffolds were created from chitosan-based hyaluronan hybrid polymer fibers. Forty-eight rabbit infraspinatus tendons and their humeral insertions were removed to create defects. Each defect was covered with a fibroblast-seeded scaffold (n = 16) or a non-fibroblast-seeded scaffold (n = 16). In the other 16 shoulders, the rotator cuff defect was left free as the control. At 4 and 12 weeks after surgery, the engineered tendons were assessed by histological, immunohistochemical (n = 2), and biomechanical (n = 6) analyses. Results: Type I collagen was only seen in the fibroblast-seeded scaffold and increased in the regenerated tissue. The tensile strength and tangent modulus in the fibroblast-seeded scaffold were significantly improved from 4 to 12 weeks postoperatively. The fibroblast-seeded scaffold had a significantly greater tangent modulus than did the non-fibroblast-seeded scaffold and the control at 12 weeks. Conclusion: This scaffold material enhanced the production of type I collagen and led to improved mechanical strength in the regenerated tissues of the rotator cuff in vivo. Clinical Relevance: Rotator cuff regeneration is feasible using this tissue engineering technique.
• Combinatorial synthesis of MUC1 glycopeptides: Polymer blotting facilitates chemical and enzymatic synthesis of highly complicated mucin glycopeptides

  M Fumoto, H Hinou, T Ohta, T Ito, K Yamada, A Takimoto, H Kondo, H Shimizu, T Inazu, Y Nakahara, SI Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 127 33 11804 - 11818 2005年08月 [査読有り][通常論文]
   

  The chemoselective polymer blotting method allows for rapid and efficient synthesis of glycopeptides based on a "catch and release" strategy between solid-phase and water-soluble polymer supports. We have developed a heterobifunctional linker sensitive to glutamic acid specific protease (BLase). The general procedure consists of five steps, namely (i) the solid-phase synthesis of glycopeptide containing BLase sensitive linker, (ii) subsequent deprotections and the release of the glycopeptide from the resin, (iii) chemoselective blotting of the glycopeptide intermediates in the presence of water-soluble polymers with oxylamino functional groups, (iv) sugar elongations using glycosyltransferases, and (v) the release of target glycopeptides from the polymer platform by selective BLase promoted hydrolysis. The combined use of the solid-phase chemical syntheses of peptides and the enzymatic syntheses of carbohydrates on water-soluble polymers would greatly contribute to the production of complicated glycopeptide libraries, thereby enhancing applicative research. We report here a high-throughput synthetic system for the various types of MUC1 glycopeptides exhibiting a variety of sugar moieties. It is our belief that this concept will become part of the entrenched repertoire for the synthesis of biologically important glycopeptides on the basis of glycosyltransferase reactions in automated and combinatorial syntheses.
• A novel glycosylation concept; microwave-assisted acetal-exchange type glycosylations from methyl glycosides as donors

  Yayoi Yoshimura, Hiroki Shimizu, Hiroshi Hinou, Shin-Ichiro Nishimura

  Tetrahedron Letters 46 28 4701 - 4705 2005年07月 [査読有り][通常論文]
  
• Versatile glycoblotting nanoparticles for high-throughput protein glycomics

  K Niikura, R Kamitani, M Kurogochi, R Uematsu, Y Shinohara, H Nakagawa, K Deguchi, K Monde, H Kondo, SI Nishimura

  CHEMISTRY-A EUROPEAN JOURNAL 11 13 3825 - 3834 2005年06月 [査読有り][通常論文]
   

  We have developed an effective and practical trap-and-release method based on chemoselective ligation of carbohydrates with reactive aminooxyl groups attached to the surface of nanoparticles (referred to as glycoblotting nanoparticles). These glycoblotting nanoparticles were synthesized by UV irradiation of diacetylene-functionalized lipids that contain the aminooxyl group. The glycoblotting nanoparticles captured carbohydrates in aqueous solution under mild conditions and were collected by simple centrifugation. ne trapped carbohydrates were effectively released from the nanoparticles under acidic conditions to give pure oligosaccharides. This glycoblotting process reduced the time required for the purification process of carbohydrates to less than 6 h, compared to the several days needed for conventional chromatographic techniques. The oligosaccharides (N-glycan) were released from ovalbumin (glyco-protein) by PNGaseF after tryptic digestion. MALDI-TOF mass spectra before purification did not show any significant signals corresponding to N-glycans because these signals were hidden by the large signals of the abundant peptides. However, after purification with the glycoblotting nanoparticles, only signals corresponding to oligosaccharides appeared. We also demonstrated a clear analysis of the oligosaccharides contained in the mice dermis by means of glycoblotting.
• Alginate and chitosan polyion complex hybrid fibers for scaffolds in ligament and tendon tissue engineering

  T Majima, T Funakosi, N Iwasaki, ST Yamane, K Harada, S Nonaka, A Minami, SI Nishimura

  JOURNAL OF ORTHOPAEDIC SCIENCE 10 3 302 - 307 2005年05月 [査読有り][通常論文]
   

  Selecting the material for a scaffold is critically important for the success of tissue engineering. To simplify complicated biosynthetic matrices and achieve a novel class of potential materials, a model of polyion complex fibers was prepared from alginate and chitosan. In the current in vitro study, we thought that alginate-based chitosan hybrid biomaterials could provide excellent supports for fibroblast adhesion. In the current study, alginate polymer fiber (alginate group) and alginate-based chitosan hybrid polymer fibers (alginate with 0.05% chitosan, alginate-chitosan 0.05% group; alginate with 0.1% chitosan, alginate-chitosan 0.1% group) were originally prepared. We investigated the adhesion behavior of rabbit tendon fibroblast onto alginate polymer fibers versus the adhesion of the fibroblast onto alginate-based chitosan hybrid polymer fibers. Furthermore, mechanical properties and synthesis of the extracellular matrix were investigated. Mechanically, the novel fiber has considerable tensile strength of more than 200MPa. We demonstrated that the alginate-based chitosan hybrid polymer fibers showed much improved adhesion capacity with fibroblast compared with alginate polymer fiber. Additionally, morphologic studies revealed the dense fiber of the type I collagen produced by the fibroblast in the hybrid polymer fibers. We concluded that an alginate-based chitosan hybrid polymer fiber has considerable potential as a desirable biomaterial scaffold for tendon and ligament tissue engineering.
• Carbohydrate analysis by a phenol-sulfuric acid method in microplate format

  T Masuko, A Minami, N Iwasaki, T Majima, SI Nishimura, YC Lee

  ANALYTICAL BIOCHEMISTRY 339 1 69 - 72 2005年04月 [査読有り][通常論文]
   

  Among many colorimetric methods for carbohydrate analysis, the phenol-sulfuric acid method is the easiest and most reliable method. It has been used for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. This method is used widely because of its sensitivity and simplicity. In its original form, it required 50-450 nmol of monosaccharides or equivalent for analysis and thus is inadequate for precious samples. A scaled-down version requiring only 10-80 nmol of sugars was reported previously. We have now modified and optimized this method to use 96-well microplates for high throughput, to gain greater sensitivity, and to economize the reagents. This modified and optimized method allows longer linear range (1-150 nmol for Man) and excellent sensitivity. Moreover, our method is more convenient, requiring neither shaking nor covering, and takes less than 15 min to complete. The speed and simplicity of this method would make it most suitable for analyses of large numbers of samples such as chromatographic fractions. (c) 2004 Elsevier Inc. All rights reserved.
• Simultaneous analysis of 2-aminopyridine-derivatized neutral and sialylated oligosaccharides from human serum in the negative-ion mode by sonic spray ionization ion trap mass spectrometry

  Y Takegawa, K Deguchi, S Ito, S Yoshioka, H Nakagawa, S Nishimura

  ANALYTICAL CHEMISTRY 77 7 2097 - 2106 2005年04月 [査読有り][通常論文]
   

  Neutral and acidic (sialylated) 2-aminopyridine-derivatized (PA) oligosaccharides were analyzed by using reversed-phase high-performance liquid chromatography/ion trap mass spectrometry (RP-HPLC/IT MS) with a sonic spray ionization (SSI) source. Under the RP-HPLC separation using a buffer of 1 mM ammonium acetate (pH4.3) at a flow rate of 0.2 mL/min, both PA-oligosaccharides in the negative-ion mode showed a comparable degree of ionization efficiency, differing from that of the positive-ion mode, which exhibits a wide gap between their ionization efficiencies. In addition, the ion intensities of both PA-oligosaccharides were higher in the negative-ion mode than in the positive-ion mode. These results strongly suggest that the negative-ion mode of SSI-MS is suitable for simultaneous analysis of neutral and acidic (sialylated) oligosaccharides in RP-HPLC/MS. In the present study, RP-HPLC/SSI-IT MS in the negative-ion mode was used in the analysis of PA-oligosaccharides from human serum and its usefulness was investigated. As a result, 32 neutral and sialylated PA-oligosaccharides from human serum were identified with differentiating isomeric oligosaccharides and relatively quantified by a single HPLC/MS run. This method is useful for simple and rapid analysis of the overall distribution of neutral and sialylated oligosaccharides in a complex sample such as serum.
• Organic Matter Released from Activated Sludge Bacteria Cells During Their Decay Process

  H. Narita, I. Isshiki, N. Funamizu, T. Takakuwa, H. Nakagawa, S-I. Nishimura

  Environmental Technology 26 4 433 - 440 2005年04月 [査読有り][通常論文]
  
• Rapid microwave-assisted solid-phase glycopeptide synthesis

  T Matsushita, H Hinou, M Kurogochi, H Shimizu, SI Nishimura

  ORGANIC LETTERS 7 5 877 - 880 2005年03月 [査読有り][通常論文]
   

  Coupling of glycosylated Fmoc-Thr or Fmoc-Ser with N-terminal amino acids on a resin proceeded smoothly under microwave irradiation for 20 min with much higher efficiency (98% yield per coupling) than found in more general conditions. Compared with a conventional protocol, the present method greatly reduces the time required for solid-phase glycopeptide synthesis from 4 days to 7 h, as is the case with the synthesis of Muc-1-related 20-residue glycopeptide carrying five core-2 trisaccharide chains.
• Thiolation of chitosan. Attachment of proteins via thioether formation

  T Masuko, A Minami, N Iwasaki, T Majima, SI Nishimura, YC Lee

  BIOMACROMOLECULES 6 2 880 - 884 2005年03月 [査読有り][通常論文]
   

  Chitosan has a variety of biological functions through conjugating of other compounds to their amino and hydroxyl groups. To further expand applicability of chitosan, we have modified the amino group of chitosan with 2-iminothiolane to bestow thiol groups and obtained about 20% yield, which is equivalent to 913 mu equiv SH/g chitosan or 457 nequiv SH/nmol chitosan. Bovine serum albumin (BSA) was reacted with N-(c-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), and maleimide-modified BSA (MaINBSA) was obtained. The yield of sulfo-EMCS addition was 12.8-36.8 mol MaIN/mol BSA. When the chitosan-SH was reacted with MalN-BSA via thioether, 97.8% of the maleimide group was reacted, and 37.2% of the SH group was consumed. The remaining SH group was quenched by bromoacetamide. This is the first report of covalent conjugation of a protein to chitosan. Our method should find many applications in developing new chitosan-based biomedical materials containing other components such as growth factors and cell adhesion molecules, known to be crucial to cells. Our thiolated chitosan will facilitate conjugation of such biomedical components to provide new types of materials for tissue engineering.
• Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering

  S Yamane, N Iwasaki, T Majima, T Funakoshi, T Masuko, K Harada, A Minami, K Monde, S Nishimura

  BIOMATERIALS 26 6 611 - 619 2005年02月 [査読有り][通常論文]
   

  In this study, we hypothesized that hyaluronic acid could provide superior biological effects on the chondrocytes in a three-dimensional culture system. To test this hypothesis, we investigated the in vitro behavior of rabbit chondrocytes oil a novel chitosan-based hyaluronic acid hybrid polymer fiber. The goal of the current study was to show the Superiority of this novel fiber as a scaffold biomaterial for cartilage tissue engineering. Chitosan polymer fibers (chitosan group) and chitosan-based hyaluronic acid hybrid polymer fibers (HA 0.04% and HA 0.07% groups, chitosan coated with hyaluronic acid 0.04% and 0.07%, respectively) were originally developed by the wetspinning method. Articular chondrocytes were isolated from Japanese white rabbits and cultured in the sheets consisting of each polymer fiber. The effects of each polymer fiber oil cell adhesivity, proliferation, morphological changes. and synthesis of the extracellular matrix were analyzed by quantitative a cell attachment test, DNA quantification, light and scanning electron microscopy, semi-quantitative RT-PCR, and immunohistochemical analysis. Cell adhesivity, proliferation and the synthesis of agrecan were significantly higher in the hybrid fiber (HA 0.04% and 0.07%) groups than in the chitosan group. On the cultured hybrid polymer materials, scanning electron microscopic observation showed that chondrocytes proliferated while maintaining their morphological phenotype and with a rich extracellular matrix synthesis around the cells. Immunohistochemical staining with an anti-type II collagen antibody demonstrated rich production of the type II collagen in the pericellular matrix from the chondrocytes. The chitosan-based hyaluronic acid hybrid polymer fibers show great potential as a desirable biomaterial for cartilaginous tissue scaffolds. (C) 2004 Elsevier Ltd. All rights reserved.
• Chemical and enzymatic synthesis of glycocluster having seven sialyl lewis X arrays using β-cyclodextrin as a key scaffold material

  Tetsuya Furuike, Reiko Sadamoto, Kenichi Niikura, Kenji Monde, Nobuo Sakairi, Shin-Ichiro Nishimura

  Tetrahedron 61 7 1737 - 1742 2005年02月 [査読有り][通常論文]
  
• Direct observation of sugar-protein, sugar-sugar, and sugar-water complexes by cold-spray ionization time-of-flight mass spectrometry

  SI Nishimura, N Nagahori, K Takaya, Y Tachibana, N Miura, K Monde

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 44 4 571 - 575 2005年 [査読有り][通常論文]
  
• High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry

  SI Nishimura, K Niikura, M Kurogochi, T Matsushita, M Fumoto, H Hinou, R Kamitani, H Nakagawa, K Deguchi, N Miura, K Monde, H Kondo

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 44 1 91 - 96 2005年 [査読有り][通常論文]
  
• Structural assignment of isomeric 2-aminopyridine-derivatized oligosaccharides using negative-ion MSn spectral matching

  Y Takegawa, K Deguchi, S Ito, S Yoshioka, H Nakagawa, SI Nishimura

  RAPID COMMUNICATIONS IN MASS SPECTROMETRY 19 7 937 - 946 2005年 [査読有り][通常論文]
   

  To investigate the possibility of structural assignment based on negative-ion MS2 spectral matching, three isomeric pairs of 2-aminopyridine (PA)-derivatized non-fucosylated, fucosylated, and sialylated oligosaccharides (complex type N-glycans) were analyzed using high-performance liquid chromatography/ion trap mass spectrometry (HPLC/ITMS) with a sonic-spray ionization (SSI) source. In the SSI negative-ion mode the deprotortated molecule [M-2H](2-) becomes prominent. Negative-ion MS2 spectra derived from such ions contain many fragment types (B and Y, C and Z, A, and D) and therefore are more informative than the positive-ion MS2 spectra derived from [M+H+Na](2+) ions, which usually consist mainly of B and Y fragment ions. In particular the internal ions (D- and E-type ions) provided useful information about the alpha 1-6 branching patterns and the bisecting GlcNAc residue. Spectral matching based on the correlation coefficients between negative-ion MS2 spectra was performed in a manner similar to the positive-ion MS2 spectral matching previously reported. It was demonstrated that negative-ion MS2 spectral matching is as useful and applicable to the structural assignment of relatively large non-fucosylated, fucosylated, and sialylated PA-oligosaccharide isomers as its positive-ion counterpart. Copyright (c) 2005 John Wiley T Sons, Ltd.
• Molecular transporter between polymer platforms: Highly efficient chemoenzymatic glycopeptide synthesis by the combined use of solid-phase and water-soluble polymer supports

  M Fumoto, H Hinou, T Matsushita, M Kurogochi, T Ohta, T Ito, K Yamada, A Takimoto, H Kondo, T Inazu, SI Nishimura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 44 17 2534 - 2537 2005年 [査読有り][通常論文]
  
• Nanometer-scale ordering in cast films of columnar metallomesogen as revealed by STM observations

  N Kakegawa, N Hoshino, Y Matsuoka, N Wakabayashi, SI Nishimura, A Yamagishi

  CHEMICAL COMMUNICATIONS 18 2375 - 2377 2005年 [査読有り][通常論文]
   

  STM observations were performed on a cast film of a columnar metallomesogen ([Cr(5C(8))(3)]; 5C(8) = 1-(3,4,5-trioctyloxyphenyl)3-( 3,4-dioctyloxyphenyl)propane-1,3-dionate anion) on a graphite surface, revealing the nanometer-scale surface ordering into an oblique lattice (a = 10.5 nm, b = 11.5 nm, α = 55&DEG;) possibly due to the &UDelta;&UDelta;-chiral interactions.
• Characteristic pH and electrolyte concentration dependences of 2-aminopyridine-derivatized oligosaccharides (N-glycans) in sonic-spray ionization mass spectrometry

  K Deguchi, Y Takegawa, A Hirabayashi, H Nakagawa, SI Nishimura

  RAPID COMMUNICATIONS IN MASS SPECTROMETRY 19 16 2325 - 2330 2005年 [査読有り][通常論文]
   

  The intensities of ion signals from neutral oligosaccharides (N-glycans) derivatized with 2-aminopyridine (PA) were analyzed by ion trap mass spectrometry with a sonic-spray ionization (SSI) source, in both positive- and negative-ion modes, while varying the pH and concentration of ammonium acetate buffer solution. Two characteristic results are reported and discussed. The first characteristic is the pH dependence of the ion intensities; on increasing the solution pH from 4.3 to 8.6, positive ion intensities increase and negative ion intensities decrease. The second characteristic concerns the dependence of ion intensities on electrolyte concentration; on increasing the electrolyte concentration, the SSI efficiency for the PA N-glycans first increases and then decreases. Assuming that the SSI mechanism essentially conforms to the statistical charging model and the charge residue model, a new model that focuses a great deal of attention on the counter (electrolyte) ion distribution surrounding the solvated analyte (PA N-glycan) is proposed, in particular to rationalize the characteristic pH dependence. Copyright (C) 2005 John Wiley & Sons, Ltd.
• Chemo-biological approach to modification of the bacterial cell wall

  Reiko Sadamoto, Shin-Ichiro Nishimura

  Handbook of Carbohydrate Engineering 495 - 506 2005年01月01日 

  G. P. Smith’s report on the display of target proteins on phage surfaces,1 a technique now generally referred to as phage display, was the precursor of many later studies that sought to apply the same technique to the display of large proteins on microbial cells. Subsequently, target proteins fused with a cellular anchor protein have been successfully displayed on the cell surface of Escherichia coli and yeast.2,3 This strategy is based on a genetic method using recombinant DNA corresponding to the target and anchor proteins.4,5.
• The elucidation of a proteoglycan biosynthesis mechanism by organic synthesis

  Ken Shimawaki, Hiroko Hoshi, Yoshinori Fujisawa, Kenji Monde, Shin-Ichiro Nishimura

  Polymer Preprints, Japan 54 1 2155  2005年 

  In the proteoglycan biosynthesis, it is pointed out that sulfation of a core linkage tetrasaccaraide may regulate the sorting of CS and HS. To verify this hypothesis, the fluorinated analogs of a core linkage tetrasaccaride part were synthesized.
• Antifreeze Glycoprotein: Elucidation of Antifreeze Mechanism

  Masakazu Hachisu, Kohta Igarashi, Sakae Tsuda, Shuhei Koshida, Kenji Monde, Shin-Ichiro Nishimura

  Polymer Preprints, Japan 54 1 2168  2005年 

  Fishes living in the cold ocean have antifreeze glycoproteins (AFGPs). Each of the Thr residues in AFGP is O-glycosylated with a disaccharide (Galβ1→3GalNAcα). Our group has synthesized AFGP and several AFGP analogs to study the relationship between the molecular structure and the antifreeze activity. These results suggested that 4- or 6-hydroxyl groups in GalNAc residues play a vital role in the antifreeze activity and both or either of them might be ice-binding site. So we synthesized the AFGP analogs in which 4- or 6- hydroxyl groups of GalNAc residue was removed. These analogs both have antifreeze activity. In this study, we synthesized an AFGP analog which removed both 4- and 6- hydroxyl groups in order to clarify the ice-binding site in AFGP molecule.
• Versatile synthesis of oligosaccharide-containing fullerenes

  Shigeaki Abe, Hideki Moriyama, Kenichi Niikura, Fei Feng, Kenji Monde, Shin-Ichiro Nishimura

  Tetrahedron: Asymmetry 16 1 15 - 19 2005年01月 [査読有り][通常論文]
  
• Direct N-glycan profiling in the presence of tryptic peptides on MALDI-TOF by controlled ion enhancement and suppression upon glycan-selective derivatization

  Y Shinohara, J Furukawa, K Niikura, N Miura, SI Nishimura

  ANALYTICAL CHEMISTRY 76 23 6989 - 6997 2004年12月 [査読有り][通常論文]
   

  Even though the formidably laborious and time-consuming nature of oligosaccharide analysis limits certain attempts to analyze the glycosylation profile, the significant elucidation of carbohydrate modifications is largely dependent on it. Aiming to substantially improve the sample preparation procedure, a novel protocol allowing glycan-specific detection in the presence of other species, such as tryptic peptides, on MALDI-TOF was proposed and then evaluated. The new protocol is based on the concept that the desorption/ionization efficiency of glycans could be selectively and substantially enhanced while drastically suppressing the other ion species upon glycan-selective derivatizadon. A series of known and novel labeling reagents, all of which carry hydrazide functionality to allow glycan-specific derivatization, were prepared and evaluated in terms of their abilities to enhance the detection sensitivity of glycans, suppress ions of other contaminants (e.g., peptides), and detect acidic oligosaccharides. Several novel reagents that possess hydrophobic residue(s) together with quaternary ammonium/pyridinium or guanidino functionalities significantly enhanced the detection sensitivity of oligosaccharides. When enzymatically deglycosylated tryptic ovalbumin digest was directly derivatized by these reagents and subjected to MALDI-TOF analysis without any prior purification, we observed that a single type of analyte ion (labeled glycan) could suppress a large majority of peptide ions while allowing a low-femtomole level detection of oligosaccharides. The efficacy of this approach was further evaluated using several other model glycoproteins, including alpha(1)-acid glycoprotein that contains a variety of sialylated oligosaccharides.
• Assignment and quantification of 2-aminopyridine derivatized oligosaccharide isomers coeluted on reversed-phase HPLC/MS by MSn spectral library

  Y Takegawa, K Deguchi, S Ito, S Yoshioka, A Sano, K Yoshinari, K Kobayashi, H Nakagawa, K Monde, SI Nishimura

  ANALYTICAL CHEMISTRY 76 24 7294 - 7303 2004年12月 [査読有り][通常論文]
   

  2-Aminopyridine (PA)-derivatized oligosaccharides from IgG were analyzed by using reversed-phase HPLC/mass spectrometry (RP-HPLC/MS) and a MSn spectral library, in particular, focusing on two pairs of isomers incompletely separated or coeluted in chromatograms. We previously reported that MSn spectral matching considering both major fragment ions (m/z) and intensities is useful and applicable to the structural assignment of PA-oligosaccharide isomers. In this study, MSn spectral matching based on the MSn spectral library was applied to the assignment of these PA-oligosaccharide isomers in IgG. Its usefulness was investigated by comparing it to the conventional two-dimensional mapping method based on retention time indexes. Specifically, we focus on the assignment and quantification of the isomers, which are coeluted in chromatograms. From this, we propose a new method using MSn spectral matching and the working curve on which are plotted the relative intensities of selected fragment ions in their MS2 spectra versus various mixtures of the isomers. This new method demonstrated that the obtained quantities coincide very well with those estimated after separating by a combination of lectin and reversed-phase columns. This means that separation by RP-HPLC/MS is greatly simplified because complete separation of the isomers is no longer required. Application of this new method was tested by using the two other pairs of fucosylated and nonfucosylated PA-oligosaccharides from IgG. The results showed that this method works for them as well.
• Glycoinsulins: Dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity

  M Sato, T Furuike, R Sadamoto, N Fujitani, T Nakahara, K Niikura, K Monde, H Kondo, SI Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 126 43 14013 - 14022 2004年11月 [査読有り][通常論文]
   

  Mono-, di-, and trisialyloligosaccharides were introduced to mutant insulins through enzymatic reactions. Sugar chains were sialylated by alpha2,6-sialyltransferase (alpha2,6-SiaT) via an accessible glutamine residue at the N-terminus of the B-chain attached by transglutaminase (TGase). Sia2,6-di-LacNAc-Ins(B-F1Q) and Sia2,6-tri-LacNAc-Ins(B-F1Q), displaying two and three sialyl-N-acetyllactosamines, respectively, were administered to hyperglycemic mice. Both branched glycoinsulins showed prolonged glucose-lowering effects compared to native or lactose-carrying insulins, showing that sialic acid is important in obtaining a prolonged effect. Sia2,6-tri-LacNAc-lns(B-F1Q), in particular, induced a significant delay in the recovery of glucose levels.
• A characteristic CH band in VCD of methyl glycosidic carbohydrates

  Tohru Taniguchi, Kenji Monde, Nobuaki Miura, Shin-Ichiro Nishimura

  Tetrahedron Letters 45 46 8451 - 8453 2004年11月 [査読有り][通常論文]
  
• Mechanism-based fluorescent labeling of beta-galactosidases - An efficient method in proteomics for glycoside hydrolases

  M Kurogochi, SI Nishimura, YC Lee

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 43 44704 - 44712 2004年10月 [査読有り][通常論文]
   

  (4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-D-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S. T., and Landry, D. Glycobiology (1995) 5, 19-28 and Taron, C. H., Benner, J. S., Hornstra, L. J., and Guthrie, E. P. (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K. (1988) Glycoconj. J. 15, 155-160) for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-off-light/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases. It was demonstrated by using X. manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of (IP)-I-56 (R) under bar AYWKD(63), was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T. (2002) J. Mol. Biol. 322, 79-91). A catalytic glutamic acid (Glu-537) of E. coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in (TL)-T-257 (E) under barE(260) was selectively labeled using B. circulans beta-galactosidase, indicating that Glu-259 is one of the nucleophiles in the active site. The present method can be readily extended to other glycosidases and should greatly aid the high throughput proteomics of many glycoside hydrolases showing both retaining- and inverting-type mechanisms.
• Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry

  M Kurogochi, SI Nishimura

  ANALYTICAL CHEMISTRY 76 20 6097 - 6101 2004年10月 [査読有り][通常論文]
   

  The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique described to date has proven to be a convenient and rapid method for identification and characterizations of proteins. However, the general MALDI-TOF MS analysis of complex carbohydrates and glycopeptides still entails special consideration of ionization and the fragmentation characteristics of labile carbohydrate moieties. In this study, an efficient and practical method we termed the matrix-dependent selective fragmentation (MDSF) technique of MALDI-TOF/TOF MS, which allows highly sensitive and reliable fragmentation of oligosaccharides and N-glycopeptides. Results from application of the MDSF technique to TOF/TOF MS analysis demonstrated that in comparison to the conventional postsource decay up to 170 times more sensitive product ion peaks could be obtained. It was also suggested that MDSF generates desired structural information based on the controlled cleavage of the singly charged precursor ion with different electronic excited states made by this method. Ideal product ion peaks observed by MDSF in TOF/TOF MS facilitated structural characterization of complex oligosaccharide derivatives including unstable Neu5Ac and Fuc residues and N-glycopeptides.
• 71(P-61) 振動円二色性(VCD)スペクトルによる複合糖質等の立体化学研究(ポスター発表の部)

  谷口 透, 三浦 信明, 内田 直人, 西村 紳一郎, 門出 健次

  天然有機化合物討論会講演要旨集 46 401 - 406 天然有機化合物討論会 2004年10月01日 

  Chiral molecules exhibit circular dichroism (CD), the differential absorption of left versus right circularly polarized light, and the spectroscopies have been useful method for determining the absolute configuration. Recently, the measurement of CD in an infrared region, i.e. vibrational circular dichroism (VCD), has been advanced instrumentally and theoretically. VCD has some advantages over conventional electronic circular dichroism (ECD) by the applicability to all organic molecules and the reliability of ab initio quantum calculations. We apply this powerful technique to the determination of the absolute stereochemistry of some cruciferous phytoalexin-related compounds and to analysis of glycoconjugates. The determination of the absolute configuration of cruciferous phytoalexin-related compounds A number of cruciferous phytoalexin studies have been reported so far, but most of their absolute stereochemistry is still to be investigated. So we applied the VCD spectroscopy to determine the absolute configuration of dioxibrassinin (1) and 3-cyanomethyl-3-hydroxyoxindole (2). Compounds 1 and 2 were synthesized and their VCD spectra were measured. The comparison of observed VCD spectra and calculated ones indicated that their stereochemistry are both S. This is the first case of the determination of absolute stereochemistry of chiral tertiary alcohols by VCD. Application of VCD to carbohydrates Recently, the biological significance of carbohydrates have been recognized and analytical studies of carbohydrates have been widely investigated. Most analyses have been based on achiral methods such as NMR or MS in spite of their chiral properties. We considered that the detailed stereochemical information could be extracted using VCD. Various carbohydrates have been measured to construct an extensive database aiming to create a methodology for VCD pattern discrimination of the carbohydrates. The database in the fingerprint region indicated that sugars with the axial glycosidic linkage showed a negative, sharp and intense VCD band, named "Glycoside band" at around 1145 cm^<-1>. The applicability of the Glycoside band was demonstrated by quantifying α-β ratio of mixture samples and monitoring of an enzymatic reaction. In the C-H stretching region, we found the stereochemistry of single chiral center in the presence of multiple such centers could be extracted through the vibrational chirality transfer phenomenon. Also, VCD in this region was indicated to be a sensitive probe of glycosidic linkage sites. In conclusion, by using the VCD database and the proposed spectra-structure correlations, VCD will be an effective tool in carbohydrate analysis.
• Vibrational circular dichroism: Chiroptical analysis of biomolecules

  T Taniguchi, N Miura, SI Nishimura, K Monde

  MOLECULAR NUTRITION & FOOD RESEARCH 48 4 246 - 254 2004年09月 [査読有り][通常論文]
  
• Sequential Glycoproteins:  Practical Method for the Synthesis of Antifreeze Glycoprotein Models Containing Base Labile Groups

  Yuki Tachibana, Kenji Monde, Shin-Ichiro Nishimura

  Macromolecules 37 18 6771 - 6779 2004年09月 [査読有り][通常論文]
  
• Specific Band Observed in VCD Predicts the Anomeric Configuration of Carbohydrates

  Kenji Monde, Tohru Taniguchi, Nobuaki Miura, Shin-Ichiro Nishimura

  Journal of the American Chemical Society 126 31 9496 - 9497 2004年08月 [査読有り][通常論文]
  
• Feasibility of polysaccharide hybrid materials for scaffolds in cartilage tissue engineering: Evaluation of chondrocyte adhesion to polyion complex fibers prepared from alginate and chitosan

  N Iwasaki, ST Yamane, T Majima, Y Kasahara, A Minami, K Harada, S Nonaka, N Maekawa, H Tamura, S Tokura, M Shiono, K Monde, SI Nishimura

  BIOMACROMOLECULES 5 3 828 - 833 2004年05月 [査読有り][通常論文]
   

  The ideal cell-carrier material for cartilage regeneration should be one that closely mimics the natural environment in a living articular cartilage matrix. In the current study, we considered that alginate-based chitosan hybrid biomaterials could provide excellent supports for chondrocyte adhesion. To test this hypothesis, we investigated the adhesion behavior of rabbit chondrocytes onto an alginate polymer versus the adhesion of the chondrocytes onto some alginate-based chitosan hybrid polymer fibers in vitro. We demonstrated that the alginate-based chitosan hybrid polymer fibers showed much improved adhesion capacity with chondrocytes in comparison with alginate polymer fiber. Additionally, morphologic studies revealed maintenance of the characteristic round morphology of the chondrocyte and the dense fiber of the type II collagen produced by the chondrocytes in the hybrid polymer. On the basis of these results, we conclude that an alginate-based chitosan hybrid polymer fiber has considerable potential as a desirable biomaterial for cartilage tissue scaffolds.
• Synthesis of 4-fluorinated UDP-MurNAc pentapeptide as an inhibitor of bacterial growth

  T Ueda, F Feng, R Sadamoto, K Niikura, K Monde, SI Nishimura

  ORGANIC LETTERS 6 11 1753 - 1756 2004年05月 [査読有り][通常論文]
   

  4-Fluorinated UDP-MurNAc pentapeptide, 2, has been synthesized. In our previous study, UDP-MurNAc pentapeptide analogue 1 was found to be incorporated into the bacterial cell wall through biosynthesis. Compound 2 showed growth-inhibition activity against Gram-positive bacteria when it was added to growth media at 0.01 mg/mL.
• Azasugar-based MMP/ADAM inhibitors as antipsoriatic agents

  H Moriyama, T Tsukida, Y Inoue, K Yokota, K Yoshino, H Kondo, N Miura, S Nishimura

  JOURNAL OF MEDICINAL CHEMISTRY 47 8 1930 - 1938 2004年04月 [査読有り][通常論文]
   

  As a part of synthetic studies on MMP (matrix metalloproteinase)/ADAM (a disintegrin and metalloproteinase) inhibitors, we have preliminarily communicated that azasugar-based compound la exhibited a potential inhibitory activity on some metalloprotease-catalyzed proteolytic reactions. To find promising candidates for the topical treatment of psoriasis, we investigated stability in aqueous solution of compound la and its derivative 1b and then optimized the P1' substuent (2-5). In the present study, we synthesized novel derivatives of compound-la and evaluated their inhibitory activity toward MMP-1, -3, and -9, TACE, and HB-EGF shedding, from a viewpoint of versatility of azasugars as a functional scaffold. As a result, it was found that compound 1b demonstrated desirable inhibitory activity as an antipsoriatic agent, and some of the derivatives showed selective inhibitory activity. In addition, it was found that compound 1b exhibited a significant therapeutic effect on a mouse TPA-induced epidermal hyperplasia. model. Therefore, compound 1b could become a promising candidate as a practical antipsoriatic agent.
• Generation of leukemia-specific T-helper type 1 cells applicable to human leukemia cell-therapy

  T Fujimura, K Chamoto, T Tsuji, T Sato, H Yokouchi, S Aiba, H Tagami, J Tanaka, M Imamura, Y Togashi, T Koda, T Nishimura

  IMMUNOLOGY LETTERS 93 1 17 - 25 2004年04月 [査読有り][通常論文]
   

  Leukemic dendritic cells (DC) were induced from the peripheral blood (PB) or bone marrow (BM) of leukemia patients by culture with (i) GM-CSF + IL-3 (neutral condition); (ii) GM-CSF + IL-3 + IL-12 + IFN-gamma (type 1-condition); or (iii) GM-CSF + IL-3 + IL-4 (type 2-condition). Although leukemic cells rapidly differentiated into adhesive leukemic DC in all culture conditions, type 1-conditions were the most suitable for inducing leukemic DC expressing high levels of HLA and costimulatory molecules. Addition of IL-2 after 2 days of culture induced a preferential growth of minor T cell populations interacting with leukemic DC. In particular, IFN-gamma-producing CD4(+) Th1 cells were efficiently expanded in type I culture conditions but nor in neutral or type 2-conditions. However, CD4+ T cells expanded in neutral conditions showed Th1-like functions if they were pulsed with IFN-gamma for 2 days before harvest. Such Th1 cells produced IFN-gamma and exhibited cytotoxicity in response to autologous leukemia cells. We further dernonstrated that IFN-gamma production of leukemia-specific Th1 cells was blocked by anti-HLA-DR mAb. Thus, we established a novel culture system for inducing leukemia-specific Thl cells. (C) 2004 Elsevier B.V. All rights reserved.
• A practical synthesis of amphiphilic cyclodextrins fully substituted with sugar residues on the primary face

  F Sallas, K Niikura, SI Nishimura

  CHEMICAL COMMUNICATIONS 5 596 - 597 2004年03月 [査読有り][通常論文]
   

  New amphiphilic cyclodextrins fully substituted with sugar residues on the primary face have been synthesised and enzymatically modified.
• Synthesis and biological activity of selective azasugar-based TACE inhibitors

  T Tsukida, H Moriyama, Y Inoue, H Kondo, K Yoshino, SI Nishimura

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 14 6 1569 - 1572 2004年03月 [査読有り][通常論文]
   

  A series of azasugar-based hydroxamic acid derivatives bearing 2R,3R,4R,5R-configuration is described. Compound 4c with 4,5-O-acetonide group showed excellent in vitro potency against TACE, with high selectivity over MMP-1 and moderate selectivity over MMP-3 and MMP-9. (C) 2004 Elsevier Ltd. All rights reserved.
• Control of bacteria adhesion by cell-wall engineering

  R Sadamoto, K Niikura, T Ueda, K Monde, N Fukuhara, SI Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 126 12 3755 - 3761 2004年03月 [査読有り][通常論文]
   

  UDP-MurNAc-pentapeptide derivative bacterial cell-wall precursors were synthesized as effective tools for surface display on living bacteria. Lactobacilli were incubated in the ketone-modified precursor-containing medium, and the ketone moiety was displayed on the bacterial surface through cell-wall biosynthesis. Oligomannose was coupled with the ketone moiety on the bacterial surface via a aminooxyl linker, thereby displaying this oligosaccharide on the surface of the bacteria. The increase in the adhesion of the sugar-displaying bacteria onto a concanavalin A-attached film compared to that of native bacteria was confirmed by microscopic observation and surface plasmon resonance measurement. The incorporation of the artificial cell-wall precursors was enhanced when incubated with fosfomycin, an inhibitor of cell-wall precursor biosynthesis.
• Fluorescent Glyconanoparticles as a Sensitive Device to Monitor Sugar-Involving Molecular Events

  Kenichi Niikura, Noriko Osuga, Noriko Nagahori, Reiko Sadamoto, Masamichi Shiono, Norimasa Iwasaki, Kenji Monde, Akio Minami, Shin-Ichiro Nishimura

  Polymer Journal 36 3 209 - 218 2004年03月 [査読有り][通常論文]
  
• IFN-gamma-induced SOCS-1 regulates STAT6-dependent eotaxin production triggered by IL-4 and TNF-alpha

  T Sato, R Saito, T Jinushi, T Tsuji, J Matsuzaki, T Koda, S Nishimura, H Takeshima, T Nishimura

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 314 2 468 - 475 2004年02月 [査読有り][通常論文]
   

  The production of eotaxin, which is a critical mediator for airway inflammation, is inhibited by IFN-gamma. Here, we investigated the precise mechanisms underlying IFN-gamma-dependent inhibition of eotaxin production using mouse embryonic fibroblasts (MEF). MEF produced high levels of eotaxin in STAT6-dependent manner when they were cultured with both IL-4 and TNF-alpha. However, the eotaxin production by MEF was strongly inhibited by addition of IFN-gamma. Western-blotting analysis demonstrated that IFN-gamma downmodulated STAT6 phosphorylation induced by IL-4 and TNF-alpha. Moreover, IFN-gamma did not exhibit its inhibitory effect on both STAT6-phosphorylation and eotaxin production in MEF obtained from deficient mice in STAT1, a key molecule of IFN-gamma signaling. We also demonstrated that SOCS-1, a potent inhibitory molecule of IL-4 signaling, was induced by IFN-gamma in STAT1-dependent manner. This indicated that SOCS-1 might be involved in IFN-gamma-mediated STAT1-dependent inhibition of cotaxin production. In SOCS-1(-/-) MEF, IFN-gamma inhibited neither STAT6 phosphorylation nor eotaxin production induced by IL-4 and TNF-a. Conversely, retroviral transduction of SOCS-1 into MEF inhibited STAT6 phosphorylation and eotaxin production induced by IL-4 and TNF-alpha, in the absence of IFN-gamma. Thus, we demonstrated that IFN-gamma-induced inhibition of STAT6 phosphorylation and eotaxin production were mediated by SOCS-1 induced in STAT1-dependent manner. (C) 2003 Elsevier Inc. All rights reserved.
• An engineered hyaluronan synthase - Characterization of recombinant human hyaluronan synthase 2 expressed in Escherichia coli

  H Hoshi, H Nakagawa, S Nishiguchi, K Iwata, K Niikura, K Monde, SI Nishimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 4 2341 - 2349 2004年01月 [査読有り][通常論文]
   

  The Class I hyaluronan synthase ( HAS) is a unique glycosyltransferase synthesizing hyaluronan ( HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.
• Persubstituted cyclodextrin-based glycoclusters as inhibitors of protein-carbohydrate recognition using purified plant and mammalian lectins and wild-type and lectin-gene-transfected tumor cells as targets

  S Andre, H Kaltner, T Furuike, SI Nishimura, HJ Gabius

  BIOCONJUGATE CHEMISTRY 15 1 87 - 98 2004年01月 [査読有り][通常論文]
   

  Multivalent glycoclusters have the potential to become pharmaceuticals by virtue of their target specificity toward clinically relevant sugar receptors. Their application can also provide fundamental insights into the impact of two spatial factors on binding, i.e., topologies of ligand (branching mode, cluster presentation) and carbohydrate recognition domains in lectins. Persubstituted macrocycles derived from nucleophilic substitution of iodide from heptakis 6-deoxy-6-iodo-beta-cyclodextrin by the unprotected sodium thiolate of 3-(3-thioacetyl propionamido)propyl glycosides (galactose, lactose and N-acetyllactosamine) were prepared. The produced glycoclusters were first tested as competitive inhibitors in solid-phase assays. A plant toxin from mistletoe and an immunoglobulin G fraction from human serum were markedly susceptible. A nearly 400-fold increase in inhibitory potency of each galactose moiety in the heptavalent form relative to free lactose (217-fold relative to free galactose) was detected. Thus, these glycoclusters can efficiently interfere, for example, with xenoantigen-dependent hyperacute rejection. Among the tested galectins selected from this family of adhesion- and growth-regulatory endogenous lectins, the substituted beta-cyclodextrins acted as sensors to delineate topological differences between the two dimeric prototype proteins. The relatively strong reactivity with chimera-type galectin-3, a mediator of tumor metastasis, disclosed selectivity for glycocluster binding among galectins. Equally important, the geometry of ligand display (maxiclusters, bi- or triantennary N-glycans) made its mark on the inhibitory potency. To further determine the sensitivity of a distinct galectin presented on the cell surface and not in solution, we established a stably transfected tumor cell clone. We detected a significant response to presence of the multivalent inhibitor. This type of chemical scaffold with favorable pharmacologic properties might thus be exploited for the design of galectin- and ligand-type-selective glycoclusters.
• Antifreeze glycoproteins: Elucidation of the structural motifs that are essential for antifreeze activity

  Y Tachibana, GL Fletcher, N Fujitani, S Tsuda, K Monde, SI Nishimura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 43 7 856 - 862 2004年 [査読有り][通常論文]
  
• Structural assignment of isomeric 2-aminopyridine-derivatized oligosaccharides using MSn spectral matching

  Y Takegawa, S Ito, S Yoshioka, K Deguchi, H Nakagawa, K Monde, SI Nishimura

  RAPID COMMUNICATIONS IN MASS SPECTROMETRY 18 4 385 - 391 2004年 [査読有り][通常論文]
   

  Two isomeric pairs of 2-aminopyridine (PA)-derivatized fucosylated and non-fucosylated oligosaccharides (complex-type N-glycans of IgG) were analyzed using liquid chromatography/ion trap mass spectrometry (LC/ITMS) with a sonic spray ionization source and by varying the collision-induced dissociation voltage. Reproducibility of MSn (n = 2) spectra obtained by LC/ITMS was tested considering both fragment ions (m/z) and intensities. A comparison of their MSn spectra and evaluation of similarities (or matching), based on correlation coefficients between MSn spectra, was investigated as a possibility for structural assignment of the isomers. It is shown that such MSn spectral matching is useful and applicable to the structural assignment of relatively large fucosylated and sialylated PA-oligosaccharides released from IgG based on Bn- and Yn-type fragmentations of the corresponding [M+H+Na](2+) ions. Copyright (C) 2004 John Wiley Sons, Ltd.
• Site-specific introduction of sialic acid into insulin

  M Sato, R Sadamoto, K Niikura, K Monde, H Kondo, SI Nishimura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 43 12 1516 - 1520 2004年 [査読有り][通常論文]
  
• Chemo-enzymatic synthesis of fluorinated 2-N-acetamidosugar nucleotides using UDP-GlcNAc pyrophosphorylase

  F Feng, K Okuyama, K Niikura, T Ohta, R Sadamoto, K Monde, T Noguchi, SI Nishimura

  ORGANIC & BIOMOLECULAR CHEMISTRY 2 11 1617 - 1623 2004年 [査読有り][通常論文]
   

  Two non-natural. uorinated 2-N-acetamidosugar nucleotides, uridine 5'-diphosphate (UDP) 2-acetamido-2,4-dideoxy-4-fluoro-alpha-D-glucopyranose (UDP-4-FGlcNAc) 1 and its galacto isomer (UDP-4-FGalNAc) 2, were enzymatically constructed by treating chemically synthesized. uorinated 2-N-acetamidosugar 1-phosphates as the donor with UDP 2-acetamido-2-deoxy-alpha-D-glucopyranose pyrophosphorylase in the presence of uridine 5'-triphosphate (UTP).
• Post-translational modifications on proteins: Facile and efficient procedure for the identification of O-glycosylation sites by MALDI-LIFT-TOF/TOF mass spectrometry

  M Kurogochi, T Matsushita, SI Nishimura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 43 31 4071 - 4075 2004年 [査読有り][通常論文]
  
• Identification of Th1- and Th2-speciric genes by microarray analysis

  J Li, T Koda, A Yamaguchi, S Yamamoto, T Sato, Y Togashi, T Nishimura

  BIOMEDICAL RESEARCH-TOKYO 24 6 299 - 307 2003年12月 [査読有り][通常論文]
   

  CD4(+) helper T (Th) cells are subdivided into Th1 and Th2 cells in terms of their differential cytokine production pattern. These cells are key immunoregulatory cells in host defense mechanisms. Therefore, identification of differential gene expression in Th1 and Th2 cells is crucial for immunomodulation. Here we describe identification of Th1- or Th2-specific genes by DNA microarray analysis. We prepared mRNA from anti-CD3 mAb stimulated or unstimulated Th1 and Th2 cells, then gene expression profiles were analyzed by using a microarray spotted with 8,700 EST clones. We detected preferential expression of 57 genes in Th1 cells and 37 genes in Th2 cells. By semi-quantitative RT-PCR analysis, we identified 11 genes specifically expressed in Th1 cells, and 7 genes in Th2 cells. These genes may contribute to the differentiation and function of helper T cells and thereby regulation of immune response. Further analysis would help to develop a method to modulate immune response.
• Functional expression of the TrkC gene, encoding a high affinity receptor for NT-3, in antigen-specific T helper type 2 (Th2) cells

  Masashi Sekimoto, Takemasa Tsuji, Jyunko Matsuzaki, Kenji Chamoto, Toshiaki Koda, Kiyomitsu Nemoto, Masakuni Degawa, Shin-ichiro Nishimura, Takashi Nishimura

  Immunology Letters 88 3 221 - 226 2003年09月 [査読有り][通常論文]
  
• Structure-activity relationships of azasugar-based MMP/ADAM inhibitors

  H Moriyama, T Tsukida, Y Inoue, H Kondo, K Yoshino, SI Nishimura

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 13 16 2737 - 2740 2003年08月 [査読有り][通常論文]
   

  In order to investigate structure-activity relationships of azasugar series toward metalloprotemases, we synthesized and evaluated several azasugar-based compounds. As a result, it was found that 4-phenoxybenzene derivative 3 having 2R,3R,4R,5S-configurations exhibited most potent inhibitory activities against matrix metalloproteinase-1, -3 and -9 and TACE. (C) 2003 Elsevier Ltd. All rights reserved.
• Design, synthesis and evaluation of novel azasugar-based MMP/ADAM inhibitors

  H Moriyama, T Tsukida, Y Inoue, H Kondo, K Yoshino, SI Nishimura

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 13 16 2741 - 2744 2003年08月 [査読有り][通常論文]
   

  In order to verify whether azasugar would be a useful scaffold for inhibitory activity against metalloproteinases, we synthesized some azasugar-based compounds. As a result, it is clarified that azasugar moiety could function as successful inhibitor of matrix metalloproteinase-1, -3 and -9 and TACE. (C) 2003 Elsevier Ltd. All rights reserved.
• Preparation of cruciferous phytoalexin related metabolites, (−)-dioxibrassinin and (−)-3-cyanomethyl-3-hydroxyoxindole, and determination of their absolute configurations by vibrational circular dichroism (VCD)

  Kenji Monde, Tohru Taniguchi, Nobuaki Miura, Shin-Ichiro Nishimura, Nobuyuki Harada, Rina K Dukor, Laurence A Nafie

  Tetrahedron Letters 44 32 6017 - 6020 2003年08月 [査読有り][通常論文]
  
• Differential regulation of VLA-2 expression on T(h)1 and T(h)2 cells: a novel marker for the classification of T-h subsets (vol 15, pg 701, 2003)

  K Sasaki, T Tsuji, T Jinushi, J Matsuzaki, T Sato, K Chamoto, Y Togashi, T Koda, T Nishimura

  INTERNATIONAL IMMUNOLOGY 15 7 893 - 893 2003年07月 [査読有り][通常論文]
  
• An efficient synthesis of a biantennary sialooligosaccharide analog using a 1,6-anhydro-β-lactose derivative as a key synthetic block

  Tetsuya Furuike, Kuriko Yamada, Takashi Ohta, Kenji Monde, Shin-Ichiro Nishimura

  Tetrahedron 59 27 5105 - 5113 2003年06月 [査読有り][通常論文]
  
• Glycosyltransferase Microarray Displayed on the Glycolipid LB Membrane

  Noriko Nagahori, Kenichi Niikura, Reiko Sadamoto, Masahiro Taniguchi, Akihiko Yamagishi, Kenji Monde, Shin-Ichiro Nishimura

  Advanced Synthesis & Catalysis 345 67 729 - 734 2003年06月 [査読有り][通常論文]
  
• Synthesis of a useful anomeric thioacetate of an N-acetyllactosamine derivative and its application

  Koji Matsuoka, Takumi Ohtawa, Hiroshi Hinou, Tetsuo Koyama, Yasuaki Esumi, Shin-Ichiro Nishimura, Ken Hatano, Daiyo Terunuma

  Tetrahedron Letters 44 18 3617 - 3620 2003年04月 [査読有り][通常論文]
  
• Design, synthesis, and evaluation of β-galactosylceramide mimics promoting β-glucocerebrosidase activity in keratinocytes

  Kyoko Fukunaga, Masahiro Yoshida, Fumio Nakajima, Rie Uematsu, Mariko Hara, Shintaro Inoue, Hirosato Kondo, Shin-Ichiro Nishimura

  Bioorganic & Medicinal Chemistry Letters 13 5 813 - 815 2003年03月 [査読有り][通常論文]
  
• Detection of genes with tissue-specific expression patterns using Akaike's information criterion procedure.

  Kadota K, Nishimura S, Bono H, Nakamura S, Hayashizaki Y, Okazaki Y, Takahashi K

  Physiological genomics 12 3 251 - 259 2003年02月 [査読有り][通常論文]
  
• Structure determination and conformational change induced by tyrosine phosphorylation of the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase

  N Fujitani, M Kanagawa, T Aizawa, T Ohkubo, S Kaya, M Demura, K Kawano, S Nishimura, K Taniguchi, K Nitta

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 300 1 223 - 229 2003年01月 [査読有り][通常論文]
   

  It has been well established that phosphotylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase. reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H+/K+-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation. and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H+/K+-ATPase. (C) 2002 Elsevier Science (USA). All rights reserved.
• Glycotentacles: Synthesis of cyclic glycopeptides, toward a tailored blocker of influenza virus hemagglutinin

  T Ohta, N Miura, N Fujitani, F Nakajima, K Niikura, R Sadamoto, CT Guo, T Suzuki, Y Suzuki, K Monde, SI Nishimura

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 42 42 5186 - 5189 2003年 [査読有り][通常論文]
  
• Protective action of reduced water against alloxan-induced cytotoxicity in pancreatic beta-cell

  YP Li, T Nishimura, K Teruya, Y Katakura, S Kabayama, K Otsubo, S Morisawa, Y Ishii, Z Gadek, S Shirahata

  ANIMAL CELL TECHNOLOGY: BASIC & APPLIED ASPECTS, VOL 13 145 - 149 2003年 [査読有り][通常論文]
   

  Previous studies showed that reduced water (RW) such as electrolyzed-reduced water (ERW) and natural reduced water (NRW) scavenge reactive oxygen species (ROS), protected DNA from oxidative damage, and stitnulated glucose uptake into myotubes and adipocytes. Here, we investigated that the protective mechanism of reduced water (RW) against alloxan-induced pancreatic beta-cell damage using HIT-T15 cells, a clonal hamster beta-cell line. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted by the production of ROS. Hydrogen peroxide was detected during the incubation of HIT-T15 cells with alloxan, and this generation of hydrogen peroxide was prevented by RW. Alloxan-treated HIT-T15 cells caused beta-cell damage with elevation of cytosolic free Ca2+, decrease of intracellular ATP level, inhibition of glucose-stimulated insulin release. RW not only completely prevented alloxan-derived ROS but also prevented the decrease of intracellular ATP level, the increase of cytosolic Ca2+ and the inhibition of insulin secretion. RW partly blocked alloxan-induced lowering of the cell viability. These results suggest that RW protected pancreatic beta-cell damage from the alloxan-derived oxygen radical generation. RW tray be useful to prevent alloxan-induced type 1 diabetes mellitus.
• Cell wall engineering of living bacteria through biosynthesis.

  Sadamoto R, Niikura K, Monde K, Nishimura S

  Methods in enzymology 362 273 - 286 2003年 [査読有り][通常論文]
  
• Synthesis of Photopolymerizable Glycolipids and their Application as Scaffolds to Immobilize Proteins with a Micron-Sized Pattern

  Noriko Nagahori, Kenichi Niikura, Reiko Sadamoto, Kenji Monde, Shin-Ichiro Nishimura

  Australian Journal of Chemistry 56 6 567 - 567 2003年 [査読有り][通常論文]
   

  Photopolymerizable glycolipids incorporating ceramide- or amido-type linkers and able to form stable monolayers were efficiently synthesized by chemical and enzymatic methods. Glycolipid polymer films served as platforms for the immobilization of proteins through specific carbohydrate–protein interactions at the air–water interface. Carbohydrate-binding proteins deposited on the glycolipid film were observed by atomic force microscopy, which showed varying submicron-sized protein patterns such as dendrites, dots, and networks, depending on the lipid structure, membrane preparation process, and sugar density of the membrane. Surface plasmon resonance measurement confirmed that the subunit-type lectins immobilized on the glycolipid membranes exhibited the ability to interact specifically with carbohydrate ligands by using unoccupied binding sites.
• Efficient and versatile synthesis of mucin-like glycoprotein mimics

  Yuki Tachibana, Naoki Matsubara, Fumio Nakajima, Tetsuro Tsuda, Sakae Tsuda, Kenji Monde, Shin-Ichiro Nishimura

  Tetrahedron 58 51 10213 - 10224 2002年12月 [査読有り][通常論文]
  
• Inhibition of adhesion of type 1 fimbriated Escherichia coli to highly mannosylated ligands

  N Nagahori, RT Lee, S Nishimura, D Page, R Roy, YC Lee

  CHEMBIOCHEM 3 9 836 - 844 2002年09月 [査読有り][通常論文]
   

  The inhibitory potencies of a number of mannosides, di- and trivalent mannosides, a set of mannose-terminating dendrimers, and five types of mannose-bearing neoglycoproteins were determined by using a binding assay that measures the binding of I-125-labeled, highly mannosylated neoglycoprotein to a type 1 fimbriated Escherichia coli (K72) strain In suspension. The IC50 values (the concentration of inhibitor that causes 50 % reduction-in the bound I-125-ligand to E. coli) obtained by this method were much lower than the equivalent values obtain hemagglutination or in assays that involve microplate immobilization. Two important factors that strongly influence the affinity to E. coli adhesin are: 1) the presence of an alpha-oriented aglycon that has a long aliphatic chain or an aromatic group immediately next to the glycosyl oxygen, and 2) the presence of multiple mannosyl residues that can span a distance of 20 nm or longer on a relatively structure. The two best inhibitors, which ore a highly mannosylated neoglycoprotein with the longest linking arm between a mannose and protein amino group and the largest mannosylated dendrimer (fourth generation), exhibited sub-nM IC50 values.
• Cell-wall engineering of living bacteria

  R Sadamoto, K Niikura, PS Sears, HT Liu, CH Wong, A Suksomcheep, F Tomita, K Monde, SI Nishimura

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 124 31 9018 - 9019 2002年08月 [査読有り][通常論文]
  
• Preparation of Self-Organized Micro-Patterned Polymer Films Having Cell Adhesive Ligands

  Jin Nishida, Kazutaka Nishikawa, Shin-Ichiro Nishimura, Shigeo Wada, Takeshi Karino, Takehiro Nishikawa, Kuniharu Ijiro, Masatsugu Shimomura

  Polymer Journal 34 3 166 - 174 2002年03月 [査読有り][通常論文]
  
• Encounter with unexpected collagenase-1 selective inhibitor: Switchover of inhibitor binding pocket induced by fluorine atom

  M Sawa, H Kondo, S Nishimura

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 12 4 581 - 584 2002年02月 [査読有り][通常論文]
   

  Phosphonamide-based inhibitors having trifluoromethyl moiety showed highly selective inhibition against MMP-1. A possible mechanism of the selectivity of MMP-1 inhibitors through the switchover of the binding pocket was speculated by computational calculations. As a consequence of the unexpected selectivity, the specific interaction of CF3 group of the inhibitor and Arg214 in the S1' pocket of MMP-1 conducted a low binding energy. (C) 2002 Elsevier Science Ltd. All rights reserved.
• Germ cell survival through carbohydrate-mediated interaction with Sertoli cells

  Tomoya O. Akama, Hiroaki Nakagawa, Kazuhiro Sugihara, Sonoko Narisawa, Chikara Ohyama, Shin Ichiro Nishimura, Deborah A. O'Brien, Kelley W. Moremen, José Luis Millán, Michiko N. Fukuda

  Science 295 5552 124 - 127 2002年01月04日 [査読有り][通常論文]
   

  Spermatogenesis is a precisely regulated process in which the germ cells closely interact with Sertoli cells. The molecular basis of this cell-cell adhesion is unknown. Here, we demonstrate that targeted disruption of Man2a2, a gene encoding α-mannosidase llx (MX), an enzyme that forms intermediate asparagine-linked carbohydrates (N-glycans), results in Man2a2 null males that are largely infertile. The Man2a2 null spermatogenic cells fail to adhere to Sertoli cells and are prematurely released from the testis to epididymis. We identified an N-glycan structure that plays a key role in germ cell-Sertoli cell adhesion and showed that a specific carbohydrate was required for spermatogenesis.
• Erythrocyte-like liposomes prepared by means of amphiphilic cyclodextrin sulfates

  T Sukegawa, T Furuike, K Niikura, A Yamagishi, K Monde, SI Nishimura

  CHEMICAL COMMUNICATIONS 5 430 - 431 2002年 [査読有り][通常論文]
   

  A novel class of sulfated glycolipids with excellent self-assembling capacity to form stable monolayers at an air-water interface and specific erythrocyte-like liposomes was synthesised from alpha, beta, and gamma-cyclodextrins as starting materials.
• An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3-N-Acetyl-D-glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose-Binding Protein

  Atsushi Toda, Kuriko Yamada, Shin-Ichiro Nishimura

  Advanced Synthesis & Catalysis 344 1 61 - 61 2002年01月 [査読有り][通常論文]
  
• Th1 cytokine-conditioned bone marrow-derived dendritic cells can bypass the requirement for Th functions during the generation of CD8(+) CTL

  M Sato, K Chamoto, T Tsuji, Y Iwakura, Y Togashi, T Koda, T Nishimura

  JOURNAL OF IMMUNOLOGY 167 7 3687 - 3691 2001年10月 [査読有り][通常論文]
   

  Bone marrow-derived dendritic cell (BMDC) subsets have distinct immunoregulatory functions. Th1 cytokine-induced BMDC (BMDC1), compared with Th2 cytokine-induced BMDC2, have superior activities for the differentiation and expansion of CTL. To evaluate the cellular interactions between dendritic cells and CD8(+) T cells for the induction of CTL, BALB/c-derived BMDC subsets were cocultured with purified CD8(+) T cells from C57BL/6 mice. Our results demonstrate that BMDC1 support the generation of allogeneic CD8(+) CTL in the absence of CD4(+) Th cells. In contrast, BMDC0 (GM-CSF- plus IL-3-induced BMDC) and BMDC2 failed to promote the differentiation of CD8(+) CTL. Using Ab-blocking experiments and studies with gene knockout mice, IL-2 and LFA-1 are demonstrated to be critical for BMDC1-induced CTL differentiation. Unexpectedly, BMDC1 were able to induce CTL from CD8(+) T cells isolated from IFN-gamma (-/-) and IFN-gamma receptor(-/-) mice. However, BMDC1 produced higher levels of IFN-beta than other BMDC subsets, and anti-IFN-beta mAb blocked BMDC1-dependent CTL generation. These results indicated an indispensable role of IFN-beta, but not IFN-gamma, during BMDC1-induced CTL differentiation. We conclude that Th1-cytokine-conditioned BMDC1 can bypass Th cell function for the differentiation of naive CD8(+) T cells into CTL.
• Characterization of a human H9N2 influenza virus isolated in Hong Kong

  Takehiko Saito, Wilina Lim, Takashi Suzuki, Yasuo Suzuki, Hiroshi Kida, Shin-Ichiro Nishimura, Masato Tashiro

  Vaccine 20 1-2 125 - 133 2001年10月 [査読有り][通常論文]
  
• Highly efficient oligosaccharide synthesis on water-soluble polymeric primers by recombinant glycosyltransferases immobilised on solid supports

  S Nishiguchi, K Yamada, Y Fuji, S Shibatani, A Toda, SI Nishimura, SI Nishimura

  CHEMICAL COMMUNICATIONS 19 1944 - 1945 2001年10月 [査読有り][通常論文]
   

  Recombinant beta -1,4-galactosyltransferase (beta1,4-GalT) and alpha -2,6-sialyltransferase (alpha2,6-SiaT) immobilised covalently with activated Sepharose beads were employed for the practical synthesis of a trisaccharide derivative, Neu5Ac alpha (2 -->6)Gal beta (1 -->4)GlcNAc beta -O-(CH2)(6)-NH2, on a water-soluble primer having GlcNAc residues through a alpha -chymotrypsin-sensitive linker.
• Combinatorial syntheses of sugar derivatives

  Shin-Ichiro Nishimura

  Current Opinion in Chemical Biology 5 3 325 - 335 2001年06月 [査読有り][通常論文]
  
• Cyclic di-t-butylsilylenediyl ether group as a convenient protective group for the glycoconjugate synthesis

  Daijyu Kumagai, Masaki Miyazaki, Shin-Ichiro Nishimura

  Tetrahedron Letters 42 10 1953 - 1956 2001年03月04日 [査読有り][通常論文]
   

  Treatment of methyl β-D-glucopyranoside, methyl α-D-glucopyranoside, 2-azido-2-deoxy-β-D-galactopyranosyl fluoride, and 1,6-anhydro-β-lactose with di-t-butyldichlorosilane gave the corresponding 4,6-cyclic di-t-butylsilylenediyl ether (4,6-CDBS) derivatives in high yields. It was suggested that the 4,6-CDBS group is quite stable under general conditions for further chemical manipulations such as the acetylation, benzylation and glycosylation reactions employed widely in the carbohydrate chemistry. This protective group was readily removed by treatment with tetrabutylammonium fluoride or triethylamine-3HF complex under mild conditions. © 2001 Elsevier Science Ltd.
• Heterobifunctional Ligands:  Practical Chemoenzymatic Synthesis of a Cell Adhesive Glycopeptide That Interacts with Both Selectins and Integrins

  Masao Matsuda, Shin-Ichiro Nishimura, Fumio Nakajima, Takashi Nishimura

  Journal of Medicinal Chemistry 44 5 715 - 724 2001年03月 [査読有り][通常論文]
  
• Anti-oxidative water improves diabetes

  S Shirahata, T Nishimura, S Kabayama, D Aki, K Teruya, K Otsubo, S Morisawa, Y Ishii, Z Gadek, Y Katakura

  ANIMAL CELL TECHNOLOGY: FROM TARGET TO MARKET 1 574 - 577 2001年 [査読有り][通常論文]
   

  Electrolyzed reduced water and natural waters Such is Hita Tenryosui water in Japan, Nordenau water in Germany and Tracote water in Mexico. which are known to improve various diseases, were all anti-oxidative waters which could scavenge intracellular reactive oxygen species. The anti-oxidative waters stimulated not only the glucose uptake of rat myotube L6 cells, but also the secretion of insulin from a pancreatic beta cell line HIT-T15. The and-oxidative Waters improved the damage in the sugar tolerance test of type 2 diabetes model mice (db/db mice). A clinical investigation demonstrated that Nordenau water could significantly improve the diabetes mellitus.
• Human N-Acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides

  Kazuhito Fujiyama, Yoshihiro Ido, Ryo Misaki, Daniel G. Moran, Itaru Yanagihara, Takeshi Honda, Shin-Ichiro Nishimura, Toshiomi Yoshida, Tatsuji Seki

  Journal of Bioscience and Bioengineering 92 6 569 - 574 2001年 [査読有り][通常論文]
   

  N-Acetylglucosaminyltransferase I (GnT-I), which catalyzes the transfer of an N-acetylglucosamine residue from UDP-N-acetylglucosamine to the α1,3-linked mannose on Man5GlcNAc2 (M5), is a critical enzyme for the synthesis of high-mannose-type to complex-type glycan structures in N-linked glycan processing. We developed a large-scale preparation system for recombinant human GnT-I (hGnT-I) using the maltose binding protein (MBP) fusion system to facilitate the chemoenzymatic route for complex-type N-linked glycan synthesis. MBP-fused GnT-I was purified by affinity chromatography on an amylose resin column. The relative activity of MBP-fused GnT-I toward high-mannose-type N-linked oligosaccharides was 100% for Man5GlcNAc2, 52% for Man3GlcNAc2, 17% for Man6GlcNAc2. MBP-fused GnT-I exhibited optimal activity at pH 6.5-9.5 and was more active between pH 6.5-9.0. The optimum temperature for MBP-fused GnTI activity was 40°C, but the enzyme was active between 0-70°C. Mn2+ and Co2+ were critical for the enzyme activity, while Zn2+ and Ca2+ inhibited the activity. Kinetic analysis of the purified enzyme showed an apparent Km value of 0.483 mM and a Vmax of 101 nmol/mg/min for M5. Immobilization of MBP-fused GnT-I on the amylose resin led to an 80% yield of the high mannose-type-of oligosaccharide.
• Tailored glycopolymers: controlling the carbohydrate-protein interaction based on template effect.

  Nagahori N, Nishimura S

  Biomacromolecules 2 1 22 - 24 2001年 [査読有り][通常論文]
  
• A highly practical synthesis of cyclodextrin-based glycoclusters having enhanced affinity with lectins

  T Furuike, S Aiba, SI Nishimura

  TETRAHEDRON 56 51 9909 - 9915 2000年12月 [査読有り][通常論文]
   

  A simple and highly practical method for the synthesis of cyclodextrin-scaffolded glycoclusters recognized specifically by lectins is described. Nucleophilic displacement of iodide from heptakis 6-deoxy-6-iodo-beta -cyclodextrin by unprotected sodium thiolates derived from 3-(3-thioacetyl propionamido)propyl glycosides proceeded smoothly in mild condition and gave novel per-glycosylated cyclodextrins (glycocyclodextrins, glycoCDs) having D-galactose, N-acetyl-D-glucosamine, lactose or N-acetyllactosamine residues in high yields (78-88%). It was demonstrated that all these per-glycosylated P-cyclodextrins showed amplified inhibitory effects on the erythrocytes agglutination induced by wheat germ (Triticum vulgaris) agglutinin (WGA) or Erythrina corallodendron lectin (ECorL). (C) 2000 Elsevier Science Ltd. All rights reserved.
• The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes

  T Nishimura, H Kitamura, K Iwakabe, T Yahata, A Ohta, M Sato, K Takeda, K Okumura, L Van Kaer, T Kawano, M Taniguchi, M Nakui, M Sekimoto, T Koda

  INTERNATIONAL IMMUNOLOGY 12 7 987 - 994 2000年07月 [査読有り][通常論文]
   

  In vivo administration of NKT cell ligand, alpha-galactosylceramide (alpha-GalCer), caused the activation of NKT cells to induce a strong NK activity and cytokine production by CD1d-restricted mechanisms. Surprisingly, we also found that alpha-GalCer induced the activation of immunoregulatory cells involved in acquired immunity. Specifically, in vivo administration of alpha-GalCer resulted in the induction of the early activation marker CD69 on CD4(+) T cells, CD8(+) T cells and B cells in addition to macrophages and NKT cells, However, no significant induction of CD69 was observed on cells from CD1d- or V(alpha)14 NKT-deficient mice, indicating an essential role for the interaction between NKT cells and CD1d-expressing dendritic cells (DC) in the activation of acquired immunity in response to alpha-GalCer, Indeed, in vivo injection of alpha-GalCer resulted not only in the activation of NKT cells but also in the generation of CD69(+)CD8(+) T cells possessing both cytotoxic T lymphocyte (CTL) activity and IFN-gamma-producing ability, Tumor-specific CTL generation was also accelerated by alpha-GalCer, The critical role of CD40-CD40 ligand (CD40L)-mediated NKT-DC interaction during the development of CD69(+)CD8(+) CTL by alpha-GalCer was demonstrated by blocking experiments using anti-CD40L mAb, These findings provide direct evidence for a critical role of CD1d-restricted NKT cells and DC in bridging innate and acquired immunity.
• The critical role of Th1-dominant immunity in tumor immunology

  Takashi Nishimura, Minoru Nakui, Marimo Sato, Kenji Iwakabe, Hidemitsu Kitamura, Masashi Sekimoto, Akio Ohta, Toshiaki Koda, Shinichiro Nishimura

  Cancer Chemotherapy and Pharmacology 46 S1 S52 - S61 2000年07月 [査読有り][通常論文]
  
• Design of fluorogenic substrates for continuous assay of sialyltransferase by resonance energy transfer

  K Washiya, T Furuike, F Nakajima, YC Lee, SI Nishimura

  ANALYTICAL BIOCHEMISTRY 283 1 39 - 48 2000年07月 [査読有り][通常論文]
   

  Glycosyltransferases are important synthetic enzymes for the construction of naturally occurring glycoconjugates as well as for the design of neoglycoconjugates. The assay methods currently available for these enzymes require tedious and time consuming procedures for separation of products and do not permit continual assay of enzyme activities. As a set of convenient fluorogenic substrates for continuous monitoring of sialyltransferase activities, we designed and synthesized a novel CMP-Neu5Ac derivative with a naphthylmethyl group at the C-9 position and N-acetyllactosamine derivative containing a dansyl group at the terminal position of aglycon. In such substrates, the emission peak of the naphthylmethyl group (lambda(em) = 340 nm) of the glycosyl donor is successfully overlapped with the excitation peak due to the dansyl group (lambda(ex) = 335 nm) of the glycosyl acceptor. A coupling reaction of these two substrates catalyzed by rat liver 2,6-sialyltransferase caused an increase of dansyl fluorescence (lambda(em) = 525 nm) and a decrease of naphthylmethyl fluorescence on the basis of resonance energy transfer between two fluorescence probes. The substrates presented here permit continuous fluorescent monitoring of enzymatic sugar combining reactions. Actually, using this time course of enzymatic reactions, kinetic constants of rat liver 2,6-sialyltransferase against glycosyl donor substrates were estimated to be K-m = 4.85 mu M and V-max = 0.119 mu mol/min, respectively. This strategy allows precise and efficient analyses of enzyme kinetics not possible with the conventional assay methods for the glycosyltransferases that usually require separation of products from the reaction mixture. (C) 2000 Academic Press.
• Indispensable role for TNF-α and IFN-γ at the effector phase of liver injury mediated by Th1 cells specific to hepatitis B virus surface antigen

  Ohta A, Sekimoto M, Sato M, Koda T, Nishimura S.-I, Iwakura Y, Sekikawa K, Nishimura T

  Journal of Immunology 165 2 956 - 961 2000年07月 [査読有り][通常論文]
  
• Interleukin-4-dependent induction of preproenkephalin in antigen-specific T helper-type 2 (Th2) cells

  T Yahata, C Yahata, A Ohta, M Sekimoto, H Kitamura, K Iwakabe, S Habu, S Azuma, M Nakui, M Sato, T Koda, T Nishimura

  JOURNAL OF NEUROIMMUNOLOGY 105 2 103 - 108 2000年06月 [査読有り][通常論文]
   

  Naive Th cells obtained from OVA(323-339)-specific DO11.10TCR-Tg mice did not express preproenkephalin (PPE) mRNA. However, culture of naive Th cells with OVA(323-339) peptide (OVA-pep) plus IL-2 under Th2-inducing conditions for 7 days resulted in an induction of PPE mRNA. The PPE mRNA was also induced by culturing with OVA-pep plus IL-2 (neutral condition). However, PPE mRNA induction under neutral conditions was totally abrogated by addition of anti-IL-4 mAb. The existence of methionine-enkephalin was also demonstrated in peptidase-digested peptides derived from Th2 cell lysate. These results demonstrate that IL-4 is a critical factor for the induction of PPE mRNA in freshly expanded antigen-specific Th2 cells. (C) 2000 Elsevier Science B.V. All rights reserved.
• Redefined substrate specificity of ST6GalNAc II: A second candidate sialyl-Tn synthase

  Mari Kono, Tetsuro Tsuda, Shunichiro Ogata, Shou Takashima, Hong Liu, Toshiro Hamamoto, Steven H. Itzkowitz, Shinichiro Nishimura, Shuichi Tsuji

  Biochemical and Biophysical Research Communications 272 1 94 - 97 2000年05月27日 [査読有り][通常論文]
   

  The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-linked oligosaccharides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala](n) polymer (n = 7-11). It has been suggested that only ST6GalNAc I can synthesize carbohydrate structures of sialyl-Tn-antigen i.e., NeuAcα2-6GalNAc-O-Thr/Ser based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity toward asialoagalacto-fetuin. In this study, we present evidence that both ST6GalNAc I and II exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala](n) polymer (n = 7-11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GalNAc I but also II are candidates for sialyl-Tn synthases. (C) 2000 Academic Press.
• Web-Structured Films of an Amphiphilic Polymer from Water in Oil Emulsion:  Fabrication and Characterization

  Takehiro Nishikawa, Jin Nishida, Ryusuke Ookura, Shin-Ichiro Nishimura, Volker Scheumann, Manfred Zizlsperger, Reinald Lawall, Wolfgang Knoll, Masatsugu Shimomura

  Langmuir 16 3 1337 - 1342 2000年02月 [査読有り][通常論文]
  
• α-Galactosylceramide induces early B-cell activation through IL-4 production by NKT cells

  Hidemitsu Kitamura, Akio Ohta, Masashi Sekimoto, Marimo Sato, Kenji Iwakabe, Minoru Nakui, Takashi Yahata, Hongxu Meng, Toshiaki Koda, Shin-Ichiro Nishimura, Tetsu Kawano, Masaru Taniguchi, Takashi Nishimura

  Cellular Immunology 199 1 37 - 42 2000年01月10日 [査読有り][通常論文]
   

  α-Galactosylceramide (α-GalCer), a glycolipid antigen, specifically activates natural killer T (NKT) cells by a CD1d-restricted mechanism. In this work, we found that in vivo administration of α-GalCer resulted in the activation of B cells in addition to NKT cells, namely, α-GalCer administration caused upregulation of the early activation marker, CD69, on both NKT and B cells. In addition, expression of B7.2 and I-A(b) on B cells was greatly upregulated by α-GalCer. However, serum levels of IgE, IgG1, and IgG2a were not significantly changed within 48 h. In the present experiments, it was also demonstrated that the upregulation of CD69 expression by α- GalCer was strongly blocked by anti-IL-4 monoclonal antibody. Moreover, B- cell activation by α-GalCer was not observed in NKT-deficient mice. These results suggested that antigen-stimulated NKT cells might play a critical role not only in early defense mechanisms but also in early B-cell activation through IL-4 production. (C) 2000 Academic Press.
• Synthetic studies on specific mucins: Antifreeze glygoprotein.

  Tachibana, Y, Tsuda, T, Matsubara, N, Tsuda, S, Nishimura, SI

  Abstracts of Papers of the American Chemical Society 219 U244 - U244 2000年 [査読有り][通常論文]
  
• Bi-fluorescence-labeled maltoheptaoside: Convenient substrate for continual assay of α-amylase

  S. I. Nishimura, N. Kimura, K. Matsuoka, Yuan Chuan Lee

  Carbohydrate Letters 4 2 77 - 84 2000年 [査読無し][通常論文]
   

  A new maltoheptaose derivative was prepared as a useful substrate for continual assay of α-amylase. The maltoheptaoside has thionaphtyl group as a fluorescent energy donor at the reducing end and dansyl group as an acceptor group at the non-reducing end. Excitation of the thionaphthyl group at 290 nm results in emission at 523 nm from the dansyl group, while the emission from the thionaphthyl group is quenched by the dansyl group. This fluorescence energy transfer is reduced by the hydrolytic action with α-amylase and a significant decrease in the dansyl emission concomitant with an increase in the thionaphthyl emission was observed. Usefulness of this substrate was demonstrated for sensitive and continuous assay of α-amylase from Aspergillus oryzae.
• Density Dependent Interaction of Polymeric Analogs ofβ-Galactosyl Ceramide with GP120 of Human Immunodeficiency Virus 1

  Fujita E, Nishimura S -I

  Carbohydr. Lett. 4 53 - 60 2000年 [査読有り][通常論文]
  
• Chemo-enzymatic synthesis of glycopolymers and sequential glycopeptides bearing lactosamine and sialyl Lewis(x) unit pendant chains

  F Sallas, SI Nishimura

  JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 1 13 2091 - 2103 2000年 [査読有り][通常論文]
   

  A variety of glycoconjugates bearing either N-acetyllactosamine or sialyl Lewis(x) units have been synthesised in a chemo-enzymatic way. This includes the synthesis of glycopolymer copolymerised with acrylamide and whose glucosamine unit was substituted with different kinds of side-chain. Glycopeptides with different spacer-arm glucosamine units have also been prepared and polymerised. The sugar chain was then elongated using glycosyl transferases to afford the novel sequential glycopeptides. In these cases, the polymeric sugar cluster effect led to enzymatic glycosylation with high efficiency. Nevertheless, some differences have been noticed depending on the reaction conditions used for each substrate.
• Synthesis and anti-influenza virus activity of novel glycopolymers having triantennary oligosaccharide branches

  T Furuike, S Aiba, T Suzuki, T Takahashi, Y Suzuki, K Yamada, SI Nishimura

  JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 1 1 17 3000 - 3005 2000年 [査読有り][通常論文]
   

  An efficient method for the synthesis of novel glycopolymers with triantennary sialooligosaccharides showing potent anti-influenza virus activity is described. Polymerisable glycoside of triantennary N-acetyllactosamine [beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-D-glucopyranose, Gal beta(1-->4)GlcNAc] is synthesised from lactose and 4-(3-hydroxypropyl)-4-nitroheptane-1,7-diol as key starting materials, and converted into water-soluble glycopolymers by radical copolymerisation with acrylamide. Subsequent enzymic sialylation using cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) with alpha-2,3-sialyltransferase from porcine liver or with alpha-2,6-sialyltransferase from rat liver gives novel glycoprotein mimics having potent inhibitory activity against influenza virus infection. It is demonstrated that the present triantennary glycoligands exhibit much higher biological activities than the effects by glycopolymers derived from the simple monovalent-type glycomonomers.
• Honeycomb-patterned thin films of amphiphilic polymers as cell culture substrates

  Takehiro Nishikawa, Jin Nishida, Ryusuke Ookura, Shin-Ichiro Nishimura, Shigeo Wada, Takeshi Karino, Masatsugu Shimomura

  Materials Science and Engineering: C 8-9 495 - 500 1999年12月 [査読有り][通常論文]
   

  Recently, we have found honeycomb patterns with sub-micron line width that form during the non-equilibrium process of cast film formation. The honeycomb-patterned films were fabricated using four macromolecular compounds (amphiphilic copolymers containing lactose units or carboxyl groups as side-chains and polyion complexes composed of anionic polysaccharides). The specific binding of lactose by lectin confirmed that the lactose moieties contained in the honeycomb films work as biologically active ligands. Bovine serum albumin (BSA) labeled with fluorescein was covalently attached to the honeycomb films using water-soluble carbodiimide (WSC) as an activator. Using fluorescence imaging of the modified film, we could show that the proteins are immobilized on the honeycomb patterns. Adhesion of bovine aorta endothelial cells (ECs) to the honeycomb films indicates that the honeycomb structure works as an adhesive site for the cells.
• Mesoscopic patterning of cell adhesive substrates as novel biofunctional interfaces

  Takehiro Nishikawa, Jin Nishida, Ryusuke Ookura, Shin-Ichiro Nishimura, Shigeo Wada, Takeshi Karino, Masatsugu Shimomura

  Materials Science and Engineering: C 10 1-2 141 - 146 1999年12月 [査読有り][通常論文]
   

  In this report, cell adhesion to honeycomb-patterned films is described with respect to the dimensions of the honeycomb structure. The honeycomb-patterned films can be fabricated by casting a dilute solution of amphiphilic polymers on solid substrates. The honeycomb structure is not homogeneous in all dimensions. Analysis of distribution of the honeycomb hole sizes demonstrates a gradual decrease in honeycomb hole diameter along the radius of the cast area. The largest holes were located near geometric center of the cast area. The diameter of the largest honeycomb holes in the cast area could be controlled by varying the cast volume of the polymer solution. Cell cultures on the honeycomb films demonstrated that cell adhesion could be inhibited at the outer region of the cast area. The extent of the inhibition of cell adhesion was influenced by the chemical properties of the polymers constituting the honeycomb films.
• Cloning and expression of a human gene encoding an N-acetylgalactosamine-alpha2,6-sialyltransferase (ST6GalNAc I): a candidate for synthesis of cancer-associated sialyl-Tn antigens.

  Ikehara Y, Kojima N, Kurosawa N, Kudo T, Kono M, Nishihara S, Issiki S, Morozumi K, Itzkowitz S, Tsuda T, Nishimura SI, Tsuji S, Narimatsu H

  Glycobiology 9 11 1213 - 1224 11 1999年11月 [査読有り][通常論文]
   

  The sialyl-Tn (sTn) antigen is a well known cancer-associated antigen, the expression of which is related to the prognosis of cancer patients. We aimed to isolate a human gene encoding an N-acetylgalactosamine α2,6-sialyltransferase which synthesizes sTn antigen, and to characterize the enzyme. Degenerate primers encoding sialyl motifs were used for the polymerase chain reaction to amplify complementary DNAs prepared from RNAs of human pyloric mucosae with intestinal metaplasia, which abundantly expressed sTn antigen, followed by screening of full-length cDNAs using the amplified DNA fragment as a probe. We isolated two human cDNA clones, long-form (2.46 kb) and short-form (2.23 kb) cDNAs. The former encodes an active enzyme with a predicted 600 amino acid sequence. The latter, a splice-variant of the long-form, encodes an inactive enzyme. HCT15 human colorectal cancer cells stably expressing the long-form cDNA expressed sTn epitopes on O-glycans. The long form cDNA was considered to encode a human homologue of chick ST6GalNAc I for the following reasons: (1) the putative amino acid sequence showed greater homology to that of chick ST6GalNAc I (55%) compared to other sialyltransferases, (2) it encodes the extraordinarily long stem region that is a typical feature of chick ST6GalNAc I, and (3) the substrate specificity was very similar to that of chick ST6GalNAc I. In situ hybridization demonstrated that the localization of transcripts correlated well with that of sTn antigen in gastric cancer cells and Goblet cells in intestinal metaplastic glands. Thus, we determined that the long-form cDNA of the human ST6GalNAc I gene encodes the probable candidate for the human sTn synthase(s).
• The natural killer T (NKT) cell ligand α-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells

  Hidemitsu Kitamura, Kenji Iwakabe, Takashi Yahata, Shin-Ichiro Nishimura, Akio Ohta, Yasushi Ohmi, Marimo Sato, Kazuyoshi Takeda, Ko Okumura, Luc Van Kaer, Tetsu Kawano, Masaru Taniguchi, Takashi Nishimura

  Journal of Experimental Medicine 189 7 1121 - 1127 1999年04月05日 [査読有り][通常論文]
   

  The natural killer T (NKT) cell ligand α-galactosylceramide (α- GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of α-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by α-GalCer. We first established, using purified subsets of various lymphocyte populations, that α-GalCer selectively activates NKT cells for production of interferon (IFN)-γ. Production of IFN-γ by NKT cells in response to α-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, α-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1(-/-) or Vα14(-/-) mice. This effect of α-GalCer required the production of IFN-γ by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of α-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-γ production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by α- GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.
• Efficient synthesis of non-natural ganglioside (pseudo-GM3) and fluorescent labelled lysoGM3 on the basis of polymer-assisted enzymatic strategy

  K Yamada, S Matsumoto, SI Nishimura

  CHEMICAL COMMUNICATIONS 6 507 - 508 1999年03月 [査読有り][通常論文]
   

  The versatility of polymer-assisted enzymatic synthesis of non-natural and biologically significant glycolipid derivatives was demonstrated by constructing pseudo-ganglioside GM3 1 having the trisaccharide sequence Neu5Ac alpha(2-->6)Gal beta(1-->4)Glc and a fluorescent labelled lysoGM3 2.
• Specific crosslinking of cell adhesive molecules by heterobifunctional glycopeptide synthesised on the basis of chemoenzymatic strategy

  SI Nishimura, M Matsuda, H Kitamura, T Nishimura

  CHEMICAL COMMUNICATIONS 15 1435 - 1436 1999年 [査読無し][通常論文]
   

  Heterobifunctional glycopeptide 1 composed of Neu5Ac alpha-(2 --> 3)Gal beta(1 --> 4)[Fuc alpha(1 --> 3)]GlcNAc (sialyl Lewis(x)) and Lys-Gly-Arg-Gly-Asp-Ser (KGRGDS) having specific activity to bind concurrently with two different types of cell adhesive molecules such as selectins and integrins was synthesised on the basis of a combined chemical and enzymatic strategy.
• Nonnatural branched polysaccharides: Synthesis and properties of chitin and chitosan having α-mannoside branches

  Keisuke Kurita, Kumi Shimada, Yasuhiro Nishiyama, Manabu Shimojoh, Shin-Ichiro Nishimura

  Macromolecules 31 15 4764 - 4769 1998年07月28日 [査読有り][通常論文]
   

  Regioselective introduction of α-mannoside branches at C-6 of chitin and chitosan has been accomplished by a series of regioselective modification reactions starting from N-phthaloyl-chitosan as a key precursor. Glycosylation of the derived acceptor with reactive groups only at C-6 with an ortho ester of D-mannose proceeded smoothly in dichloromethane in the presence of trimethylsilyl trifluoromethanesulfonate, and the degree of branching was up to 0.6. Full deprotection gave chitosans with α-mannoside branches, which were subsequently transformed into the corresponding branched chitins by N-acetylation. The resulting branched polysaccharides showed a remarkable solubility in neutral water in sharp contrast to the insoluble linear chitin and chitosan. Concanavalin A exhibited a specific affinity for these products, which was ascribable to the presence of α-mannoside groups. Though nonnatural, the branched chitins were susceptible to lysozyme, and the enzymatic degradation was heavily dependent on the extent of branching. Furthermore, the branched chitosan exhibited considerable antimicrobial activity.
• Mesoscopic pattern formation of nanostructured polymer assemblies

  Norihiko Maruyama, Olaf Karthaus, Kuniharu Ijiro, Masatsugu Shimomura, Takeo Koito, Shinnichiro Nishimura, Tetsuro Sawadaishi, Norio Nishi, Seiichi Tokura

  Supramolecular Science 5 3-4 331 - 336 1998年07月 [査読有り][通常論文]
  
• Amphiphilic poly(sugar amino acid)s: a novel class of glycoclusters for supramolecular materials

  SI Nishimura, S Nomura, K Yamada

  CHEMICAL COMMUNICATIONS 5 617 - 618 1998年03月 [査読有り][通常論文]
   

  A poly(sugar amino acid) having self-assembling properties was efficiently synthesised by simple polymerisation of 1-O-dodecyl-2-amino-2-deoxy-beta-D-glucopyranosiduronic acid 7 derived from D-glucofuranurono-6,3-lactone as the key starting material.
• Synthesis and self-organisation of new cyclodextrin amphiphile

  T Sukegawa, M Matsuda, SI Nishimura, M Shimomura, K Ijiro, O Karthaus

  MOLECULAR RECOGNITION AND INCLUSION 519 - 522 1998年 [査読無し][通常論文]
   

  A novel amphiphilic cyclodextrin derivative having sialic acid residues was systematically synthesised. Hexakis (2,3-di-O-palmitoyl)-di-6-O-toluenesulfonyl-alpha-cyclodextrin cyclodextrin was converted to hexakis (2,3-di-O-palmitoyl)-6-di-(2-aminoethyl)-amino-6-dideoxy-alpha-cyclodextrin by reaction with ethylenediamine as a reactive spacer. Coupling of a sialic acid intermediate and alpha-cyclodextrin derivative gave an amphiphilic cyclodextrin derivative having two sialic acid residues. This unique compound showed an excellent ability to form monolayer membrane.
• Regioselective syntheses of sulfated polysaccharides: specific anti-HIV-1 activity of novel chitin sulfates

  Shin-Ichiro Nishimura, Hideaki Kai, Katsuhiko Shinada, Takashi Yoshida, Seiichi Tokura, Keisuke Kurita, Hideki Nakashima, Naoki Yamamoto, Toshiyuki Uryu

  Carbohydrate Research 306 3 427 - 433 1998年01月 [査読有り][通常論文]
  
• Convenient fluorescence probes for the characterisation of glycosyltransferases related to the acrosome reaction

  Nishimura S. -I, Washiya K

  Zygote 6 35 - 36 1998年 [査読有り][通常論文]
  
• Endotherial cell responses to chitin and its derivatives.

  Mori T, Irie Y, Nishimura SI, Tokura S, Matsuura M, Okumura M, Kadosawa T, Fujinaga T

  J Biomed Mater Res 43 4 469 - 472 1998年 [査読無し][通常論文]
  
• High performance polymer supports for enzyme-assisted synthesis of glycoconjugates

  K Yamada, E Fujita, SI Nishimura

  CARBOHYDRATE RESEARCH 305 3-4 443 - 461 1997年12月 [査読有り][通常論文]
   

  Efficient and practical methodology for the construction of carbohydrates, including oligosaccharide derivatives and sphingoglycolipids, was established on the basis of a water-soluble polymer supports having unique linkers that can be cleaved by specific conditions. Novel glycomonomers for the construction of polymer supports were synthesized and copolymerized with acrylamide to give three types of water-soluble glycopolymers having primer sugars through the specific linkers containing (i) p-substituted benzyl group, (ii) L-phenylalanine residue, and (iii) ceramide-mimetic L-serine derivative, respectively. These glycopolymers were employed for sugar elongation reactions with glycosyl transferases such as GlcNAc beta 1,4-galactosyl transferase, beta Gal1-->3/4GlcNAc alpha-2,6-sialyl transferase, and beta Gal1-->3/4GlcNAc alpha-2,3-sialyl transferase in the presence of each sugar nucleotide as glycosyl donor to afford polymers having N-acetyllactosamine, sialyl alpha-(2-->6) N-acetyllactosamine, and sialyl alpha-(2-->3) lactose residues in excellent yield. Subsequent hydrogenolysis, hydrolysis with alpha-chymotrypsin, or transglycosylation to ceramide with ceramide glycanase proceeds smoothly to give N-acetyllactosamine, a versatile sialyl alpha-(2-->6) N-acetyllactosamine derivative having a terminal amino group, and ganglioside GM3 in high yield. (C) 1998 Elsevier Science Ltd.
• Transfer of ganglioside GM3 oligosaccharide from a water soluble polymer to ceramide by ceramide glycanase. A novel approach for the chemical-enzymatic synthesis of glycosphingolipids

  S Nishimura, K Yamada

  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 119 43 10555 - 10556 1997年10月 [査読有り][通常論文]
  
• Swine influenza virus strains recognize sialylsugar chains containing the molecular species of sialic acid predominantly present in the swine tracheal epithelium

  T Suzuki, G Horiike, Y Yamazaki, K Kawabe, H Masuda, D Miyamoto, M Matsuda, SI Nishimura, T Yamagata, T Ito, H Kida, Y Kawaoka, Y Suzuki

  FEBS LETTERS 404 2-3 192 - 196 1997年03月 [査読有り][通常論文]
   

  We determined the ratio of N-glycolylneuraminic acid (Neu5Gc) to N-acetylneuraminic acid (Neu5Ac) in swine respiratory epithelia by fluorometric high-performance liquid chromatography, and examined the binding specificity of swine influenza virus strains for gangliosides containing different molecular species of sialic acid (Neu5Ac and Neu5Gc), and for bovine erythrocyte sialoglycoprotein 2 (GP-2) containing Neu5Ge as its predominate sialic acid (96% of total sialic acids), The presence of Neu5Gc, which had not been detected in human tracheal epithelia, and Neu5Ac in swine tracheal epithelia was observed in a 1:1 ratio, The swine influenza virus H1 and H3 isolates tested, except for A/swine/Iowa/15/30 (H1N1), displayed a marked binding ability for sialylsugar chains containing Neu5Gc compared with that of the human influenza virus strains, These results suggest that swine influenza viruses recognize sialylsugar chains containing the molecular species of sialic acid present predominantly in the swine tracheal epithelium. (C) 1997 Federation of European Biochemical Societies.
• Mercapto-chitins: A new type of supports for effective immobilization of acid phosphatase

  Keisuke Kurita, Hitoshi Yoshino, Shin-Ichiro Nishimura, Shigeru Ishii, Tomonori Mori, Yasuhiro Nishiyama

  Carbohydrate Polymers 32 3-4 171 - 175 1997年 [査読有り][通常論文]
   

  Acid phosphatase was immobilized on two kinds of mercapto-chitins, 2-mercapto-chitin and 6-mercapto-chitin, and assayed with 4-nitrophenyl phosphate as the substrate. The optimal pH values for immobilization were 4.5 and 4.8, respectively. The resulting immobilized enzymes showed maximum activities at pH 6.0 and 5.5, almost the same as that for the soluble enzyme. 6-Mercaptochitin/enzyme conjugate retained high activity even after repeated uses in batch systems, suggesting effective immobilization through covalent bond formation, while 2-mercapto-chitin/enzyme and chitin/enzyme conjugates showed decreases in activity after a few runs. © 1997 Elsevier Science Ltd.
• Preparation of fluorescence-labeled neoglycolipids for ceramide glycanase assays

  Matsuoka K, Nishimura S. I, Lee Y. C

  Fluorescence Spectroscopy 278 519 - 528 1997年 [査読有り][通常論文]
  
• Preparation of chitin/polystyrene hybrid materials by efficient graft copolymerization based on mercaptochitin

  Keisuke Kurita, Soichiro Hashimoto, Hitoshi Yoshino, Shigeru Ishii, Shin-Ichiro Nishimura

  Macromolecules 29 6 1939 - 1942 1996年03月11日 [査読有り][通常論文]
   

  Graft copolymerization of styrene onto mercaptochitin has been examined. Chitin was first tosylated to give tosylchitin, which was subsequently transformed into mercaptochitin. Athough the graft copolymerization was carried out in suspension, it proceeded efficiently to give chitin derivatives having polystyrene branches, a novel type of hybrid materials composed of a natural polysaccharide and a synthetic polymer. Under appropriate conditions, the grafting percentage reached 970%, indicating the high efficiency of the mercaptochitin as an initiator for the polymerization of styrene. The resulting graft copolymers exhibited glass transition phenomena at 115°C and showed high swelling in organic solvents as a result of the introduction of polystyrene branches. Hydrolytic degradation of the chitin main chain allowed the isolation of the side chains, and the polystyrene isolated from the graft copolymer with a grafting percentage of 940% had Mn, Mw, and Mw/Mn values of 9.74 × 104, 2.55 × 105, and 2.62. These values indicate that the ratio of the mercapto groups actually used for initiating graft copolymerization was 4%, and a polystyrene chain attached on average to every 45 pyranose units.
• Preparation of novel 1,6-anhydro-beta-lactose derivatives for the synthesis of N-acetyllactosamine-containing oligosaccharides

  T Tsuda, T Furuike, SI Nishimura

  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 69 2 411 - 416 1996年02月 [査読有り][通常論文]
   

  Versatile intermediates for the syntheses of cell-surface oligosaccharides bearing N-acetyllactosamine units have been prepared from a readily available 1,6-anhydro-beta-lactose as a key starting material. Regioselective chemical manipulations of 1,6-anhydro-beta-lactose were carried out in a stepwise procedure and gave fully-protected lactosamine derivatives. It was clearly suggested that modification at C-3' position of the galactose residue and the following introduction of a sterically hindered leaving group at C-2 position of the reducing glucose remarkably accelerated and facilitated further derivatization of this potential disaccharide material. Selective generation of hydroxyl groups at C-3, C-3', and/or C-6' positions proceeded smoothly in mild conditions and afforded a series of fully-functionalized disaccharide-acceptors in high yields.
• Synthetic glycogels for affinity electrophoresis: A facile and efficient method for investigating sugar-lectin interaction

  M Ohmae, S Suzuki, S Tokura, N Nishi, SI Nishimura

  JOURNAL OF BIOCHEMISTRY 119 2 367 - 371 1996年02月 [査読有り][通常論文]
   

  A novel and efficient method for analyzing sugar-lectin interaction using affinity electrophoresis (AEP) is described, Polyacrylamide gels covalently conjugated with 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) residues were successfully prepared by radical copolymerization of highly reactive 3-(N-acryloylamino)propyl 2-acetamido-2-deoxy-beta-D-glucopyranoside with acrylamide in the presence of N,N-methylenebisacrylamide (BIS), When the glycogels carrying various densities of GlcNAc branches were employed for polyacrylamide gel electrophoresis (PAGE) of lectins, the mobilities of wheat germ agglutinin (WGA) were specifically reduced by increasing the concentrations of the GlcNAc residues in gels, although concanavalin A (Con A) showed no significant change in the mobility, It was also demonstrated that the association constant of WGA with immobilized GlcNAc residue can be determined by combined use of this stable glycogel and an automated gel-scanning system associated with fluorometric spectroscopy, The association constant of WGA with the GlcNAc moiety was estimated to be 1.24X10(4) M(-1).
• Utilization of DNA as functional materials: Preparation of filters containing DNA insolubilized with alginic acid gel

  Norio Nishi, Kazumichi Iwata, Tetsuro Sawadaishi, Shin-Ichiro Nishimura, Seiichi Tokura

  International Journal of Biological Macromolecules 18 1-2 149 - 150 1996年 

  Thin films composed of DNA and alginic acid were prepared by casting their mixed solution on glass plate followed by coagulation with aqueous solution of calcium chloride. DNA could be conveniently insolubilized by this method. DNA in the films adsorbed intercalating materials, such as ethidium bromide. This phenomenon was successfully applied to the preparation of filters for the selective removal or accumulation of harmful intercalating pollutants.
• Linear polybiurets: Facile synthesis and characteristic properties

  Keisuke Kurita, Shin-Ichi Watabe, Shin-Ichiro Nishimura, Shigeru Ishii

  Journal of Polymer Science, Part A: Polymer Chemistry 34 3 429 - 438 1996年 [査読有り][通常論文]
   

  Linear polybiurets have been synthesized by polyaddition of benzyloxyamine and diisocyanates, and properties of the novel polymeric materials have been elucidated. Prior to polymerization, model reactions between benzyloxyamine and phenyl isocyanate were examined in detail and proved to be controlled by the molar ratio of reagents and by catalysts to give the urea (1 : 1 adduct) and/or biuret (1 : 2 adduct). Under appropriate conditions, the biuret was synthesized in a quantitative yield. Polymerization of equimolar amounts of benzyloxyamine and diphenylmethane or hexamethylene diisocyanate proceeded smoothly to give polybiurets with inherent viscosities up to 0.52 dL/g. The benzyl group of the model biuret and polybiuret could be removed by catalytic hydrogenation. Both the N-benzyloxy-type and N-hydroxy-type polybiurets showed excellent solubility in common organic solvents. The two kinds of polybiurets as well as model biurets adsorbed metal cations efficiently. The N-hydroxybiuret structure exhibited particularly high affinity for iron(III) and was useful for selective removal of iron from metal cation mixtures. © 1996 John Wiley & Sons, Inc.
• Synthesis of an antifreeze glycoprotein analogue: Efficient preparation of sequential glycopeptide polymers

  Tetsuro Tsuda, Shin-Ichiro Nishimura

  Chemical Communications 24 2779 - 2780 1996年 [査読有り][通常論文]
   

  A sequential glycopeptide polymer, antifreeze glycoprotein (AFGP) 1, was efficiently synthesised by simple polymerisation of the repeating glycopeptide unit of AFGP 10 with diphenylphosphoryl azide (DPPA) as a convenient promotor.
• Efficient preparation of β-(1←6)-(GlcNAc)2 by enzymatic conversion of chitin and chito-oligosaccharides

  Kayo Shimoda, Kohji Nakajima, Yoshiharu Hiratsuka, Shin-Ichiro Nishimura, Keisuke Kurita

  Carbohydrate Polymers 29 2 149 - 154 1996年 [査読有り][通常論文]
   

  A chitin-degrading marine bacterium strain, OK2607, was isolated from the sea water of Okinawa, Japan. The bacterium was identified as Alteromonas sp. The extracellular enzyme preparation obtained from the culture media of the bacterium effectively degraded chitin, and the degradation behavior was examined in detail. Unexpectedly, the major product was a β-(1→6)-linked disaccharide of N-acetylglucosamine (GlcNAc), and only small amounts of N,N′-diacetylchitobiose and GlcNAc were detected. High transglycosylation activity of the enzyme preparation was also confirmed with N-acetyl-chito-oligosaccharides giving rise to the same β-(1→6) disaccharide. The results indicate that the enzyme preparation is quite efficient to synthesize the unusual β-(1→6) oligosaccharide starting from the β-(1→6) polysaccharide or the oligosaccharides and would serve as a useful tool to prepare various β-(1→6) oligosaccharides. Copyright © 1996 Published by Elsevier Science Ltd.
• Efficient graft copolymerization of 2-methyl-2-oxazoline onto tosyl- and iodo-chitins in solution

  Keisuke Kurita, Soichiro Hashimoto, Shigeru Ishii, Tomonori Mori, Shin-Ichiro Nishimura

  Polymer Journal 28 8 686 - 689 1996年 [査読有り][通常論文]
   

  Graft copolymerization of 2-methyl-2-oxazoline onto tosyl- and iodo-chitins has been examined. The reaction proceeded efficiently with tosyl-chitin in solution to give chitin derivatives having poly(N-acetylethyleneimine) side chains, and the grafting percentage was 280% under appropriate conditions. Graft copolymerization was also possible with iodo-chitin, but rather sluggish. Hydrolytic degradation of the chitin main chain allowed the isolation of the introduced side chains. The number-average molecular weight of the polyethyleneimine isolated from a graft copolymer with grafting percentage of 160% was 2700, indicating that 18% of the carbon atoms having a tosyloxy group were actually utilized for initiating the graft copolymerization. The resulting graft copolymers showed high affinity for both organic solvents and water, and may be useful as a new type of chitin-derived hybrid materials.
• An efficient synthesis of sialoglycoconjugates on a peptidase-sensitive polymer support

  K Yamada, SI Nishimura

  TETRAHEDRON LETTERS 36 52 9493 - 9496 1995年12月 [査読有り][通常論文]
   

  A novel method for the enzymatic synthesis of oligosaccharide derivatives on a alpha-chymotrypsin-sensitive polymer support is described. The primer polymer having N-acetyl-D-glucosamine (GlcNAc) residue through a phenylalanine-containing spacer moiety was successfully elongated with galactosyl and sialyltransferases to give a glycopolymer bearing sialyl alpha(2 --> 6) N-acetyllactosamine branches in high yield. Subsequent hydrolysis with alpha-chymotrypsin proceeded smoothly and afforded a versatile sialotrisaccharide derivative having a terminal amino group which can he used for creating neoglycoconjugates.
• Polyisoimides: Synthesis and Evaluation as Efficient Precursors for Polyimides

  Keisuke Kurita, Yukiko Suzuki, Takahito Enari, Shigeru Ishii, Shin-Ichiro Nishimura

  Macromolecules 28 6 1801 - 1806 1995年11月01日 [査読有り][通常論文]
   

  Polyisoimides have been evaluated as potential precursors for polyimides since they are expected to be tractable and isomerized to polyimides without producing water. Detailed studies on isoimides were carried out first with model compounds. Compared to N-phenyl- and N-benzylphthal-isoimides, N-(benzyloxy)phthalisoimide proved to be superior in terms of ease of synthesis, hydrolytic stability, and quantitative isomerization to the imide. Polymerizations of p- and m-xylylenebis(oxyamine)s with tetracarboxylic dianhydrides such as pyromellitic dianhydride, benzophenonetetracarboxylic dianhydride, and 2,2-bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride proceeded efficiently in N-methyl-2-pyrrolidone, and the resulting poly(amic acid)s had inherent viscosities up to 0.61 dL/g. The poly(amic acid)s were then converted into polyisoimides by chemical dehydration. Under appropriate conditions with N,N'-dicyclohexylcarbodiimide, isoimide formation was preferred to imide formation, and the isoimide contents of the polymers were about 90%. The polyisoimides obtained exhibited high solubility and low glass transition temperatures unlike the corresponding polyimides. Furthermore, on heating or treating with a base catalyst, the polyisoimides were isomerized to polyimides quantitatively, indicating the high potential of the polyisoimides as precursors for polyimides. © 1995, American Chemical Society. All rights reserved.
• SYNTHETIC GLYCOCONJUGATES .6. PREPARATION AND BIOCHEMICAL EVALUATION OF NOVEL CLUSTER-TYPE GLYCOPOLYMERS CONTAINING GAL BETA(1-]4)GLCNAC (N-ACETYLLACTOSAMINE) RESIDUE

  T FURUIKE, N NISHI, S TOKURA, SI NISHIMURA

  MACROMOLECULES 28 21 7241 - 7247 1995年10月 [査読有り][通常論文]
   

  A simple and facile method for the syntheses of cluster-type homopolymers from omega-(acrylamido)alkyl glycosides of N-acetyllactosamine [beta-D-Galp-(1-->4)-beta-n-GlcpNAc] is described. An efficient procedure for the preparation of poly[omega-(acrylamido)alkyl O-(beta-D-galactopyranosyl)-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside] was established on the basis of radical polymerization of the novel glycosides in the presence of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine. These synthetic cluster glycopolymers exhibited good solubility in water and had sugar densities much higher than the glycopolymers derived from known n-pentenyl glycosides. The association constants of Erythrina corallodendron lectin (ECorL) with these cluster glycopolymers were evaluated by measuring the changes in fluorescence intensity of fluorescent-labeled ECorL induced by the addition of the polymeric ligands. Addition of the cluster-type glycopolymers having different degrees of N-acetyllactosamine density to ECorL induced a significant decrease of the fluorescence intensity at 520 nm, and this phenomenon was used for the determination of binding constants between the glycopolymers and ECorL.
• FLUORESCENCE-LABELED SYNTHETIC GLYCOPOLYMERS - A NEW-TYPE OF SUGAR LIGANDS OF LECTINS

  K NAGATA, T FURUIKE, SI NISHIMURA

  JOURNAL OF BIOCHEMISTRY 118 2 278 - 284 1995年08月 [査読有り][通常論文]
   

  Radical copolymerization of a polymerizable dansyl derivative, N-2-propenyl-(5-dimethylamino)-1-naphthalene sulfonamide, with sugar monomers and acrylamide proceeded smoothly in aqueous solution in the presence of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine and afforded a novel type of water-soluble glycopolymers having fluorescent side-chains, Fluorescence emission spectra of these polymeric sugar-ligands by excitation at 340 nm revealed maxims at 448 and 528 nm, When the glycopolymer carrying galactose residues was saturated with Ricinus communis agglutinin (RCA(60)), the fluorescence emission maxima at 448 and 528 nm were not shifted significantly, although the fluorescence intensities were decreased by 20 and 14%, respectively. Polymeric sugar-cluster effects drastically enhanced the association constants of galactose residues with RCA(60) in the order of 10(8) M(-1). The significance for efficient binding of galactose density on the glycopolymer was also demonstrated by using glycopolymers with different degrees of galactose branching.
• Three-dimensional display of surface cortical perfusion by SPECT: Application in assessing Alzheimer's disease

  K. Hashikawa, M. Matsumoto, H. Moriwaki, N. Oku, Y. Okazaki, Y. Seike, T. Uehara, H. Tanabe, Y. Ohie, T. Kamada, T. Nishimura

  Journal of Nuclear Medicine 36 690 - 696 1995年01月01日 [査読無し][通常論文]
  
• A new approach to assay endo-type carbohydrases: Bifluorescent-labeled substrates for glycoamidases and ceramide glycanases

  Kyung Bok Lee, Koji Matsuoka, Shin-Ichiro Nishimura, Yuan Chuan Lee

  Analytical Biochemistry 230 1 31 - 36 1995年 [査読有り][通常論文]
   

  Glycoamidases and ceramide glycanases are important 'endo-type' enzymes for structural elucidations of glycoconjugates as well as for construction of neoglycoconjugates. The assay methods currently available for these enzymes are tedious and do not permit continual assay of the enzyme activities. We modified a desialylated biantennary glycopeptide with 2-naphthylacetic acid at the N-terminus and at the nonreducing terminal galactosyl residues with mono-N-dansylethylenediamine, via a specific oxidation of the C-6 hydroxyl group with galactose oxidase. In such a substrate, the naphthyl fluorescence (λem = 335 nm) is quenched due to absorption of its emitted light by the dansyl group, which in turn results in emission of fluorescence (λex = 520 nm) by the latter. However, when the link between the two fluorophores is severed by glycoamidase (PNGase), the energy transfer ceases to occur. Consequently the emission of the dansyl fluorescence and the quenching of naphthyl fluorescence diminish or disappear. Likewise, the energy transfer between the fluorophores in an alkyl lactoside containing a dansyl group at the terminal position of aglycon and a 2-naphthylmethyl group on the galactosyl residue is also eliminated by the glycosidic cleavage by a ceramide glycanase from American leech, Macrobdella decora, resulting in enhancement of the naphthyl emission and decrease in the dansyl emission. The substrates presented here permit continuous fluorescent monitoring of the enzymatic reaction. This allows precise analyses of enzyme kinetics not possible with the conventional assay methods for the endo-type enzymes which usually require separation of reaction products. © 1995 Academic Press, Inc.
• GLYCOCONJUGATES .5. POLYMERIC SUGAR LIGANDS AVAILABLE FOR DETERMINING THE BINDING-SPECIFICITY OF LECTINS

  MATSUOKA K, NISHIMURA SI

  MACROMOLECULES 28 8 2961 - 2968 1995年 [査読有り][通常論文]
  
• A FACILE AND QUANTITATIVE PREPARATION OF ACTIVATED CYCLIC SUGAR-DERIVATIVES USING HGBR2 AND 2,4,6-COLLIDINE

  MATSUOKA K, NISHIMURA SI, LEE YC

  BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 68 6 1715 - 1720 1995年 [査読有り][通常論文]
  
• A BI-FLUORESCENCE-LABELED SUBSTRATE FOR CERAMIDE GLYCANASE BASED ON FLUORESCENCE ENERGY-TRANSFER

  MATSUOKA K, NISHIMURA SI, LEE YC

  CARBOHYDRATE RESEARCH 276 1 31 - 42 1995年 [査読有り][通常論文]
  
• Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties

  Keisuke Kurita, Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, Shin-Ichiro Nishimura

  Macromolecules 27 26 7544 - 7549 1994年12月01日 [査読有り][通常論文]
   

  Synthesis of 6,6′-diamino-6,6′-dideoxy-α,α-d-trehalose, the polyaddition with diisocyanates, and the characteristics of the resulting synthetic carbohydrate polymers have been disclosed. Among four preparative approaches to efficient preparation of diaminotrehalose starting from trehalose, that involving first protection of the C-6,6′ hydroxyl groups with trityl groups, acetylation of the remaining hydroxyl groups, detritylation, tosylation, azidation, deacetylation, and finally catalytic reduction proved to be superior to the others, and the overall yield was as high as 26%. The resulting diaminotrehalose was subjected to polyaddition with diisocyanates to give polyureas containing trehalose residues in the main chain. The polyureas showed high solubility in organic solvents. The derived membranes were evaluated in dialysis with urea and vitamin B12 and exhibited marked permeability. The polyureas were also susceptible to trehalase and α-amylase, suggesting the high potential as a novel type of biodegradable synthetic carbohydrate polymers. © 1994, American Chemical Society. All rights reserved.
• SPLIT-DOSE IODINE-123-IMP SPECT - SEQUENTIAL QUANTITATIVE REGIONAL CEREBRAL BLOOD-FLOW CHANGE WITH PHARMACOLOGICAL INTERVENTION

  K HASHIKAWA, M MATSUMOTO, H MORIWAKI, N OKU, Y OKAZAKI, T UEHARA, N HANDA, H KUSUOKA, T KAMADA, T NISHIMURA

  JOURNAL OF NUCLEAR MEDICINE 35 7 1226 - 1233 1994年07月 [査読無し][通常論文]
   

  At least two quantitative rCBF measurements are needed to evaluate rCBF changes with pharmacological intervention. We have developed the split dose I-123-IMP SPECT method, which enables measurement of rCBF to be repeated in a short time. Methods: Thirty-one cerebrovascular disease patients were investigated to assess reproducibility and vasoreactivity to acetazolamide. During 44-min dynamic SPECT imaging, I-123-IMP injection and respective arterial sampling were performed twice at an interval of about 25 min. The rCBF values were calculated using a microsphere model in which the washout of I-123-IMP from the brain can be negligible in the first several minutes after injection. For the second rCBF measurement, the remaining activity due to the first I-123-IMP injection was estimated and subtracted from the total brain activity. Results: In ten patients, two consecutive resting mean rCBF values in the MCA territory (CBF1 and CBF2) had good correlation (CBF1 = 47.4 +/- 4.0 (ml/min/100 mi: mean +/- s.d.), CBF2 = 45.2 +/- 8.2, CBF2 = 0.900*CBF1 + 2.9, r = 0.915). In 11 patients with occlusive lesions in the unilateral ICA system, mean rCBF in the MCA territory was increased by only 27.7% +/- 14.0% in the affected side by al-g intravenous acetazolamide injection, while 44.5% +/- 12.3% increase was found in the nonaffected side. In 10 patients without a major arterial lesion, a 49.7% +/- 17.0% increase of rCBF was demonstrated. Conclusions: This split dose method I-123-IMp SPECT can be useful to estimate vascular reserve.
• CARBON-DIOXIDE REACTIVITY BY CONSECUTIVE TECHNETIUM-99M-HMPAO SPECT IN PATIENTS WITH A CHRONICALLY OBSTRUCTED MAJOR CEREBRAL-ARTERY

  N OKU, M MATSUMOTO, K HASHIKAWA, H MORIWAKI, Y OKAZAKI, Y SEIKE, N HANDA, T UEHARA, T KAMADA, T NISHIMURA

  JOURNAL OF NUCLEAR MEDICINE 35 1 32 - 40 1994年01月 [査読無し][通常論文]
   

  In the management of major cerebral artery obstruction, cerebral perfusion reserve is key to introducing cerebral revascularization surgery. The purpose of this study was to evaluate the feasibility of assessing cerebral perfusion reserve by consecutive Tc-99m-hexamethyl-propyleneamine oxime (Tc-99m-HMPAO) SPECT with 5% carbon dioxide (CO2) inhalation. Methods: The CO2 inhalation and consecutive Tc-99m-HMPAO SPECT study was performed on 30 chronic ischemic cerebrovascular disease patients with unilateral major cerebral artery obstruction and on 27 patients without. CO2 reactivity was expressed as the percent increase of (99m)Tcc-HMPAO accumulation from the baseline (%Change) and as a constant k' that was the ratio of Tc-99m-HMPAO accumulation per 1 mmHg change of end-tidal CO2 tension by exponential curve fitting. Results: The mean %Change and k' in the middle cerebral artery (MCA) territory on the side without an obstructive lesion or in the cerebellum ranged from 10.0% to 11.1% and from 0.98% to 1.13% per mmHg, respectively. In the MCA territory, an obstructive lesion was noted in 5.9% versus 0.54% per mmHg in the contralateral MCA territory (9 < 0.01). Eleven of 30 patients with major cerebral artery obstruction revealed significant asymmetry in the k' value between bilateral MCA territories. Conclusion: The results showed compromised cerebral perfusion reserve in the obstructed major cerebral artery territory. The present method was proven clinically useful for evaluating cerebral perfusion reserve in patients with unilateral major cerebral artery obstruction.
• PREPARATION OF GLYCOPROTEIN MODELS - PENDANT-TYPE OLIGOSACCHARIDE POLYMERS

  S NISHIMURA, T FURUIKE, K MATSUOKA

  NEOGLYCOCONJUGATES, PT A 242 235 - 246 1994年 [査読有り][通常論文]
  
• Biosynthesis of a novel polysaccharide by Acetobacter xylinum

  Akihiro Shirai, Mikio Takahashi, Hiroaki Kaneko, Shin-Ichiro Nishimura, Masato Ogawa, Norio Nishi, Seiichi Tokura

  International Journal of Biological Macromolecules 16 6 297 - 300 1994年 [査読有り][通常論文]
   

  An Acetobacter xylinum adapted to a medium containing N-acetylglucosamine (GlcNAc) has been used to prepare a novel polysaccharide containing residual GlcNAc in cellulose. The maximum amount of incorporation was found to be 4 mol% in cellulose, when a mixed medium containing 1.4% glucose (Glc) and 0.6% GlcNAc was used for the culture of A. xylinum. The resulting polysaccharide was lysozyme-susceptible. The aminosugar residue incorporated into bacterial cellulose was found to be only GlcNAc, even if galactosamine (GalN) and glucosamine (GlcN) were applied, whereas there was little effect by mannosamine (ManN). As the major component of the resulting polysaccharide was Glc residues, even if the only carbon source in the culture medium was GlcNAc, it was suggested that there must be several enzyme systems to convert GlcNAc into Glc in the bacteria. Several ammonium salts were also found to be effective for the incorporation of GlcNAc residues when the incubation system was converted to rotatory and aerobic incubation from static incubation. The amount of residual GlcNAc was remarkably increased by the addition of lysozyme-susceptible phosphoryl-chitin (P-chitin) and increased slightly with addition of P-chitin that was less lysozyme-susceptible. However, little effect was found on addition of highly substituted P-chitin. © 1994.
• Reactivity characteristics of squid β‐chitin as compared with those of shrimp chitin: High potentials of squid chitin as a starting material for facile chemical modifications

  Keisuke Kurita, Shigeru Ishii, Koji Tomita, Shin‐Ichiro Nishimura, Kayo Shimoda

  Journal of Polymer Science Part A: Polymer Chemistry 32 6 1027 - 1032 1994年 [査読有り][通常論文]
   

  Chemical reactivity of β‐chitin isolated from squid pens has been examined in various reactions to elucidate the possibility of facile modifications in simple manners leading to the preparation of derivatives with well‐defined structures. β‐Chitin swelled in common solvents such as methanol and pyridine unlike the ordinary α‐chitin and exhibited much higher reactivity than β‐chitin. Free amino groups present in β‐chitin were easily and selectively acetylated with acetic anhydride in methanol to give chitin with a uniform structure, poly(N‐acetyl‐D‐glucosamine). When acetylation reaction was carried out in pyridine, O‐acetylation proceeded smoothly besides N‐acetylation. In the presence of 4‐dimethylaminopyridine as the catalyst, even full acetylation was achieved under mild conditions. Tosylation was also quite efficient in pyridine without side reactions such as N‐deacetylation which is unavoidable in the tosylation of α‐chitin. β‐Chitin also enabled direct tritylation in pyridine in the presence of 4‐dimethylaminopyridine. All these reactions were quite sluggish with β‐chitin, and no reactions or only very low extents of substitution were observed, indicating the high potential of β‐chitin as a versatile starting material for facile modification reactions. © 1994 John Wiley & Sons, Inc. Copyright © 1994 John Wiley & Sons, Inc.
• A novel synthetic approach to a polybiuret from benzyloxyamine and 4,4′‐diphenylmethane diisocyanate

  Shigeru Ishii, Shin‐Ichi Watabe, Shin‐Ichiro Nishimura, Keisuke Kurita

  Journal of Polymer Science Part A: Polymer Chemistry 32 3 575 - 577 1994年 [査読有り][通常論文]
  
• Induction of drug specific antibody and the controlled release of drug by 6-O-carboxymethyl-chitin

  Tokura Seiichi, Miura Yoshiaki, Johmen Masayoshi, Nishi Norio, Nishimura Shin-Ichiro

  Journal of Controlled Release 28 1-3 235 - 241 1994年 [査読有り][通常論文]
   

  Biopolymeric properties of a water-soluble and biodegradable chitin derivative, 6-O-carboxymethyl-chitin (CM-chitin), have been investigated to demonstrate the immunological function serving to induce a hapten-specific antibody and the chemotherapeutic function as a drug carrier of controlled release. When CM-chitin was linked by methamphetamine (MA) through a nonbiodegradable spacer, 1-aminobutane (MABA-CM-chitin), MA-specific antibody was produced by the subcutaneous injection, in rabbits, of MABA-CM-chitin in combination with Freund's complete adjuvant, and when injected without intense immunoadjuvant, MABA-CM-chitin oligomer was secreted into blood for more than 120 h. Two-step hydrolysis of pendant-type of polymeric drug was also investigated in order to design a more sophisticated drug delivery system. Phenylalanine-containing peptide spacers were designed to be hydrolysed by enzymes other than lysozyme as a model drug. In the model system, the chromophore-bound phenylalanine peptide spacer was stabilized against enzymatic hydrolysis until CM-chitin was hydrolysed by lysozyme. © 1994.
• Isoimides and polyisoimides: Facile preparation and quantitative isomerization

  Keisuke Kurita, Yukiko Suzuki, Takahito Enari, Masaki Kikuchi, Shin‐Ichiro Nishimura, Shigeru Ishii

  Journal of Polymer Science Part A: Polymer Chemistry 32 2 393 - 396 1994年 [査読有り][通常論文]
  
• SYNTHETIC GLYCOCONJUGATES .4. USE OF OMEGA-(ACRYLAMIDO)ALKYL GLYCOSIDES FOR THE PREPARATION OF CLUSTER GLYCOPOLYMERS

  NISHIMURA SI, FURUIKE T, MATSUOKA K

  MACROMOLECULES 27 18 4876 - 4880 1994年 [査読有り][通常論文]
  
• SYNTHETIC GLYCOCONJUGATES .3. AN EFFICIENT SYNTHESIS OF A GLYCOPROTEIN MODEL HAVING A LE(X)-TYPE TRISACCHARIDE SEQUENCE OF TUMOR-ASSOCIATED CARBOHYDRATE ANTIGEN

  NISHIMURA SI, MATSUOKA K, FURUIKE T

  MACROMOLECULES 27 1 157 - 163 1994年 [査読有り][通常論文]
  
• SYNTHESIS OF BI-FLUORESCENCE-LABELED LACTOSIDE - A SUBSTRATE FOR CONTINUAL ASSAY OF CERAMIDE GLYCANASE

  MATSUOKA K, NISHIMURA SI, LEE YC

  TETRAHEDRON-ASYMMETRY 5 12 2335 - 2338 1994年 [査読有り][通常論文]
  
• CHEMOENZYMIC PREPARATION OF A GLYCOCONJUGATE POLYMER HAVING A SIALYLOLIGOSACCHARIDE - NEU5AC-ALPHA(2-]3)GAL-BETA(1-]4)GLCNAC

  NISHIMURA SI, LEE KB, MATSUOKA K

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 199 1 249 - 254 1994年 [査読有り][通常論文]
  
• CHEMOENZYMATIC OLIGOSACCHARIDE SYNTHESIS ON A SOLUBLE POLYMERIC CARRIER

  NISHIMURA SI, MATSUOKA K, LEE YC

  TETRAHEDRON LETTERS 35 31 5657 - 5660 1994年 [査読有り][通常論文]
  
• An Efficient Method for the Syntheses of Novel Amphiphilic Polysaccharides by Regio- and Thermoselective Modifications of Chitosan

  NISHIMURA Shin-ichiro, MIURA Yoshiaki, REN Longdi, SATO Manabu, YAMAGISHI Akihiko, NISHI Norio, TOKURA Seiichi, KURITA Keisuke, ISHII Shigeru

  Chemistry letters 1993 9 1623 - 1626 The Chemical Society of Japan 1993年09月05日 

  The first example of an unique and efficient method for the preparation of amphiphilic polysaccharides having self-assembling nature is described. Regio- and thermospecific chemical manipulations of a standardized intermediate derived from chitosan markedly facilitated the synthetic procedures of novel types of polysaccharide architectures. Artificial glycolipid-type polymers showed excellent properties to form stable monolayer membranes at air/water interface.
• Reactivity characteristics of a new form of chitosan - Facile N-phthaloylation of chitosan prepared from squid β-chitin for effective solubilization

  Keisuke Kurita, Koji Tomita, Tomoyoshi Tada, Shin-Ichiro Nishimura, Shigeru Ishii

  Polymer Bulletin 30 4 429 - 433 1993年04月 [査読有り][通常論文]
   

  A new form of chitosan prepared by deacetylating squid β-chitin was subjected to N-phthaloylation to evaluate the reactivity as compared with that of the conventional chitosan prepared from shrimp α-chitin. The reaction proceeded much more efficiently with squid chitosan than shrimp chitosan to give N-phthaloylchitosan, indicating considerable differences in higher-order structures between the two kinds of chitosans. Squid chitosan thus proved to show enhanced reactivity and be superior to shrimp chitosan as a starting material for controlled regioselective chemical modifications of chitin. © 1993 Springer-Verlag.
• Quantitative assessment of cerebral blood flow by 123I-IMP SPECT: Venous sampling method with hand warming in the water bath

  H. Moriwaki, M. Matsumoto, K. Hashikawa, N. Oku, Y. Okazaki, N. Handa, K. Kimura, T. Kozuka, T. Kamada, T. Nishimura

  Kakuigaku 30 481 - 488 1993年01月01日 [査読無し][通常論文]
  
• Studies on Functionalized Chitosan Beads. I. Selective Adsorption of Bovine Lactoferrin onto Partially Sulfated Chitosan Beads.

  Itoyama Koki, Nishimura Shin-ichiro, Kawamura Yoshihide, Seo Hiroshi, Sato Kaoru, Dosako Shun-ichi, Nishi Norio, Tokura Seiichi

  繊維学会誌 49 11 580 - 585 The Society of Fiber Science and Technology, Japan 1993年 

  Porous chitosan beads preparing under the wet condition showed specific capacity to adsorb lactoferrin directly from bovine milk by the partial sulfation reactions. Crosslinking of chitosan beads followed by sulfation was found to give much improved stability and the material exhibited excellent property as an adsorbent material for the affinity chromatography of lactoferrin even in case for higher degrees of sulfation. The amount of lactoferrin bound to the column of chitosan beads was effectively enhanced by increasing CSV value (salt splitting capacity per bed volume of beads) untill 100μeq/mL.
• β‐chitin as a convenient starting material for acetolysis for efficient preparation of N‐acetylchitooligosaccharides

  Keisuke Kurita, Koji Tomita, Shigeru Ishii, Shin‐Ichiro Nishimura, Kayo Shimoda

  Journal of Polymer Science Part A: Polymer Chemistry 31 9 2393 - 2395 1993年 [査読有り][通常論文]
  
• Squid chitin as a potential alternative chitin source: Deacetylation behavior and characteristic properties

  Keisuke Kurita, Koji Tomita, Tomoyoshi Tada, Shigeru Ishii, Shin‐Ichiro Nishimura, Kayo Shimoda

  Journal of Polymer Science Part A: Polymer Chemistry 31 2 485 - 491 1993年 [査読有り][通常論文]
   

  β‐Chitin was isolated from squid pens, and the characteristic chemical and physical properties were elucidated in comparison with those of shrimp chitin, α‐chitin. Deacetylation behavior of the squid chitin was first studied to look into the reactivity of β‐chitin and also to establish an efficient procedure for preparing squid chitosan. The squid chitin proved to show much higher reactivity in alkaline deacetylation than shrimp chitin. Although it was deacetylated quite easily, the product assumed a dark brown color under the ordinary reaction conditions for shrimp chitosan. Squid chitosan was successfully prepared by repeated alkaline treatments under mild conditions, particularly with high concentration alkali at low temperatures, without appreciable discoloration. The structural characteristics of the squid chitin were discussed on the basis of the IR and x‐ray analysis data. The crystalline structure of squid chitin was destroyed easily on deacetylation compared to that of shrimp chitin, and moreover, the resulting squid chitosan was amorphous unlike crystalline shrimp chitosan. The squid chitin was characterized by the remarkable affinity for organic solvents and water. Squid chitin and chitosan also showed much higher hygroscopicity and retention of the absorbed water than shrimp chitin and chitosan and are considered to be useful as highly hydrophilic materials. © 1993 John Wiley & Sons, Inc. Copyright © 1993 John Wiley & Sons, Inc.
• Preparation and biodegradability of chitin derivatives having mercapto groups

  Keisuke Kurita, Hitoshi Yoshino, Shin-Ichiro Nishimura, Shigeru Ishii

  Carbohydrate Polymers 20 4 239 - 245 1993年 [査読有り][通常論文]
   

  Two kinds of chitin derivatives having mercapto groups have been prepared, and the properties, including solubility and susceptibility to lysozyme, are discussed. Chitosan was first modified by N-acylation with S-protected mercaptoacetic acid, followed by selective acetylation of the remaining amino groups and deprotection to give derivatives having mercaptoacetyl groups at the C-2 amino groups. In an alternative preparation of mercapto-chitins, tosylchitin was treated with potassium thioacetate and then with alkali to afford derivatives having mercapto groups at the C-6 positions. The derivatives showed much enhanced swelling and solubility in organic solvents. Biodegradability of the mercapto derivatives was evaluated with lysozyme, and they turned out to be more susceptible to enzyme hydrolysis than the original chitin. © 1993.
• Preparation of Tosylchitins as Precursors for Facile Chemical Modifications of Chitin

  Keisuke Kurita, Hitoshi Yoshino, Koji Yokota, Motonari Ando, Satoshi Inoue, Shigeru Ishii, Shin-Ichiro Nishimura

  Macromolecules 25 14 3786 - 3790 1992年07月01日 [査読有り][通常論文]
   

  Tosylation of chitin was accomplished by interfacial condensation to give tosylchitins expected to be useful as soluble and reactive precursors for chemical manipulations under mild conditions. The reaction was efficient even at low temperatures. Tosylation was quantitative with a 20-fold excess of tosyl chloride. Free amino groups in the tosylchitins were acylated to form well-defined structures. The tosylchitins with substitution degrees above 0.4 were soluble in common polar organic solvents. The resulting tosylchitins underwent efficiently reactions such as acetylation and iodination to fully acetylated tosylchitins and iodochitins. Iodochitins were more soluble than tosylchitins. Reduction of tosyl- and iodochitins with sodium borohydride led to deoxychitins. © 1992, American Chemical Society. All rights reserved.
• Cationic and Radical Graft Copolymerization of Styrene onto Iodochitin

  Keisuke Kurita, Satoshi Inoue, Kiichi Yamamura, Hitoshi Yoshino, Shigeru Ishii, Shin-Ichiro Nishimura

  Macromolecules 25 14 3791 - 3794 1992年07月01日 [査読有り][通常論文]
   

  Iodochitin was evaluated as a precursor for graft copolymerization of styrene both by a cationic and a free-radical mechanism. In the presence of Lewis acids, cationic species were formed at the carbons bearing an iodo group, and graft copolymerization of styrene proceeded efficiently in nitrobenzene or somewhat less efficiently in nitromethane. As a catalyst, SnCl4 was superior to TiC4. Grafting percentages were as high as 800 % under appropriate conditions. The polystyrene branches were isolated by hydrolytic degradation of the chitin main chains and characterized by GPC. On irradiation of the iodochitin by an excimer laser, the C-I bonds of iodochitin were cleaved to free radicals which initiated styrene grafting. The grafting percentages were not high in the radical graft copolymerization, but very little homopolystyrene was formed. The resulting chitin-graft-polystyrenes were much more soluble or swollen in organic solvents. © 1992, American Chemical Society. All rights reserved.
• Introduction of poly(L‐alanine) side chains into chitin as versatile spacer arms having a terminal free amino group and immobilization of nadh active sites

  Keisuke Kurita, Seiji Iwawaki, Shigeru Ishii, Shin‐Ichiro Nishimura

  Journal of Polymer Science Part A: Polymer Chemistry 30 4 685 - 688 1992年 [査読有り][通常論文]
  
• Polymers Based on p-Aminophenol. 7. New Synthetic Approaches to the Simplest Wholly Aromatic Poly(imide ester) via the Corresponding Poly(amic acid) Precursor

  Keisuke Kurita, Naohiro Mikawa, Shigeru Ishii, Shin-Ichiro Nishimura

  Macromolecules 24 26 6853 - 6855 1991年12月01日 [査読有り][通常論文]
  
• Chemospecific Manipulations of a Rigid Polysaccharide: Syntheses of Novel Chitosan Derivatives with Excellent Solubility in Common Organic Solvents by Regioselective Chemical Modifications

  Shin-Ichiro Nishimura, Osamu Kohgo, Keisuke Kurita, Hiroyoshi Kuzuhara

  Macromolecules 24 17 4745 - 4748 1991年08月01日 [査読有り][通常論文]
   

  Efficient procedures for the preparations of soluble chitosan derivatives have been established on the basis of the regioselective chemical modifications. Selective and quantitative N-phthaloylation of chitosan proceeds smoothly by the reaction of chitosan with phthalic anhydride in N,N-dimethylformamide (DMF) at 130 °C. The resulting phthaloylchitosan exhibits much improved solubility in common organic solvents such as DMF, N,N-dimethylacetamide, dimethyl sulfoxide, and pyridine. The enhanced solubility of new types of chitosan derivatives under homogeneous reaction conditions. Facile conversions of phthaloylchitosan, a key starting material, into several 6-O-substituted derivatives are carried out by the reactions with bulky substituents such as triphenylmethyl (trityl) and (p-tolylsulfonyl) oxy (tosyloxy) groups. These reactions also proceed in homogeneous solution under mild conditions, and the degrees of substitution are estimated to be 1.0. Subsequent 3-O-acetylation of the secondary hydroxyl groups of 6-O-substituted materials gives rise to regioselectively modified chitosan derivatives showing much better solubility. Specific N-dephthaloylation or O-detritylation of 6-O-trityl derivatives affords versatile intermediates, which permit the introduction of additional functional groups regioselectively. © 1991, American Chemical Society. All rights reserved.
• Graft copolymerization of vinyl monomers onto chitin with cerium (IV) ion

  Keisuke Kurita, Mitsuo Kawata, Yoshiyuki Koyama, Shin‐Ichiro Nishimura

  Journal of Applied Polymer Science 42 11 2885 - 2891 1991年 [査読有り][通常論文]
   

  Graft copolymerization of vinyl compounds onto chitin was studied, and an efficient and reproducible procedure has been established, cerium (IV) being used as the initiator. The reactions with acrylamide and acrylic acid onto powdery chitin were carried out under various conditions to elucidate the polymerization behavior in terms of grafting percentage. The amount of cerium (IV) affected the polymerization most strikingly, and grafting percentages showed maxima with suitable amounts of initiator for both the monomers. As a solvent water proved to be superior to aqueous nitric acid except the reaction with a small amount of initiator. Under appropriate conditions, around 240 and 200% grafting percentages were achieved for acrylamide and acrylic acid, respectively. The resulting graft copolymers showed much improved affinity for solvents and hygroscopicity compared to the original chitin. Copyright © 1991 John Wiley & Sons, Inc.
• Preparation of soluble chitin derivatives as reactive precursors for controlled modifications: Tosyl‐ and iodo‐chitins

  Keisuke Kurita, Satoshi Inoue, Shin‐Ichiro Nishimura

  Journal of Polymer Science Part A: Polymer Chemistry 29 6 937 - 939 1991年 [査読有り][通常論文]
  
• Solubilization of a rigid polysaccharide: Controlled partial N-Acetylation of chitosan to develop solubility

  Keisuke Kurita, Mami Kamiya, Shin-Ichiro Nishimura

  Carbohydrate Polymers 16 1 83 - 92 1991年 [査読有り][通常論文]
   

  The solubility-structure relationship was studied in the acetylation of chitosan to elucidate key factors for solubilization of rigid polysaccharides. N-Acetylation of chitosan proceeded smoothly and reproducibly in a highly swollen state, and the degree of substitution was easily controlled. Evaluation of the properties of the resulting acetylated chitosans indicated the importance of the distribution mode of acetyl groups as well as the extent of substitution for solubilization. The chitosan derivatives with about 50% substitution prepared under appropriate conditions exhibited high solubility in water, and the presence of half a molar amount of randomly distributed substituents on the amino groups was confirmed to be an essential requirement in developing solubility. The present procedure is much simpler to prepare the water-soluble chitin than the conventional method involving partial deacetylation of chitin. © 1991.
• SYNTHETIC GLYCOCONJUGATES .2. NORMAL-PENTENYL GLYCOSIDES AS CONVENIENT MEDIATORS FOR THE SYNTHESES OF NEW TYPES OF GLYCOPROTEIN MODELS

  NISHIMURA SI, MATSUOKA K, FURUIKE T

  MACROMOLECULES 24 15 4236 - 4241 1991年 [査読有り][通常論文]
  
• Synthesis of a peripheral trisaccharide sequence of lutropin, a pituitary glycoprotein hormone; use of chitobiose as a key starting material

  Shin-Ichiro Nishimura, Hiroyoshi Kuzuhara

  Carbohydrate Research 206 2 207 - 217 1990年10月10日 [査読有り][通常論文]
   

  A chitobiose derivative, methyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→4)-3,6-di-O-acetyl-2-deoxy-2-phthalimido-β-d- glucopyranoside, was derived from the corresponding N-acetyl derivative and this was converted into the glycosyl bromide (5). Glycosidation reaction between 5 and methyl 3,4,6-tri-O-benzyl-α-d-mannopyranoside in the presence of silver trifluoromethanesulfonate gave a β-d-linked trisaccharide derivative. Replacement of the N,N-phthaloyl group by acetyl groups resulted in a product that was converted into methyl O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β-d-glucopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β-d- glucopyranosyl)-(1→2)-3,4,6-tri-O-benzyl-α-d-mannopyranoside (11) by use of a few reaction steps. The 43-hydroxyl group of 11 was methanesulfonylated, and the product subjected to SN2 replacement with acetate anion, to give the d-galactosamine-containing trisaccharide derivative (12). After basic hydrolysis of 12, the 43-hydroxyl group was sulfated, and all benzyl groups were removed by hydrogenolysis, giving methyl O-(2-acetamido-2-deoxy-4-O-sulfo-β-d-galactopyranosyl)-(1→4)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→2)-α-d- mannopyranoside monosodium salt, the methyl α-glucoside derivative of the peripheral trisaccharide sequence of the pituitary glycoprotein hormone lutropin. © 1990.
• Preparations of soluble chitin derivatives and the modifications to branched chitins

  Keisuke Kurita, Shin Ichiro Nishimura, Yoshiyuki Koyama, Satoshi Inoue, Osamu Kohgo

  American Chemical Society, Polymer Preprints, Division of Polymer Chemistry 31 1 624 - 625 1990年04月 

  We have been examining various modes of chemical modifications of chitin. With regard to solubilization, we reported the formation of water-soluble chitin by controlled partial deacetylation of chitin or reversely, by controlled partial acetylation of chitosan. Introduction of some bulky groups at specific positions is anticipated to develop solubility in rather common organic solvents, which would allow further efficient modification reactions in regioselective manners in homogeneous solution. Among the possible modifications, introduction of pendant sugar units is especially of importance. Here we report the preparation of new chitin derivatives having solubility in common organic solvents, and modifications of the soluble derivatives to give chitins with sugar branches.
• Synthetic Glycoconjugates: Simple and Potential Glycoprotein Models Containing Pendant N-Acetyl-D-glucosamine and N.N’-Diacetylchitobiose

  Shin-Ichiro Nishimura, Koji Matsuoka, Keisuke Kurita

  Macromolecules 23 18 4182 - 4184 1990年 [査読有り][通常論文]
  
• ((Diethylamino) ethyl) chitins: Preparation and Properties of Novel Aminated Chitin Derivatives

  Keisuke Kurita, Yoshiyuki Koyama, Satoshi Inoue, Shin-Ichiro Nishimura

  Macromolecules 23 11 2865 - 2869 1990年 [査読有り][通常論文]
   

  Procedures for (diethylamino)ethylation of chitin with (diethylamino)ethyl chloride have been established. ((Diethylamino)ethyl)chitins (DEAE-chitins) were prepared in dispersion in organic solvents with a small amount of aqueous sodium hydroxide. Among the solvents examined, dimethyl sulfoxide was confirmed to be most suitable, and degrees of substitution above 1.2 were achieved. The substitution reactions in solution in aqueous sodium hydroxide also proceeded smoothly, and DEAE-chitins with substitution degrees above 1.4 were obtained. Addition of a phase-transfer catalyst to the aqueous solution enhanced the reaction efficiency considerably. The resulting DEAE-chitins exhibited highly improved affinity to water and organic solvents, and these properties were dependent on both the mode of preparation and substitution extent. The derivatives prepared in aqueous solution showed much better solubility and swelling they were readily water soluble and swelled remarkably in common solvents such as benzene and alcohols. The separation behavior of these DEAE-chitins for organic dyes was studied, and those with high substitution degrees proved useful as adsorbents and separating materials. © 1990, American Chemical Society. All rights reserved.
• Polymers Based on p-Aminophenol. 6. Facile Synthesis of the Simplest Wholly Aromatic Poly(imide-ester) by Pyrolytic Polymerization of Monomers Containing Preformed Ester Linkages

  Keisuke Kurita, Naohiro Mikawa, Yoshiyuki Koyama, Shin-Ichiro Nishimura

  Macromolecules 23 10 2605 - 2609 1990年 [査読有り][通常論文]
   

  An improved synthetic procedure for a wholly aromatic poly(imide-ester) with the simplest structure has been examined. The polymer was synthesized through imide formation polymerization by pyrolytic condensation of monomers containing preformed ester linkages and evaluated as a high performance polymer. The monomers were prepared by first N-protection of p-aminophenol with phenyl chloroformates followed by acylation of the remaining hydroxyl group with trimellitic anhydride acid chloride. Thermal analysis revealed that these monomers underwent pyrolysis to split off the corresponding phenols and carbon dioxide giving rise to the poly(imide-ester). Pyrolysis behavior was closely associated with the substituents on the phenol moiety the monomers having electron-withdrawing groups showed improved thermal dissociation and hence resulted in the formation of poly(imide-esters) with higher viscosities. This procedure was much superior to the previous one involving the ester-forming polymerization. The resulting poly(imide-esters) had inherent viscosities up to 0.38 dL/g in concentrated sulfuric acid. Thermogravi-metric analysis showed high thermal stability with a weight loss of only 2% at 500 °C with a heating rate of 5 °C/min in air. © 1990, American Chemical Society. All rights reserved.
• N-hydroxyacylation of chitosan with lactones

  Kurita Keisuke, Takanobu Takeda, Shinichiro Nishimura

  Polymer Journal 22 5 429 - 434 1990年 [査読有り][通常論文]
   

  Although γ-butyrolactone is not reactive enough toward the amino groups of chitosan, the β-form has been found to give N-hydroxyacylated derivatives. The conditions for the reactions were studied in detail, and efficient procedures have been established. The reaction was highly dependent on the kind of solvent and confirmed to proceed only in dimethyl sulfoxide among ordinary solvents either in dispersion of powdery chitosan or in a highly swollen state of chitosan. The degree of substitution could easily be controlled by reaction temperature and time, and reached 0.65–0.67 after about 48 h at 100°C. The resulting derivatives showed much improved hygroscopicity, and those prepared in the swollen state were superior to those prepared in solid dispersion. © 1990 The Society of Polymer Science, Japan.
• Peracetylated chitobiose: Preparation by specific degradations of chitin, and chemical manipulations

  Shin-Ichiro Nishimura, Hiroyoshi Kuzuhara, Yasuyuki Takiguchi, Kenzo Shimahara

  Carbohydrate Research 194 C 223 - 231 1989年12月01日 [査読有り][通常論文]
   

  Chitobiose octaacetate (3) was preparable in moderate yield from chitin by microbial degradation followed by acetylation, or by modified chemical degradation. Compound 3 was chemically manipulated to give various compounds, including an oxazoline derivative, glycosides, and partially O-benzylated derivatives. The conformation of the oxazoline derivative is discussed. © 1989.
• TRANSFORMATION OF NIH3T3 CELLS WITH SYNTHETIC C-HA-RAS GENES

  H KAMIYA, K MIURA, N OHTOMO, T KODA, M KAKINUMA, S NISHIMURA, E OHTSUKA

  JAPANESE JOURNAL OF CANCER RESEARCH 80 3 200 - 203 1989年03月 [査読有り][通常論文]
  
• PREPARATION AND ANTITHROMBOGENIC ACTIVITIES OF HEPARINOID FROM 6-O-(CARBOXYMETHYL)CHITIN

  S NISHIMURA, S TOKURA

  INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 9 4 225 - 232 1987年08月 [査読有り][通常論文]
  
• Induction of methamphetamine-specific antibody using biodegradable carboxymethyl-chitin

  Seiichi Tokura, Osatoshi Hasegawa, Shin-Ichiro Nishimura, Norio Nishi, Takehiko Takatori

  Analytical Biochemistry 161 1 117 - 122 1987年02月15日 

  Induction of a specific antibody for methamphetamine, an antihypnotic drug, has been studied using carboxymethylchitin (CM-chitin) as a hapten carrier. The hapten-specific antiserum was induced by only a couple of hypodermal injections every 2 weeks. Inhibition of antigen-antibody complex formation was linearly related to methamphetamine concentration in the range 0.5 to 50 ng/50 μl when an enzyme-linked immunosorbent assay was performed using an avidin-biotin-peroxidase complex. Little antibody directed against CM-chitin and CM-chitin oligomer was detected. Thus it seems to be advantageous in the hapten-bovine serum albumin system. The specificity of the antibody was high as shown by the use of various methamphetamine analogs. © 1987.
• Effect of multiporous microspheres derived from chitin and partially deacetylated chitin on the activation of mouse peritoneal macrophages

  Keiko Nishimura, Shin-ichiro Nishimura, Hiroshi Seo, Norio Nishi, Seiichi Tokura, Ichiro Azuma

  Vaccine 5 2 136 - 140 1987年 

  Multiporous microspheres were prepared from 80% deacetylated chitin (DAC-80) and chitin, and their effects on the activation of murine peritoneal macrophages in vivo and on the production of monokines such as colony-stimulating factor (CSF) and interleukin 1 (IL-1) were examined. Multiporous DAC-80 microspheres of mean diameter 2.5 μm [MS-DAC-80(2.5)] enhanced the cytolytic activity of peritoneal macrophages and the production of CSF in vitro by macrophages, spleen cells and bone marrow cells, and in vivo. MS-DAC-80(2.5) also stimulated the production of IL-1 by both resident and thioglycolate-induced peritoneal macrophages. Multiporous chitin microspheres [MS-chitin(2.5)] showed no effect on the activation of peritoneal macrophages in vivo and on the production of IL-1 in vitro, but slightly enhanced the production of CSF in serum in vivo. © 1987.
• Interaction of chitin heparinoids with bovine antithrombin III

  Shin-ichiro Nishimura, Norio Nishi, Seiichi Tokura

  International Journal of Biological Macromolecules 9 5 305 - 307 1987年 

  The binding of chitin heparinoids to bovine antithrombin III was investigated by means of fluorescence spectroscopy and gel chromatography. An N,O-sulphated derivative of partially N-deacetylated O-(carboxymethyl)chitin showed a high affinity to antithrombin III (Kd = 3.5 × 10-7) m), which was comparable with that of heparin (Kd = 2.5 × 10-7 m). It was suggested that 1:1 complex (apparent molecular weight = 74 K) was enough to achieve the maximum activity of antithrombin III and that the additional binding of heparinoid to antithrombin III did not enhance the activity anymore. This observation seemed to explain the results of kinetic study on the anticoagulant activity. © 1987.
• Inhibition of the hydrolytic activity of thrombin by chitin heparinoids

  Shin-Ichiro Nishimura, Norio Nishi, Seiichi Tokura, Wesley Okiei, Oyin Somorin

  Carbohydrate Research 156 C 286 - 292 1986年11月15日
• Bioactive chitin derivatives. Activation of mouse-peritoneal macrophages by O-(carboxymethyl)chitins

  Shin-Ichiro Nishimura, Norio Nishi, Seiichi Tokura, Keiko Nishimura, Ichiro Azuma

  Carbohydrate Research 146 2 251 - 258 1986年02月01日 

  The effect of O-(carboxymethyl)chitins (CM-chitins) on the activation of mouse-peritoneal macrophages in vivo and their mitogenic activity on mouse spleen-cells were investigated. The induction of cytotoxic macrophages is enhanced by an increase of negative charge at O-6 and decreased by further modification at O-3 of the GlcNAc residue. CM-Chitins had a minor effect on mitogenic activity that was independent of the site of modification partially N-deacetylated chitins had little activity. Although there was remarkable enhancement of accessibility to lysozyme upon modification at O-6 of the GlcNAc residue, the accessibility was decreased by further substitution at O-3. © 1986.
• Highly phosphorylated derivatives of chitin, partially deacetylated chitin and chitosan as new functional polymers: preparation and characterization

  Norio Nishi, Akira Ebina, Shin-ichiro Nishimura, Akihiro Tsutsumi, Osatoshi Hasegawa, Seiichi Tokura

  International Journal of Biological Macromolecules 8 5 311 - 317 1986年 

  New functional chitin derivatives, i.e. variously phosphorylated derivatives of chitin and deacetylated chitins (DA-chitins), were prepared by the reaction of chitin or DA-chitins with phosphorus pentoxide in methanesulphonic acid. Degree of substitution (DS) of the products estimated from i.r. spectra, 13C n.m.r. spectra and elemental analyses increases with increase in the amount of phosphorus pentoxide employed, and products become nearly fully substituted when 4.0 mol. equiv. of phosphorus pentoxide to the residue is employed. 13C n.m.r. study suggested that the hydroxyl groups of C-3 and C-6 positions are equally substituted with the phosphate group. Chitin phosphates (P-chitins) are easily soluble in water independent of DS. However DA-chitin phosphates (P-DA-chitins) of high DS are insoluble in water owing to the amphoteric property, while those of low DS are water soluble. The water-soluble products behave as typical polyelectrolyte viscometrically. Two pKa values of the introduced phosphate groups were estimated to be around 3.7 and 8.2-8.5 by potentiometric titration. Preliminary experiment for adsorption of Cu2+ ion onto P-chitin (DS 1.0) by gel filtration showed that one copper ion was chelated to two phosphated N-acetyl-d-glucosamine residues, suggesting its high metal-binding ability. © 1986.
• Macrophage activation with multi‐porous beads prepared from partially deacetylated chitin

  Keiko Nishimura, Shin‐Ichiro Nishimura, Hiroshi Seo, Norio Nishi, Seiichi Tokura, Ichiro Azuma

  Journal of Biomedical Materials Research 20 9 1359 - 1372 1986年 

  The effect of multi‐porous beads prepared from 80% deacetylated chitin on the activation of mouse peritoneal macrophages was examined. Deacetylated chitin bead (DAC‐bead) preparations were shown to activate macrophages for tumoricidal activity depending on the increasing concentration of acetic acid used for the pretreatment of beads. The large DAC‐bead was more susceptible to treatment with acetic acid than small DAC‐bead, and showed more potent capacity for the activation of macrophages under the same pretreatment conditions with acetic acid. Reacetylated bead preparations, on the other hand, showed less activities. In addition, DAC‐bead pretreated with acetic acid stimulated macrophages to produce interleukin 1. The possibilities of multi‐porous beads as cancer chemotherapeutic‐carrier were examined by the method of column chromatography and of in vitro antitumor experiment. Forty‐four percent of adriamycin adsorbed on the surface of and in bead was released within the first 60 min. of elution, and then adriamycin was released more slowly in proportion to the elution time. Antitumor activity of adriamycin‐adsorbed bead was less effective than that of free adriamycin if they were compared on the basis of total content of adriamycin. Copyright © 1986 John Wiley & Sons, Inc.
• Adjuvant activity of chitin derivatives in mice and guinea-pigs

  Keiko Nishimura, Shin-ichiro Nishimura, Norio Nishi, Fumio Numata, Yukiko Tone, Seiichi Tokura, Ichiro Azuma

  Vaccine 3 5 379 - 384 1985年 

  Adjuvant activity of chitin derivatives was examined in guinea-pigs and mice. Among derivatives of chitin tested, 30 and 70% deacetylated chitin (DAC-30 and DAC-70), were active as adjuvants for the circulating-antibody formation to bacterial α-amylase and for the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-l-tyrosine. DAC-70 enhanced the helper T cell function, the generation of alloreactive cytotoxic T lymphocytes, and the activity of natural killer cells in mice, however, it was inactive as mitogen. Other derivatives of chitin showed weaker or no adjuvant activity compared with DAC-70. Carboxymethyl-, hydroxyethyl and dihydroxypropyl-chitin showed weak mitogenic activities on normal spleen cells. © 1985.
• Adhesion behaviour of murine lymphocytes on the surface of fibrous chitin and its derivatives

  Shin-ichiro Nishimura, Norio Nishi, Seiichi Tokura

  International Journal of Biological Macromolecules 7 2 100 - 104 1985年 

  Adhesion behaviour of lymphocytes of mouse spleen on the surfaces of chitin and its derivatives was studied. The amount of adhering B lymphocytes was enhanced by the deacetylation of N-acetyl groups and depressed by the introduction of carboxyl groups to the GlcNAc residues of chitin. B lymphocytes have been shown to adhere more effectively to the surface of chitin derivatives than IgG negative cells such as T lymphocytes. However, some morphological change in lymphocyte cells has been observed on adhesion to the basic surface of deacetylated chitin, in spite of little change on neutral or acidic surfaces of chitin derivatives. © 1985.
• Preparation and characterization of water-soluble chitin phosphate

  Norio Nishi, Shin-ichiro Nishimura, Akira Ebina, Akihiro Tsutsumi, Seiichi Tokura

  International Journal of Biological Macromolecules 6 1 53 - 54 1984年 

  New water-soluble chitin derivatives, chitin phosphate of various degrees of substitution, were successfully prepared by the reaction of chitin with phosphorus pentoxide in methanesulphonic acid. These materials behaved hydrodynamically as typical polyelectrolyte, and showed high ability to adsorb metal ions. © 1984.
• THE ADSORPTION OF BOVINE BLOOD PROTEINS ONTO THE SURFACE OF O-(CARBOXYMETHYL)CHITIN

  S NISHIMURA, Y IKEUCHI, S TOKURA

  CARBOHYDRATE RESEARCH 134 2 305 - 312 1984年 [査読有り][通常論文]
  
• Studies on Chitin. VII. Preparations of p-Substituted Benzoylchitins

  Norio Nishi, Hiroyuki Ohnuma, Shin-Ichiro Nishimura, Oyin Somorin, Seiichi Tokura

  Polymer Journal 14 11 913 - 919 1982年 [査読有り][通常論文]
  

MISC

• スパイクタンパク質の「動的エピトープ」は新型コロナウイルスの弱点？

  西村紳一郎, 涌井初, 横井康広 臨床化学 50 (4) 2021年 [査読無し][招待有り]
  
• 理学部で自由に研究できるという最高の幸運に感謝

  西村紳一郎 彩：理学部創立90周年記念誌 2020年 [査読無し][招待有り]
  
• 現場のアンメットニーズを考える

  西村紳一郎 臨床化学 47 342 -343 2018年07月 [査読無し][招待有り]
  
• 血清N-glycanの質量解析による抗体関連拒絶反応予知因子の探索

  野呂 大輔, 米山 徹, 畠山 真吾, 飛澤 悠葵, 盛 和行, 橋本 安弘, 古家 琢也, 田中 誠一, 西村 紳一郎, 佐々木 秀郎, 齋藤 満, 原田 浩, 力石 辰也, 石田 英樹, 田邉 一成, 佐藤 滋, 大山 力 日本臨床腎移植学会雑誌 5 (1) 71 -82 2017年07月 [査読無し][通常論文]
   

  【研究目的】血清N-glycanプロファイル解析による抗体関連拒絶(ABMR)予測の有用性について検討した。【方法】197症例の生体腎移植後1日目の血清10μLから質量解析によりN-glycanプロファイルを決定した。説明変数をABMRの有無、目的変数を36種のN-glycan、レシピエント性別、移植時年齢として判別分析し、得られた関数より作成したABMR予測式からN-glycan scoreを算出した。ROC曲線およびKaplan-Meier曲線によりABMR診断精度を評価した。【結果】ABMR予測に関するN-glycan scoreのAUCは、0.8916とDSA(0.7619)よりも有意に高く、陽性診断率81.25%、陰性診断率86.74%であった。N-glycan score陽性症例は、有意にABMR発症期間が短かった。【結論】N-glycan scoreは、ABMR予測に寄与することが示唆された。(著者抄録)
• NOTCH1EGFドメインにおけるO-結合型糖鎖修飾の構造および機能

  横井康広, 西村紳一郎 日本化学会春季年会講演予稿集(CD-ROM) 97th 2017年
• グライコブロッティング法に基づくリポソーム及びリポたんぱく質中酸化脂質の迅速解析

  古川貴之, 比能洋, 武田晴治, 千葉仁志, 西村紳一郎, HUI Shu-Ping JSBMS Letters 42 (Supplement) 2017年
• グライコブロッティング法によるリポタンパク質中の脂質アルデヒド分析

  古川貴之, 比能洋, 武田晴治, 千葉仁志, 西村紳一郎, 惠淑萍 臨床化学 46 2017年
• 網羅的糖鎖解析による肝細胞癌の浸潤能と糖鎖異常の検討

  高橋秀徳, 神山俊哉, 柿坂達彦, 相山健, 島田慎吾, 若山顕治, 永生高弘, 折茂達也, 蒲池浩文, 横尾英樹, 西村紳一郎, 武冨紹信 日本外科学会定期学術集会(Web) 117th ROMBUNNO.PS‐076‐4 (WEB ONLY) 2017年 [査読無し][通常論文]
  
• 脂質アルデヒド類の新規分析法 グライコブロッティング法によるアプローチ 合成標品からリポソームまで

  古川 貴之, 惠 淑萍, 比能 洋, 西村 紳一郎, 千葉 仁志 臨床化学 45 (Suppl.1) 167 -167 2016年10月 [査読無し][通常論文]
  
• 糖尿病網膜症の硝子体および血漿におけるN型糖鎖プロファイル

  稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋 日本糖尿病眼学会誌 20 114 -114 2016年03月 [査読無し][通常論文]
  
• 網羅的糖鎖解析による肝細胞癌の浸潤能と糖鎖異常の検討

  高橋秀徳, 神山俊哉, 柿坂達彦, 相山健, 島田慎吾, 永生高広, 若山顕治, 折茂達也, 蒲池浩文, 横尾英樹, 西村紳一郎, 武冨紹信 日本消化器癌発生学会総会プログラム・抄録集 27th 104 2016年 [査読無し][通常論文]
  
• 肝癌細胞株におけるuPAの発現変化に伴う浸潤能変化と糖鎖異常解析

  高橋秀徳, 柿坂達彦, 神山俊哉, 相山健, 島田慎吾, 若山顕治, 折茂達也, 敦賀陽介, 蒲池浩文, 横尾英樹, 西村紳一郎, 武冨紹信 日本外科学会定期学術集会(Web) 116th OP‐090‐1 (WEB ONLY) 2016年 [査読無し][通常論文]
  
• グライコブロッティング法を用いた脂質アルデヒドの定量測定法の開発

  古川貴之, 惠淑萍, 比能洋, 西村紳一郎, 千葉仁志 臨床化学 44 209 2015年10月07日 [査読無し][通常論文]
  
• Glycoblotting法を用いた牛乳中遊離オリゴ糖の網羅的定量解析

  山本 祐輔, 古川 貴之, 惠 淑萍, 比能 洋, 西村 紳一郎, 千葉 仁志 日本未病システム学会学術総会抄録集 22回 134 -134 2015年09月 [査読無し][通常論文]
  
• ブロッティング技術に基づく脂質アルデヒド類の網羅的定量解析

  古川 貴之, 惠 淑萍, 比能 洋, 西村 紳一郎, 千葉 仁志 日本未病システム学会学術総会抄録集 22回 136 -136 2015年09月 [査読無し][通常論文]
  
• 私の自慢 継続は力なり 点が線になるとき

  西村 紳一郎 化学と工業 = Chemistry & chemical industry 68 (8) 726 -728 2015年08月 [査読無し][通常論文]
  
• O-mannosylated glycan induced conformational alteration of alpha-dystroglycan fragment

  Hiroshi Hinou, Shin-Ichiro Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 249 2015年03月 [査読無し][通常論文]
  
• Structural insight into glycosylated human Notch1 EGF12 analogs

  Shun Hayakawa, Hiroshi Hinou, Shin-Ichiro Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 249 2015年03月 [査読無し][通常論文]
  
• Intracellular traffic of cell surface mimetic quantum dots-anchored glycopeptides

  Shin-Ichiro Nishimura, Roger Tan, Kentaro Naruchi, Maho Amano, Hiroshi Hinou ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 249 2015年03月 [査読無し][通常論文]
  
• 糖尿病網膜症の硝子体および血漿におけるN型糖鎖の網羅的解析

  稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 吉澤 史子, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋 日本眼科学会雑誌 119 (臨増) 160 -160 2015年03月 [査読無し][通常論文]
  
• 糖負荷刺激によるシアル酸転移酵素の誘導

  稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋 日本眼科学会雑誌 119 (臨増) 199 -199 2015年03月 [査読無し][通常論文]
  
• 糖尿病モデルマウスの比較糖鎖解析

  坂西裕介, 比能洋, 天野麻穂, 幸田敏明, 上村佳子, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 49th 2015年
• アルコールを用いたペプチド環化反応のpKa依存性について

  酒井隆史, 比能洋, 西村紳一郎 高分子学会予稿集(CD-ROM) 64 (1) 2015年
• タウタンパクフラグメントの合成および立体構造解析

  横井康広, 早川瞬, 比能洋, 西村紳一郎 高分子学会予稿集(CD-ROM) 64 (1) 2015年
• CMPアジド化シアル酸の合成と糖タンパク質末端の改変

  飯田優香, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 49th 2015年
• 糖鎖固定化電界効果トランジスタによるインフルエンザウイルスから分離したヘマグルチニンの検出

  南部谷俊介, 前川達洋, 秀島翔, 比能洋, 西村紳一郎, 迫田義博, 黒岩繁樹, 中西卓也, 逢坂哲彌, 逢坂哲彌 Chemical Sensors 31 (Supplement A) 2015年
• 糖鎖固定化電界効果トランジスタによるインフルエンザウイルスから分離したヘマグルチニンの検出

  南部谷俊介, 前川達洋, 秀島翔, 比能洋, 西村紳一郎, 迫田義博, 黒岩繁樹, 中西卓也, 逢坂哲彌 電気化学会大会講演要旨集(CD-ROM) 82nd 2015年
• 肝癌細胞株における浸潤能と糖鎖異常の解析

  高橋秀徳, 柿坂達彦, 神山俊哉, 相山健, 島田慎吾, 若山顕治, 折茂達也, 敦賀陽介, 蒲池浩文, 横尾英樹, 田中誠一, 西村紳一郎, 武冨紹信 日本癌治療学会学術集会(Web) 53rd P131‐1 (WEB ONLY) 2015年 [査読無し][通常論文]
  
• 肝疾患の病態と糖鎖抗原の意義 網羅的糖鎖解析による肝細胞癌バイオマーカーの検討

  宮原 孝治, 能祖 一裕, 森元 裕貴, 衣笠 秀明, 和田 望, 竹内 康人, 安中 哲也, 桑木 健志, 大西 秀樹, 池田 房雄, 三宅 康弘, 中村 進一郎, 白羽 英則, 高木 章乃夫, 天野 麻穂, 西村 紳一郎, 山本 和秀 肝臓 55 (Suppl.1) A154 -A154 2014年04月 [査読無し][通常論文]
  
• 量子ドットに提示したCMPシアル酸のライブセルイメージング

  平根望巳, 比能洋, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 48th 2014年
• O-Mannose型糖鎖を有する糖ペプチドライブラリの構築及び機能解析に関する研究

  菊地星矢, 松下隆彦, 比能洋, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 48th 2014年
• ヒトIgG1抗体に存在するBisect型N-Glycanの効率合成と糖ペプチド合成への応用

  石川明香里, RAVI KUMAR H.V., 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 48th 2014年
• 糖タンパク質の糖鎖を付け替える 均一な糖鎖構造をもつ糖タンパク質の効率的合成

  比能洋, 西村紳一郎 化学 69 (1) 2014年
• Prediction of Survival in Patients with Hepatocellular Carcinoma Treated with Sorafenib by Comprehensive Serum Glycan Analysis

  Kazuhiro Nouso, Koji Miyahara, Yuuki Morimoto, Yasuto Takeuchi, Hiroaki Hagihara, Kenji Kuwaki, Hideki Onishi, Fusao Ikeda, Yasuhiro Miyake, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Koichi Takaguchi, Takahisa Sato, Shinpei Sato, Shuntaro Obi, Kazuko Hirose, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto HEPATOLOGY 58 1242A -1242A 2013年10月 [査読無し][通常論文]
  
• Highly efficient synthesis of hyperbranched N-glycans from locust bean gum

  H. V. Ravi Kuma, Kentaro Naruchi, Risho Miyoshi, Hiroshi Hinou, Shin-Ichiro Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 246 2013年09月 [査読無し][通常論文]
  
• 血清糖鎖マーカーによる進行肝細胞癌の予後予測

  宮原 孝治, 能祖 一裕, 森元 裕貴, 和田 望, 竹内 康人, 萩原 宏明, 桑木 健志, 大西 秀樹, 中村 進一郎, 白羽 英則, 天野 麻穂, 西村 紳一郎, 山本 和秀 肝臓 54 (Suppl.2) A638 -A638 2013年09月 [査読無し][通常論文]
  
• High-Throughput Antigen Profiling for MUC1 Glycoprotein Antibody

  F. Garcia-Martin, T. Matsushita, H. Hinou, S-I Nishimura BIOPOLYMERS 100 (3) 269 -269 2013年05月 [査読無し][通常論文]
  
• ABERRANT GLYCOSYLATION OF N-GLYCAN ON IMMUNOGLOBULIN CLOSELY RELATES SURVIVAL OF THE PATIENTS WITH IN RENAL CELL CARCINOMA

  Shingo Hatakeyama, Yuki Tobisawa, Tohru Yoneyama, Yuichiro Suzuki, Hayato Yamamoto, Kazuyuki Mori, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Norihiko Tsuchiya, Tomonori Habuchi, Maho Amano, Shin-Ichiro Nishimura, Chikara Ohyama JOURNAL OF UROLOGY 189 (4) E186 -E187 2013年04月 [査読無し][通常論文]
  
• ぶどう膜炎における網羅的糖鎖解析

  南場 研一, 北市 伸義, 安藤 亮, 竹本 裕子, 水内 一臣, 堀江 幸弘, 大野 重昭, 天野 麻穂, 西村 紳一郎, 石田 晋 日本眼科学会雑誌 117 (臨増) 318 -318 2013年03月 [査読無し][通常論文]
  
• ケイ素保護基を用いた糖ペプチドの迅速固相合成法

  藤井宏圭, 松下隆彦, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 47th 2013年
• 糖残基を有するヒトNotch-1EGF-12ドメインの合成と配座解析

  脇本行彦, 松下隆彦, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 47th 2013年
• O-Mannose型糖鎖を有するαジストログリカン糖ペプチドライブラリの合成と相互作用解析

  菊地星矢, 比能洋, 西村紳一郎 日本化学会講演予稿集 93rd (3) 2013年
• O-マンノース含有ジストログリカン関連糖ペプチドの合成と相互作用解析

  比能洋, 比能洋, 菊地星矢, 松下隆彦, 西村紳一郎, 西村紳一郎 高分子学会予稿集(CD-ROM) 62 (1) 2013年
• ムチン型糖タンパク質におけるTTXモチーフのNMR構造解析

  早川瞬, 松下隆彦, GARCIA-MARTIN Fayna, 成地健太郎, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 47th 2013年
• O-Mannose型糖鎖を有するαジストログリカン糖ペプチドライブラリの合成と相互作用解析

  菊地星矢, 比能洋, 比能洋, 松下隆彦, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 47th 2013年
• Autoantibodies to cancer-associated MUC1 fragments in healthy human sera discovered by high performance glycopeptide microarray platform

  Nishimura S. I, Matsushita T, Takada W, Igarashi K, Naruchi K, Garcia-Martin F, Amano M, Hinou H Abstracts of Papers of the American Chemical Society 246 2013年 [査読有り][通常論文]
  
• Serum N-glycan profiling has potential to predict cardiovascular disease (CVD) event and prognosis in hemodialysis (HD) patients

  Shingo Hatakeyama, Yuki Tobisawa, Tohru Yoneyama, Maho Amano, Shin-Ichiro Nishimura, Chikara Ohyama GLYCOBIOLOGY 22 (11) 1558 -1559 2012年11月 [査読無し][通常論文]
  
• 糖鎖化学 それぞれの大志を抱け! : 留学先での新渡戸稲造博士との出会い (特集 日本の化学 : その元気度は?) -- (各分野の元気度は?)

  西村 紳一郎 化学 67 (4) 25 -27 2012年04月 [査読無し][通常論文]
  
• グライコブロッティング法を用いた異なる種類のヒアルロン酸分解酵素生成ヒアルロン酸断片の同定

  荒井みさき, 古川貴之, ガルシア ファイナ, 比能洋, 天野麻穂, 西村紳一郎 日本薬学会年会要旨集 132nd (3) 185 2012年03月05日 [査読無し][通常論文]
  
• ¹C₄配座を有する新奇グルクロン酸糖供与体に関する研究

  古川貴之, 比能洋, 西村紳一郎 日本薬学会年会要旨集 132nd (2) 238 2012年03月05日 [査読無し][通常論文]
  
• Rapid Discovery of MUC1 Epitopes by Glycopeptide Combinatorial Chemistry

  GARCIA-MARTIN Fayna, MATSUSHITA Takahiko, HINOU Hiroshi, NISHIMURA Shin-Ichiro Peptide science : proceedings of the ... Japanese Peptide Symposium 2011 67 -68 2012年03月01日
• 糖鎖捕捉反応を用いた新規糖脂質ライブラリの構築と溶液中における構造解析

  石田純也, 比能洋, 西村紳一郎 日本薬学会年会要旨集 132nd (2) 2012年
• ムチン型糖ペプチドにおけるTTXモチーフのNMR構造解析

  早川瞬, 松下隆彦, 比能洋, 西村紳一郎 日本薬学会年会要旨集 132nd (2) 2012年
• Exploration of Disease-Specific Epitopes by using MUC1 Glycopeptide Microarray

  Wataru Takada, Takahiko Matsushita, Hiroshi Hinou, Shin-Ichiro Nishimura GLYCOBIOLOGY 21 (11) 1509 -1510 2011年11月 [査読無し][通常論文]
  
• Development and Application of Versatile Glycan Microarray

  Kohta Igarashi, Wataru Takada, Hiroki Abe, Takahiko Matsushita, Shin-Ichiro Nishimura GLYCOBIOLOGY 21 (11) 1510 -1510 2011年11月 [査読無し][通常論文]
  
• Glycoblotting 法による健常成人血清中の糖鎖プロファイル解析

  木村 聡, 天野 麻穂, 西村 紳一郎 臨床病理 59 (6) 2011年06月25日 [査読無し][通常論文]
  
• HIGH THROUGH PUT, COMPREHENSIVE AND QUANTITATIVE GLYCOMICS OF WHOLE SERUM GLYCOPROTEINS FOR DIAGNOSIS OF RENAL CELL CARCINOMA

  Shingo Hatakeyama, Maho Amano, Megumi Tsushima, Hatato Yamamoto, Tohru Yoneyama, Yusuke Ishibashi, Hiromi Murasawa, Kengo Imanishi, Teppei Okamoto, Noriko Tokui, Yuichiro Suzuki, Naoki Sugiyama, Sigemasa Kudo, Takahiro Yoneyama, Takuya Koie, Norihiko Tsuchiya, Tomonori Habuchi, Shin-Ichiro Nishimura, Chikara Ohyama JOURNAL OF UROLOGY 185 (4) E385 -E385 2011年04月 [査読無し][通常論文]
  
• 配座変換を鍵戦略とした新規グルクロン酸及びイズロン酸糖供与体の開発

  古川貴之, 比能洋, 西村紳一郎 日本化学会講演予稿集 91st (4) 1146 2011年03月11日 [査読無し][通常論文]
  
• Novel Coupling Method for the Incorporation of 'Complex' Amino acid

  GARCIA-MARTIN Fayna, HINOU Hiroshi, MATSUSHITA Takahiko, NISHIMURA Shin-Ichiro Peptide science : proceedings of the ... Japanese Peptide Symposium 2010 204 -204 2011年03月01日
• グライコブロッティング法が明らかにする翻訳後修飾の意義と大規模糖鎖解析への展開

  天野 麻穂, 三浦 嘉晃, 西村 紳一郎 生化學 83 (1) 5 -12 2011年01月25日
• 不凍糖タンパク質の構造と機能の解明

  八須匡和, 西村紳一郎 中部化学関係学協会支部連合秋季大会講演予稿集 42nd 2011年
• APP-013 腎細胞癌における血清N-結合型糖鎖の網羅的解析による新規バイオマーカーの検索(総会賞応募ポスター,第99回日本泌尿器科学会総会)

  畠山 真吾, 舟生 富壽, 天野 麻穂, 西村 紳一郎, 大山 力, 山本 隼人, 米山 徹, 盛 和行, 工藤 茂将, 米山 高弘, 古家 琢也, 神村 典孝, 對馬 惠 日泌尿会誌 102 (2) 2011年 [査読無し][通常論文]
  
• プラスチド局在糖タンパク質NPPのグライコミクス解析

  金古 堅太郎, 甲州 努, 梅澤 幸歩, 石本 卓也, 天野 麻穂, 西村 紳一郎, 三ツ井 敏明 日本プロテオーム学会大会要旨集 2011 (0) 139 -139 2011年 [査読無し][通常論文]
  
• イネプラスチド局在型ヌクレオチドピロホスファターゼ／ホスホジエステラーゼのN-グライコーム解析：機能糖タンパク質がゴルジ装置の後期区画からプラスチドに輸送される

  三ツ井 敏明, ポズエタ ロメロハビア, 金古 堅太郎, 甲州 努, 梅澤 幸歩, 古賀 彩, 白矢 武士, 広瀬 和子, 天野 麻穂, 西村 紳一郎 日本プロテオーム学会大会要旨集 2011 (0) 65 -65 2011年 [査読無し][通常論文]
  
• 新規糖脂質ライブラリの構築と構造解析

  石田純也, 比能洋, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 45th 2011年
• シアリダーゼ阻害剤の探索技術に関する研究

  三好利尚, 甲斐宏一, 高須康明, 比能洋, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 45th 2011年
• 新規糖脂質類縁体ライブラリの構築と構造解析

  石田純也, 比能洋, 西村紳一郎 日本化学会講演予稿集 91st (3) 2011年
• 環状構造を有する新奇シアリダーゼ自殺基質型阻害剤の開発

  甲斐宏一, 比能洋, 西村紳一郎 日本化学会講演予稿集 91st (3) 2011年
• 疾患特異的エピトープ探索を指向したMUC1糖ペプチドマイクロアレイの開発

  松下隆彦, 高田渉, 比能洋, 西村紳一郎 日本糖質学会年会要旨集 30th 2011年
• バイオサイエンス グライコブロッティング法と糖鎖自動抽出装置(SweetBlot)による一滴の血清からの糖鎖バイオマーカー探索

  古川 潤一, 西村 紳一郎 未来材料 10 (11) 48 -54 2010年11月 [査読無し][通常論文]
  
• Systematic Determination of Multiple O-Glycosylation Sites in Proteins using Electron Capture Dissociation (ECD) in a Linear Ion Trap

  Takeshi Sakamoto, Naomi Manri, Akihito Kaneko, Atsumu Hirabayashi, Takashi Baba, Shin-Ichiro Nishimura GLYCOBIOLOGY 20 (11) 1467 -1468 2010年11月 [査読無し][通常論文]
  
• Serum N-Glycan Profile in Renal Cell Carcinoma

  Shingo Hatakeyama, Maho Amano, Naoki Fujita, Noriko Tokui, Toru Yoneyama, Kengo Imanishi, Hayato Yamamoto, Shigemasa Kudo, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Noritaka Kamimura, Shin-Ichiro Nishimura, Chikara Ohyama GLYCOBIOLOGY 20 (11) 1501 -1501 2010年11月 [査読無し][通常論文]
  
• 高反応性かつ高立体選択性を有する新規グルクロン酸供与体の開発

  古川貴之, 比能洋, 西村紳一郎 日本化学会講演予稿集 90th (4) 1027 2010年03月12日 [査読無し][通常論文]
  
• Chemo-enzymatic synthesis of mucin-type glycopeptides using sugar-protective groups and ppGalNAcT-2

  Yayoi Yoshimura, Takahiko Matsushita, Naoki Fujitani, Fujihira Haruhiko, Yasuhiro Takegawa, Naomi Manri, Akihito Kaneko, Takeshi Sakamoto, Xiao-Dong Gao, Hiroshi Hinou, Shin-Ichiro Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 239 2010年03月 [査読無し][通常論文]
  
• 化学-酵素法による複合糖質の自動合成

  八須匡和, 松下隆彦, 清水弘樹, 長島生, 作田智美, 畑中研一, 西村紳一郎, 西村紳一郎 日本化学会講演予稿集 90th (3) 2010年
• 癌特異的糖ペプチド抗原の探索と疾患マーカーの開発－Anti-KL-6 抗体のエピトープマッピングから学んだこと－

  西村 紳一郎 日本分子腫瘍マーカー研究会誌 25 3 -4 2010年 [査読無し][通常論文]
  
• 新奇シアリダーゼ自殺基質型阻害剤の開発

  甲斐宏一, 比能洋, 西村紳一郎 日本化学会講演予稿集 90th (3) 2010年
• Synthesis, Folding and Structural Insights Into Glycosylated EGF12 on Mouse Notch-1 Receptor

  Kensaku Hosoguchi, Kazumi Shimizu, Naoki Fujitani, Shin-Ichiro Nishimura GLYCOBIOLOGY 19 (11) 1350 -1350 2009年11月 [査読無し][通常論文]
  
• Large-Scale Serum Glycomics of CFG KO Mice Based on the Glycoblotting Method

  Maho Amano, Shin-Ichiro Nishimura GLYCOBIOLOGY 19 (11) 1303 -1303 2009年11月 [査読無し][通常論文]
  
• Glycoform-Focused Reverse Genomics (GFRG) in Mouse Ovulation

  Maho Amano, Mari Yokoyama, Masaki Kurogochi, Yasuhiro Takegawa, Noriko Nagahori, Tadashi Yamashita, Shin-Ichiro Nishimura GLYCOBIOLOGY 19 (11) 1340 -1340 2009年11月 [査読無し][通常論文]
  
• NMRによるリニア不凍糖タンパク質(AFGP)のコンフォメーション解析

  泉龍孝, 藤谷直樹, 松下隆彦, 比能洋, 高道学, 西宮佳志, 津田栄, 西村紳一郎 日本糖質学会年会要旨集 29th 85 2009年08月10日 [査読無し][通常論文]
  
• 【ますます重要になる細胞周辺環境(細胞ニッチ)の最新科学技術 細胞の生存、増殖、機能のコントロールから創薬研究、再生医療まで】細胞周辺環境のための材料科学技術 高分子 糖鎖による軟骨細胞機能制御の可能性 軟骨変性に伴うN-結合型糖鎖の変化より

  岩崎 倫政, 松橋 智弥, 瓜田 淳, 高畑 雅彦, 三浪 明男, 西村 紳一郎 遺伝子医学MOOK 別冊 (ますます重要になる細胞周辺環境(細胞ニッチ)の最新科学技術) 55 -59 2009年08月 [査読無し][通常論文]
   

  軟骨組織変性を基本病態とする変形性関節症の発症早期よりN-結合型糖鎖に変化が生じた。この変化は,本疾患の発症病因である軟骨細胞のアポトーシスや細胞外マトリクス分解酵素の分泌に関与していると考えられる。更なる研究により,糖鎖による軟骨細胞の機能制御や関節疾患への関与などが明らかになっていくものと考えられる。これらが明らかになると,糖鎖生物学および糖鎖工学的アプローチによる新たな治療法の開発や,再生医療用マテリアルの開発が可能になるであろう。(著者抄録)
• BIO R&D 糖鎖チップ・マイクロアレイの開発と応用展開--疾患マーカー探索へのアプローチ

  五十嵐 幸太, 松下 隆彦, 西村 紳一郎 バイオインダストリー 26 (7) 63 -70 2009年07月 [査読無し][通常論文]
  
• “Chemoenzymatic Synthesis of Glycosylated Glucagon-like Peptide 1: Effect of Glycosylation on Proteolytic Resistance and in vivo Blood Glucose- lowering Activity”

  UEDA Taichi, TOMITA Kazuyoshi, NOTSU Yoshihide, ITO Takaomi, ITO Takaomi, FUMOTO Masataka, TAKAKURA Tomoaki, NAGATOME Hirofumi, TAKIMOTO Akio, MIHARA Shin‐Ichi, TOGAME Hiroko, KAWAMOTO Keiko, IWASAKI Takanori, ASAKURA Kenji, OSHIMA Takeo, HANASAKI Kohji, NISHIMURA Shin‐Ichiro, KONDO Hirosato J. Am. Chem. Soc 131 (17) 6237 -6245 2009年05月 [査読無し][通常論文]
   

  Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogues with N-acetylglucosamine (GIcNAc), N-acetyllactosamine (LacNAc), and alpha 2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepared by chemoenzymatic approaches. We assessed the receptor binding affinity and CAMP production activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addition of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic analysis of the degradation reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addition, the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogues with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogues of other bioactive peptides as desirable pharmaceuticals.
• 新規自殺基質を用いた蛋白質のラベル化

  古川貴之, 比能洋, FENG Fei, 西村紳一郎 日本化学会講演予稿集 89th (2) 1529 2009年03月13日 [査読無し][通常論文]
  
• Notch‐1受容体EGF12のフォールディング構造における糖修飾の影響について

  細口健作, 蛭間(清水)和美, 藤谷直樹, 比能洋, 西村紳一郎 日本化学会講演予稿集 89th (2) 1314 2009年03月13日 [査読無し][通常論文]
  
• ペプチド型プローブの迅速探索法

  古川貴之, 比能洋, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 43rd 51 2009年02月03日 [査読無し][通常論文]
  
• ppGalNAcT阻害剤に関する研究

  阿部皓基, 比能洋, 比能洋, 古川潤一, 前田哲宏, 細口健作, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 43rd 2009年
• ヒトシアリダーゼの発現と機能解析

  奥山力, 高暁冬, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 43rd 2009年
• 環境調和型グリコシル化反応~固体超強酸プロモーターの改質~

  齋藤直裕, 前田哲宏, 松田匡雄, 比能洋, 西村紳一郎 日本化学会講演予稿集 89th (1) 2009年
• 新規骨格をベースとしたシアリダーゼ阻害剤の開発研究

  佐藤公法, 比能洋, 高須康明, 黒河内政樹, 西村紳一郎 日本化学会講演予稿集 89th (2) 2009年
• ppGalNAcT阻害剤に関する研究

  阿部皓基, 比能洋, 古川潤一, 前田哲宏, 細口健作, 西村紳一郎 日本化学会講演予稿集 89th (2) 2009年
• グライコミミックを用いた糖鎖生合成機構の制御

  日向寺慧, 羽藤愛美, 天野麻穂, FENG Fei, 三浦嘉晃, 篠原康郎, 比能洋, 西村紳一郎 日本化学会講演予稿集 89th (2) 2009年
• ケイ素保護基を用いた糖ペプチドの固相合成研究

  川崎慶, 嶋脇健, 松下隆彦, 比能洋, 西村紳一郎 日本化学会講演予稿集 89th (2) 2009年
• 糖ペプチドチップを用いた糖ペプチド-タンパク質間相互作用の解析

  松下隆彦, 越智里香, 五十嵐幸太, 比能洋, 西村紳一郎 繊維学会予稿集 64 (1) 2009年
• α-ジストログリカン糖ペプチドの効率的合成および機能解析

  越智里香, 比能洋, 五十嵐幸太, 松下隆彦, 西村紳一郎 日本薬学会年会要旨集 129th (2) 2009年
• ケイ素保護体アミノ酸ビルディングブロックを用いた糖ペプチド固相合成

  川崎慶, 嶋脇健, 比能洋, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 43rd 2009年
• O-マンノース型糖ペプチドの効率的合成および機能解析

  越智里香, 比能洋, 五十嵐幸太, 松下隆彦, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 43rd 2009年
• 環状不凍糖タンパク質の合成および不凍活性

  八須匡和, 高道学, 比能洋, 清水弘樹, 津田栄, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 43rd 2009年
• 癌関連糖ペプチドライブラリーの構築と抗体エピトープマッピングへの応用

  松下隆彦, 比能洋, 泉龍孝, 西村紳一郎, 大藪巨樹, 川本敬子, 沼田義人, 十亀弘子, 武本浩, 近藤裕郷 メディシナルケミストリーシンポジウム講演要旨集 28th 2009年
• 新規不凍糖タンパク質誘導体の合成と性質

  八須匡和, 比能洋, 高道学, 三浦信明, 清水弘樹, 津田栄, 西村紳一郎, 西村紳一郎 日本糖質学会年会要旨集 29th 2009年
• “Mechanism-Based Probing, Characterization, and Inhibitor Design of Glycosidases and Glycosyltransferases”

  Hiroshi Hinou, Shin-Ichiro Nishimura Curr. Top. Med. Chem 9 (1) 106 -116 2009年01月 [査読無し][通常論文]
   

  Recent structural and kinetic studies indicated that enzymes shift their peripheral structures of the catalytic sites dynamically to modify the substrate structures. Molecules which disturb such mechanism of specific enzymes may become potent candidates for therapeutic reagent. This article describes a versatile strategy to synthesize new class of mechanism-based inhibitors for glycosyltransferases and glycoside hydrolases. Combination of irreversible tagging and proteomic analysis of crucial amino acid residues using MALDI-TOF/TOF mass spectrometry allowed a promising method to probe such invisible transitional state in the enzymatic reactions. Feasibility of the fluorescence energy resonance transfer (FRET) is also documented as novel protocol for the real-time and continuous monitoring of glycosyltransferase catalyzed reactions. It was demonstrated that FRET method greatly facilitates discovery research of selective inhibitors in combination with click chemistry.
• WS-3-6 血清中糖鎖の網羅的解析による肝細胞癌新規バイオマーカーの開発(第108回日本外科学会定期学術集会)

  横尾 英樹, 三浦 嘉晃, 神山 俊哉, 中西 一彰, 濱口 純, 工藤 岳秋, 古川 潤一, 中川 隆公, 田原 宗徳, 中馬 誠, 髭 修平, 篠原 康郎, 尾崎 倫孝, 多田 光宏, 松下 通明, 浅香 正博, 西村 紳一郎, 藤堂 省 日本外科学会雑誌 109 (2) 2008年04月25日 [査読無し][通常論文]
  
• P-92 マイクロ波を活用した糖鎖・糖ペプチド合成研究(ポスター発表の部)

  清水 弘樹, 松下 隆彦, 長島 生, 吉村 弥生, 清水 和美, 西村 紳一郎 天然有機化合物討論会講演要旨集 50 (0) 813 -818 2008年 [査読無し][通常論文]
   

  Although microwave irradiation has been used in chemical reactions as an efficient heating tool since 1986, we have found that microwave also contribute other factors while studying carbohydrate-related syntheses. 1) With microwave irradiation under Lewis acid conditions, methyl glycosides could behave as glycosyl donors. 2) In a case of synthesis Lewis X, microwave irradiation even at low temperatures was very effective as reducing byproducts and side-reactions. Especially, galactosylation for 4-OH in a fucosyl glucosamine acceptor has proceeded in high yield without producing byproducts, e.g., cleavage of the fucosyl bond or nucleophilic rearrangement of methylthio group. 3) Based on a preliminary study of microwave effect for coupling Fmoc-threonine having acetylated-Core2 to alanine onto supported resin, we successfully synthesized oligopeptide which consisted of twenty of amino acid residues with five of trisaccharides, a partial structure of MUC1 glycoprotein, in shorter time and higher yields. In addition, we also revealed that benzyl protection of glutamic acid was not suitable for basic microwave condition. 4) Microwave could activate azido groups and resulted to accelerate reductions by PPh_3 and Staudinger Ligations.
• シアリダーゼ自殺基質におけるアグリコンフォーカスドライブラリーの作製と評価

  甲斐宏一, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 42nd 2008年
• 多糖類誘導体の整形外科領域での高次利用

  比能洋, 西村紳一郎, 岩崎倫政, 三浪明男 繊維学会予稿集 63 (1/2) 2008年
• 酵素の動的構造に着目したシアリダーゼ阻害剤の開発研究

  高須康明, 黒河内政樹, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 42nd 2008年
• フォーカスドライブラリを用いた特異的シアル酸転移酵素阻害剤の探索

  細口健作, 古川潤一, 前田哲宏, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 42nd 2008年
• 大規模高速糖鎖構造解析法の開発と応用

  天野麻穂, 比能洋, 古川潤一, 篠原康郎, 西村紳一郎 日本化学会講演予稿集 88th (2) 2008年
• O-マンノース型糖ペプチドの効率的合成および機能解析に関する研究

  越智里香, 比能洋, 五十嵐幸太, 西村紳一郎 日本化学会講演予稿集 88th (2) 2008年
• BlotGlyco (R) and glycoblotting for large scale, high throughput glycomics

  Yoshiaki Miura, Shin-Ichiro Nishimura TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY 20 (111) 17 -27 2008年01月 [査読無し][通常論文]
   

  Expression patterns of glycoforms are known to be dependent on the various individual states, such as development, ageing, and disease. Transcriptomics and proteomics have been generally standardized to analyze the expression of certain DNAs and proteins, facilitating potential applications for diagnosis and disease treatment. However, no such convenient methods capable of analyzing glycomics are available currently. Glycobiology is one of the most important subjects in post-genomic era, and methods for glycan analyses have been steadily improved by advancement in mass spectrometry (MS). Nevertheless, a practical procedure for large number of glycan samples that fulfills the requirements of the high speed and high yield was unavailable, thus, limiting development of useful glycomics of certain biological materials, e.g. serum and tissue biopsy. To alleviate this problem, it is highly desirable to develop a standardized methodology to achieve high-speed quantitative and qualitative profiling of glycan expression patterns in the biological materials. Recently, a chemoselective glycan enrichment technology, termed glycoblotting, has been developed to purify oligosaccharides derived from glycoproteins in highly effective quantitative manner, enabling profiling of serum glycans in a simple manner. This method is also applicable to automated analysis of multiple samples simultaneously. It flawlessly combines isolation and labeling of oligosaccharides suited for conventional analytical methods including MS analysis. We believe that the glycoblotting technique and the beads specifically designed for it (BlotGlyco) is an innovative approach in the "real world". glycan preparation. Glycoblotting and its related technologies would make a great contribution not only to the glycobiology community, but also for glycan-related biomarker search as well as for glycan-related drug development.
• マイクロ波を利用した糖鎖・糖ペプチド精密合成

  清水 弘樹, 松下 隆彦, 西村 紳一郎 高分子論文集 64 (12) 883 -896 2007年12月25日 [査読無し][通常論文]
  
• 糖鎖ケミカルバイオロジー (ケミカルバイオロジー) -- (生理活性物質のケミカルバイオロジー 化学と生物の接点)

  長堀 紀子, 西村 紳一郎 蛋白質核酸酵素 52 (13) 1742 -1750 2007年10月 [査読無し][通常論文]
  
• TAK1 represses transcription of the human telomerase reverse transcriptase gene

  T. Fujiki, T. Miura, M. Maura, H. Shiraishi, S. Nishimura, Y. Imada, N. Uehara, K. Tashiro, S. Shirahata, Y. Katakura ONCOGENE 26 (36) 5258 -5266 2007年08月 [査読無し][通常論文]
   

  In human cells, telomerase activity is tightly regulated by the expression of its catalytic subunit, namely, the human telomerase reverse transcriptase ( hTERT). However, the molecular mechanisms involved in the regulation of hTERT expression have not been completely clarified. We have previously reported that transforming growth factor beta (TGF-beta) represses the expression of the hTERT gene. In the present study, we demonstrated that TGF-beta-activated kinase 1 (TAK1), originally identified as a mitogen-activated kinase kinase kinase, represses the hTERT core promoter activity in an E-box-independent manner, and it also represses the transcription of the hTERT gene in human lung adenocarcinoma cell line, A549 cells. This TAK1-induced repression was found to be caused by the recruitment of histone deacetylase to Sp1 at the hTERT promoter and a consequent reduction in the amount of acetylated histone H4 at the hTERT promoter. Finally, we demonstrated that TAK1 induces cellular senescence programs in normal human diploid cells. Thus, we assume that TAK1 triggers the repression mechanisms of the hTERT gene as a result of evoking cellular senescence programs. Considered together, TAK1 is thought to play a causative role in the determination of a finite replicative lifespan of normal and cancer cells.
• 糖鎖研究からバイオ産業育成への挑戦

  西村 紳一郎 Journal of applied glycoscience 54 2007年07月20日 [査読無し][通常論文]
  
• 高速化が進む 糖鎖解析--糖鎖構造研究を加速する新しい解析法:グライコブロッティング法

  天野 麻穂, 西村 紳一郎 現代化学 (435) 55 -61 2007年06月 [査読無し][通常論文]
  
• 腫瘍マーカーに対する糖鎖修飾が誘導する立体構造変化

  藤谷直樹, 黒河内政樹, GAO Xiao-Dong, 松下隆彦, 比能洋, 篠原康郎, 西村紳一郎 Abstracts. Annual Meeting of the NMR Society of Japan 46th 2007年
• ポリラクトサミン含有複合糖質のライブラリー構築と構造解析

  成地健太郎, 浜本智樹, 黒河内政樹, 松下隆彦, 藤谷直樹, 比能洋, 比能洋, 西村紳一郎, 西村紳一郎 高分子学会北海道支部研究発表会講演要旨集 41st 2007年
• 平成18年度第37回繊維学会夏季セミナー

  西村 紳一郎 繊維学会誌 62 (12) "P -366"-"P-370" 2006年12月10日 [査読無し][通常論文]
  
• Construction of tumor - Related gkycpeptided library

  Shin Ichiro Nishimura, Hiroshi Hinou, Hiroki Shimizu, Takahiko Matsushita, Naoki Oyabu, Masataka Fumoto, Hirosato Kondo Polymer Preprints, Japan 55 5508 2006年12月01日 [査読無し][通常論文]
   

  Combined use of a solid-phase chemical synthesis of peptide and enzymatic synthesis of carbohydrate on water soluble polymer allowed for the construction of complicated glycopeptide library which contributes to functional analysis of the glycopeptides. This study was supported by NEDO.
• Identification of N-glycans related with cartilage deterioration

  Tomoya Matsuhashi, Norimasa Iwasaki, Hiroaki Nakagawa, Megumi Hato, Masaki Kurogochi, Kisaburo Deguchi, Tokifumi Majima, Akio Minami, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1152 -1153 2006年11月 [査読無し][通常論文]
  
• A glycomic approach to drug resistance: Direct interaction between Cisplatin and N-glycans

  Hiroaki Nakagawa, Shinji Hayashi, Shigeaki Abe, Noriko Nagahori, Kenji Monde, Miki Ichikawa-Ohira, Hirosato Kondo, Shin-Ichi Akiyama, Kisaburo Deguchi, Akira Nakagawara, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1150 -1150 2006年11月 [査読無し][通常論文]
  
• All-in-one processing of oligosaccharides on solid-support (A1POS); A general protocol with Glycoblotting for functional glycomics (part 1)

  Yoshiaki Miura, Jun-ichi Furukawa, Yasuro Shinohara, Hiromitsu Kuramoto, Masaki Kurogochi, Hideyuki Shimaoka, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1135 -1135 2006年11月 [査読無し][通常論文]
  
• An efficient, rapid, differential N-glycan profiling from mouse tissues

  Masaki Kurogochi, Yasuro Shinohara, Yoshiaki Miura, Jun-Ichi Furukawa, Hideyuki Shimaoka, Hiromitsu Kuramoto, Yoko Kita, Mika Nakano, Hiroki Ito, Hiroaki Nakagawa, Kisaburo Deguchi, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1137 -1137 2006年11月 [査読無し][通常論文]
  
• Alterations in N-glycans seen in drug-resistant human hepatocellular carcinoma

  Takeaki Kudo, Hiroaki Nakagawa, Masato Takahashi, Jun Hamaguchi, Naoya Kamiyama, Hideki Yokoo, Kazuaki Nakanishi, Takahito Nakagawa, Toshiya Kamiyama, Kisaburo Deguchi, Satoru Todo, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1151 -1151 2006年11月 [査読無し][通常論文]
  
• Increased alpha 1,6-fucosylation of N-glycan in serum glycoprotein of db/db mice

  Naofumi Itoh, Shinji Sakaue, Hiroaki Nakagawa, Masaki Kurogochi, Kisaburo Deguchi, Shin-Ichiro Nishimura, Masaharu Nishimura GLYCOBIOLOGY 16 (11) 1152 -1152 2006年11月 [査読無し][通常論文]
  
• Combined use of hydrazide functionalized polymer and sequential tag exchange; A general protocol with Glycoblotting for functional glycomics (part 2)

  Jun-ichi Furukawa, Yoshiaki Miura, Hiromitsu Kuramoto, Hideyuki Shimaoka, Masaki Kurogochi, Mika Nakano, Yasuro Shinohara, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1139 -1139 2006年11月 [査読無し][通常論文]
  
• Unusual N-glycans from alpha-mannosidase II/IIx double knockout mice identified by a systematic glycomic approach using MDSF method in MALDI-TOF/TOF-MS

  Megumi Hato, Hiroaki Nakagawa, Masaki Kurogochi, Kisaburo Deguchi, Tomoya O. Akama, Jamey D. Marth, Michiko N. Fukuda, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1124 -1124 2006年11月 [査読無し][通常論文]
  
• GnT-V expression correlates with patient survival in bladder cancer

  Shingo Hatakeyama, Hirofumi Ishimura, Toshiko Takahashi, Hiroaki Nakagawa, Shin-Ichiro Nishimura, Yohei Horikawa, Eiji Miyoshi, Atsushi Kyan, Shigeru Hagisawa, Tomonori Habuchi, Yoichi Arai, Chikara Ohyama GLYCOBIOLOGY 16 (11) 1151 -1152 2006年11月 [査読無し][通常論文]
  
• Novel chiroptical analysis of glycoconjugates by Vibrational Circular Dichroism (VCD)

  Kenji Monde, Tohru Taniguchi, Masami Fukuzawa, Mai Hashimoto, Atsufumi Nakahashi, Nobuaki Miura, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1136 -1137 2006年11月 [査読無し][通常論文]
  
• N-glycans correlate 5-FU resistance in colon 26 and derived cell lines

  Jun Hamaguchi, Hiroaki Nakagawa, Masato Takahashi, Takeaki Kudo, Naoya Kamiyama, Bailong Sun, Yuji Sato, Kisaburo Deguchi, Satoru Todo, Shin-Ichiro Nishimura GLYCOBIOLOGY 16 (11) 1151 -1151 2006年11月 [査読無し][通常論文]
  
• Efficient syntheses and characterizations of MUC1 related high-molecular weight glycopeptides

  Takahiko Matsushita, Toshiki Ohyabu, Toshiki Ohyabu, Hiroshi Hinou, Hiroki Shimizu, Shin Ichiro Nishimura, Shin Ichiro Nishimura Polymer Preprints, Japan 55 1857 2006年10月19日 [査読無し][通常論文]
   

  The mucin-type glycoprotein MUC1 is known to be aberrant glycosylated in various cancer cells. We report the efficient method for the syntheses of MUC1-related glycopeptides having different sugar chains based on core2-type trisaccharide structure by combination of microwave-assisted solid-phase syntheses and enzymatic sugar elongation. Characterizations by MUC1 antibody assay will also be discussed.
• トリスキレート型金属錯体の光学分割とVCD

  佐藤久子, 谷口透, 門出健次, 西村紳一郎, 山岸皓彦 錯体化学討論会講演要旨集 56th 326 2006年09月08日 [査読無し][通常論文]
  
• トリスおよびビスアセチルアセトナト金属錯体のVCD測定と理論計算

  佐藤久子, 谷口透, 門出健次, 西村紳一郎, 山岸晧彦 錯体化学討論会講演要旨集 56th 34 2006年09月08日 [査読無し][通常論文]
  
• CARB 63-One-pot solid phase glycoblotting and labeling by trans-oximization for high-throughput glycomics and glycoproteomics

  Hideyuki Shimaoka, Jun-ichi Furukawa, Yoshiaki Miura, Masaki Kurogochi, Hiroshi Hinou, Yasuro Shinohara, Kisaburo Deguchi, Shin-Ichiro Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 232 2006年09月 [査読無し][通常論文]
  
• 北海道で発想の転換を : 地球温暖化という大ピンチを新たなビジネスチャンス到来と考えることはできないだろうか?

  西村 紳一郎 繊維学会誌 62 (8) P231 -P231 2006年08月 [査読無し][通常論文]
  
• 糖鎖自動分析システムの医療への応用

  西村 紳一郎 臨床化学 35 1 -2 2006年07月14日 [査読無し][通常論文]
  
• colon26における5FU耐性株のN型糖鎖の変化

  濱口 純, 工藤 岳秋, 神山 直也, 孫 白龍, 高橋 将人, 佐藤 裕二, 出口 喜三郎, 中川 裕幸, 西村 紳一郎, 藤堂 省 日本外科学会雑誌 107 (2) 2006年03月05日 [査読無し][通常論文]
  
• ダイナミックな作用機序に基づく糖転移酵素阻害剤の分子設計法

  西村紳一郎, 西村紳一郎, 高谷健二, 前田哲宏, 細口健作, 古川潤一, 比能洋 日本糖質学会年会要旨集 26th 2006年
• 癌関連糖ペプチドライブラリの構築

  西村紳一郎, 比能洋, 清水弘樹, 松下隆彦, 大藪巨樹, 麓昌高, 近藤裕郷 高分子学会予稿集(CD-ROM) 55 (2 Disk1) 2006年
• MUC1関連高分子糖ペプチドの効率的合成と機能評価

  松下隆彦, 大藪巨樹, 大藪巨樹, 比能洋, 清水弘樹, 西村紳一郎, 西村紳一郎 高分子学会予稿集(CD-ROM) 55 (1 Disk1) 2006年
• 酵素連続反応による糖鎖自動合成法 糖鎖自動合成装置Golgiの開発

  西村紳一郎, 清水弘樹, 比能洋 産総研TODAY 6 (2) 2006年
• 自殺基質によるシアリダーゼ解析

  比能洋, 黒河内政樹, 秋場聡, 武川泰啓, 西村紳一郎 日本糖質学会年会要旨集 26th 2006年
• 質量分析法による糖鎖構造解析

  出口 喜三郎, 西村 紳一郎 化学と工業 = Chemistry and chemical industry 58 (12) 1422 -1425 2005年12月01日 [査読無し][通常論文]
  
• キトサン骨格を有する機能性糖鎖クラスターの開発

  李 学兵, 渡辺 慎也, 鈴木 隆, 鈴木 康夫, 渡邊 美帆, 名取 泰博, 西川 喜代孝, 喜多 英二, 門出 健次, 西村 紳一郎 キチン・キトサン研究 = Chitin and chitosan research 11 (2) 120 -121 2005年07月01日 [査読無し][通常論文]
  
• 合成不凍糖タンパクを用いた膵島凍結保存法の開発(第105回日本外科学会定期学術集会)

  松本 秀一朗, 松下 通明, 築山 周作, 森田 恒彦, 蒲池 浩文, 古川 義純, 西村 紳一郎, 藤堂 省 日本外科学会雑誌 106 (0) 2005年04月05日 [査読無し][通常論文]
  
• colon26における5FU耐性株のN型糖鎖の変化(第105回日本外科学会定期学術集会)

  濱口 純, 中川 裕章, 工藤 岳秋, 高橋 将人, 佐藤 裕二, 出口 喜三郎, 中川原 章, 西村 紳一郎, 藤堂 省 日本外科学会雑誌 106 (0) 2005年04月05日 [査読無し][通常論文]
  
• 不凍糖タンパク質(AFGP)不凍活性メカニズムの解明

  八須匡和, 五十嵐幸太, 津田栄, 越田周平, 門出健次, 西村紳一郎 高分子学会予稿集(CD-ROM) 54 (1 Disk1) 2005年
• 糖鎖改変技術の創薬応用への可能性(話題)

  西村 紳一郎 ファルマシア 41 (4) 325 -330 2005年 [査読無し][通常論文]
  
• 微量糖鎖構造解析法の進歩と将来糖鎖を開始点とするリバースゲノミクス (GFRG) を目指して

  西村 紳一郎 臨床化学 34 (4) 297 -308 2005年 [査読無し][通常論文]
  
• 微量糖鎖構造解析法の進歩と将来 : 糖鎖を開始点とするリバースゲノミクス(GFRG)を目指して

  西村 紳一郎 臨床化学 34 (4) 297 -308 2005年 [査読無し][通常論文]
  
• 糖鎖自動合成装置“Golgi”用糖転移酵素の開発と利用

  岩田一道, 太田尚志, 太田尚志, 山田久里子, 山田久里子, 戸田篤志, 戸田篤志, 比能洋, 西村紳一郎, 西村紳一郎 バイオテクノロジーシンポジウム予稿集 23rd 2005年
• 糖ペプチド合成用高分子プライマーの開発とその応用

  麓昌高, 比能洋, 太田尚志, 伊藤孝臣, 瀧本明生, 近藤裕郷, 稲津敏行, 中原義昭, 西村紳一郎 日本化学会講演予稿集 85th (2) 2005年
• 非可逆的阻害機構を有するノイラミニダーゼプローブの開発

  比能洋, 黒河内政樹, 長堀紀子, 西村紳一郎 日本化学会講演予稿集 85th (2) 2005年
• マイクロ波照射下における糖ペプチド固相合成

  松下隆彦, 比能洋, 清水弘樹, 西村紳一郎 日本化学会講演予稿集 85th (2) 2005年
• 糖鎖自動合成装置“Golgi”の開発

  山田久里子, 山田久里子, 太田尚志, 太田尚志, 岩田一道, 麓昌高, 麓昌高, 大藪巨樹, 大藪巨樹, 比能洋, 清水弘樹, 西村紳一郎 バイオテクノロジーシンポジウム予稿集 23rd 2005年
• 糖ペプチド合成用プライマーの開発と糖ペプチドライブラリーへの応用

  麓昌高, 麓昌高, 比能洋, 比能洋, 清水弘樹, 太田尚志, 太田尚志, 松下隆彦, 岩田一道, 山田久里子, 山田久里子, 伊藤孝臣, 大藪巨樹, 大藪巨樹, 瀧本明生, 近藤裕郷, 稲津敏行, 中原義昭, 西村紳一郎, 西村紳一郎 バイオテクノロジーシンポジウム予稿集 23rd 2005年
• キトサンを主鎖骨格とした機能性糖鎖クラスター

  李 学兵, 渡辺 慎也, 鈴木 隆, 鈴木 康夫, 名取 泰博, 西川 喜代孝, 門出 健次, 西村 紳一郎 キチン・キトサン研究 = Chitin and chitosan research 10 (2) 204 -205 2004年07月01日
• インタビュー 与えられたチャンスを活かせ! 大学院で何を学び,何をスキルアップさせるか (特集 新大学院時代の成功プログラム)

  西村 紳一郎 化学 59 (4) 30 -32 2004年04月 [査読無し][通常論文]
  
• Development of Mechanism-Based Fluorescence Affinity Labeling Reagent for β-Galactosidase as Proteomics Tool : A Detailed Analysis Using MALDI-TOF Mass Spectrometry

  KUROGOCHI Masaki, IWATA Kazumichi, MONDE Kenji, NIIKURA Kenichi, LEE Yuan Chuan, NISHIMURA Shin-Ichiro Peptide science : proceedings of the ... Japanese Peptide Symposium 2003 113 -114 2004年03月01日
• 糖鎖のDFT法によるコンフォメーション解析と振動円二色性(VCD)スペクトルによる検証

  三浦 信明, 谷口 透, 門出 健次, 西村 紳一郎 基礎有機化学討論会要旨集（基礎有機化学連合討論会予稿集） 17 (0) 51 -51 2004年 [査読無し][通常論文]
   

  糖鎖は不斉炭素の集合体であり、その配座解析の確固たる方法論が未だにない。我々は配座解析の強力なツールとして分子軌道法を用い、不斉性を検出可能な振動領域円二色性スペクトルを計算することによって、測定されたスペクトルを再現することによって糖鎖の配座を混合比のレベルでの解析を行う。
• 糖鎖自動合成装置“Golgi”による複合糖質の合成

  山田久里子, 戸田篤志, 西口進, 岩田一道, 麓昌高, 比能洋, 西村紳一郎 バイオテクノロジーシンポジウム予稿集 22nd 2004年
• 糖鎖自動合成装置“Golgi”用固定化糖転移酵素の開発

  戸田篤志, 山田久里子, 比能洋, 西村紳一郎 バイオテクノロジーシンポジウム予稿集 22nd 2004年
• N-glycan patterns of human serum in rheumatoid arthritis

  M Hato, H Nakagawa, Y Takegawa, K Deguchi, K Monde, N Iwasaki, A Minami, H Kondo, SI Nishimura GLYCOBIOLOGY 13 (11) 870 -870 2003年11月 [査読無し][通常論文]
  
• Structural assignment of isomeric 2-aminopyridine derivatized oligosaccharides using Ms-n spectral matching

  Y Takegawa, S Ito, S Yoshioka, K Deguchi, H Nakagawa, K Monde, SI Nishimura GLYCOBIOLOGY 13 (11) 844 -845 2003年11月 [査読無し][通常論文]
  
• Structures of N-glycan from human dermis

  R Uematsu, H Nakagawa, K Deguchi, M Ota, K Yokota, H Shimizu, K Monde, SI Nishimura GLYCOBIOLOGY 13 (11) 846 -846 2003年11月 [査読無し][通常論文]
  
• Glycoblotting: Synthetic nanoparticles for rapid isolation and analysis of glycopatterns

  SI Nishimura, K Niikura, N Nagahori, R Uematsu, H Nakagawa, S Abe, H Moriyama, R Sadamoto, K Deguchi, K Monde, H Kondo GLYCOBIOLOGY 13 (11) 843 -844 2003年11月 [査読無し][通常論文]
  
• 次世代創薬のための糖鎖工学 (特集 革新的医薬品開発のための糖鎖科学)

  新倉 謙一, 西村 紳一郎 化学工業 54 (10) 745 -747 2003年10月 [査読無し][通常論文]
  
• Synthesis and characterization of functional glycolipid LB membrane.

  N Nagahori, R Sadamoto, K Niikura, K Monde, SI Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 226 U200 -U200 2003年09月 [査読無し][通常論文]
  
• Stereochemical chiral analysis of carbohydrates by vibrational circular dichroism.

  K Monde, T Taniguchi, N Miura, SI Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 226 U213 -U213 2003年09月 [査読無し][通常論文]
  
• 生合成プロセスを模倣した糖鎖自動合成装置"Golgi" (糖鎖機能--第3の生命鎖) -- (構造生物学・新たな構造機能解析法)

  西村 紳一郎 蛋白質核酸酵素 48 (8) 1220 -1225 2003年06月 [査読無し][通常論文]
  
• 糖鎖工学が拓く新しい医薬開発

  新倉 謙一, 西村 紳一郎 化学と工業 = Chemistry and chemical industry 56 (3) 223 -224 2003年03月01日 [査読無し][通常論文]
  
• 糖鎖自動合成装置の開発 (特集 糖鎖工学の最前線)

  西村 紳一郎 バイオインダストリ- 20 (1) 44 -49 2003年01月 [査読無し][通常論文]
  
• 糖鎖エンジニアリングプロジェクト/糖鎖構造解析技術開発 糖鎖自動合成装置のさらなる改良に向けて

  西村紳一郎, 比能洋 バイオテクノロジーシンポジウム予稿集 21st 2003年
• 解説 基礎研究から応用までのキーテクノロジー 糖類自動合成装置"Golgi"

  西村 紳一郎 化学 57 (8) 18 -21 2002年08月 [査読無し][通常論文]
  
• 糖鎖自動合成装置の開発

  西村 紳一郎 バイオサイエンスとインダストリー = Bioscience & industry 60 (8) 23 -26 2002年08月01日 [査読無し][通常論文]
  
• Chemical modification of the living bacterial cell wall.

  R Sadamoto, SI Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 223 U100 -U100 2002年04月 [査読無し][通常論文]
  
• 機能化を目指した糖脂質クラスターの構築 (マイクロドメイン形成と細胞のシグナリング--スフィンゴ脂質の新しい生物機能) -- (第5部 スフィンゴ脂質プローブの開発と応用)

  新倉 謙一, 西村 紳一郎 蛋白質核酸酵素 47 (4) 543 -550,292 2002年03月 [査読無し][通常論文]
  
• 解説記事 糖鎖クラスターの構築と機能

  西村 紳一郎 未来材料 2 (2) 26 -31 2002年02月 [査読無し][通常論文]
  
• Protective mechanism of reduced water against alloxan-induced pancreatic beta-cell damage: Scavenging effect against reactive oxygen species

  YP Li, T Nishimura, K Teruya, T Maki, T Komatsu, T Hamasaki, T Kashiwagi, S Kabayama, SY Shim, Y Katakura, K Osada, T Kawahara, K Otsubo, S Morisawa, Y Ishii, Z Gadek, S Shirahata CYTOTECHNOLOGY 40 (1-3) 139 -149 2002年 [査読無し][通常論文]
   

  Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic beta cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca(2+) concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca(2+) concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2-3.5 times and 2-4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of beta-cells. The protective activity of RW was stable at 4 degreesC for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic beta-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus.
• 21 氷晶成長阻害活性を有する糖ペプチドポリマーの合成と構造活性相関(口頭発表の部)

  立花 裕樹, 五十嵐 幸太, 門出 健次, 西村 紳一郎, 津田 栄 天然有機化合物討論会講演要旨集 44 (0) 121 -126 2002年 [査読無し][通常論文]
   

  Antifreeze glycoproteins [AFGPs: {Ala-Thr(Galβ1-3GalNAcα)-Ala}_n, n=4-50] in the serum of polar fishes have been known to depress the freezing point of their blood and help them to survive at the temperature below-1.9℃[1]. Though it is widely accepted that AFGPs bind to the ice surface and prevent its growth, the definitive molecular mechanism of the action has not been solved yet. In this study, structure-activity relationships in AFGPs were studied toward understanding of the mechanism, thorough chemical syntheses, activity evaluations, and conformational analyses of the AFGP and its analogues, in which sugar or peptide moieties were different from those of the wild-type AFGP. The AFGP (1) and its analogues (2-8) were successfully synthesized via simple polymerization of their repeating unit using a DPPA method without any protection of sugar -OH groups [2]. Additionally, sialyl T-bearing glycopolypeptide (9) was successfully prepared through enzymatic elongation of a sialic acid residue to C-3 position of the galactose residue in the AFGP (1), indicating the broad applicability of this methodology for the syntheses of various glycopeptide polymers. In the evaluation assay of the antifreeze activity, artificial AFGP (1) showed similar activity as that of the wild-type AFGP. On the contrary, neither an AFGP repeating unit glycopeptide (19) nor a sugar-lacked polypeptide (poly-ATA, 8) shows the activity. Interestingly, AFGP analogs having GalNAc (2) or LacNAc (3) as sugar moiety showed similar activities as AFGP, while that of Galactose (4), Lactose (5), β-O-linked (6), and ASA (7) did not. These results clearly indicate the importance of i) repeating structure of glycopeptide, ii) NHAc group at C-2 position of the sugar moiety directly attached to the peptide, iii) α-glycosidic linkage between the sugar and the threonyl residue, for the specific interaction with ice. Furthermore, secondary structural analyses of the synthetic compounds were carried out by means of a CD spectroscopic method, in order to clarify the relationships between their conformations and their activities. The CD spectra of active compounds (1-3) were obviously different from those of inactive analogues (4-8), suggesting the presence of an ordered structure, but not α-helix.
• 糖鎖の自動合成に挑戦(「バイオ」分子を作る化学)

  西村 紳一郎 化学と教育 50 (3) 181 -183 2002年 [査読無し][通常論文]
   

  次世代ポストゲノム研究の重要な柱となることが予想される糖鎖工学の基盤技術の一つは, 糖鎖をいかにスピーディーに確実に合成するかであろう。我々は酵素(糖転移酵素)の反応を基本とする自動合成法の開発を目指して研究を進めてきた。有機溶剤・保護基などを用いない環境低負荷型の合成プロセスとしても評価されつつある。
• Combinatorial syntheses of sugar derivatives

  Shin-Ichiro Nishimura Current Opinion in Chemical Biology 5 (3) 325 -335 2001年06月01日 [査読無し][通常論文]
   

  There has been an exponential growth in interest of the functional roles of carbohydrates and cell surface glycoconjugates in the past 10 years. The importance of glycoconjugates as mediators of biosignals has stimulated investigation into simple and versatile methods for their synthesis. The synthesis of carbohydrates and glycoconjugates by combinatorial chemistry has gained considerable interest.
• Protein patterning on thin film.

  R Sadamoto, K Niikura, N Nagahori, K Yamada, SI Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 221 U351 -U351 2001年04月 [査読無し][通常論文]
  
• 生合成を模倣した糖鎖自動合成法の構築

  西村 紳一郎 日本農芸化学会誌 75 2001年03月05日 [査読無し][通常論文]
  
• The critical role of Th1-dominant immunity in tumor′ immunology.

  Nishimura T., Nakui M., Sato M., Iwakabe K., Kitamura ′ H., Sekimoto M., Ohta A., Koda T., Nishimura S. Collected papers from Institute for Genetic Medicine Hokkaido University 2 418 -426 2001年
• 853 Streptococcus agalactiae type Ia由来のβ1,3-N-アセチルグルコサミニルトランスフェラーゼのマルトース結合タンパクとしての大腸菌における発現と糖鎖合成への応用

  戸田 篤志, 柴谷 滋郎, 西口 進, 中川 裕章, 山田 久里子, 黒河内 政樹, 西村 紳一郎 日本生物工学会大会講演要旨集 13 337 -337 2001年 [査読無し][通常論文]
  
• 854 マウス由来のα2,8-シアル酸転移酵素(GD3合成酵素)のMBP融合タンパクとしての大腸菌における発現

  千代 直樹, 戸田 篤志, 柴谷 滋郎, 西口 進, 西村 紳一郎 日本生物工学会大会講演要旨集 13 338 -338 2001年 [査読無し][通常論文]
  
• 糖鎖自動合成への挑戦--水溶性高分子への糖転移反応を利用する (糖鎖分子の設計と生理機能) -- (酵素を用いた糖鎖合成)

  西村 紳一郎 季刊化学総説 (48) 3 -9 2001年 [査読無し][通常論文]
  
• Controlling the topology of the biomembrane model by modulating the surface protein.

  N Nagahori, K Nikura, K Yamada, SI Nishimura GLYCOBIOLOGY 10 (10) 1120 -1120 2000年10月 [査読無し][通常論文]
  
• Biomimetic strategy of glycoconjugates synthesis: Combined use of immobilized enzymes and designed primers

  SI Nishimura, K Yamada, M Kurogochi, S Nishiguchi, S Shibatani, A Toda, H Nakagawa GLYCOBIOLOGY 10 (10) 1100 -1100 2000年10月 [査読無し][通常論文]
  
• 多孔性ポリマーフィルムを利用した肝細胞の形態及び機能制御

  西川 和孝, 西川 雄大, 西田 仁, 大倉 隆介, 西村 紳一郎, 下村 政嗣, 斉藤 孝則, 藤本 哲也, 大久保 尚, 蒲池 浩文 北海道外科雑誌 45 (1) 90 -90 2000年06月 [査読無し][通常論文]
  
• キトサンオリゴマーとアルギン酸塩の混合紡糸

  田村 裕, 鶴田 行彦, 戸倉 清一, 西村 紳一郎 キチン・キトサン研究 = Chitin and chitosan research 6 (2) 102 -103 2000年05月01日 [査読無し][通常論文]
  
• 機能性キトオリゴ糖ハイブリッドポリマーのデザイン

  野中 佐智子, 熊谷 大樹, 前川 宜彦, 太田 尚志, 西村 紳一郎 キチン・キトサン研究 = Chitin and chitosan research 6 (2) 106 -107 2000年05月01日 [査読無し][通常論文]
  
• 複合糖質科学の研究拠点を設立--自然環境・産業構造にマッチした新バイオサイエンスエリアへの展望 (寒木春華のくに……北海道)

  西村 紳一郎 季刊河川レビュ- 29 (4) 8 -13 2000年 [査読無し][通常論文]
  
• Controlling the topology of glyco-ligand in a biological model system

  N Nagahori, M Taniguchi, A Yamagishi, SI Nishimura GLYCOBIOLOGY 9 (10) 1116 -1116 1999年10月 [査読無し][通常論文]
  
• 糖鎖自動合成法の開発を目指して

  山田 久里子, 西村 紳一郎 バイオサイエンスとインダストリー = Bioscience & industry 57 (9) 35 -36 1999年09月01日 [査読無し][通常論文]
  
• 不凍化タンパク質

  津田 哲郎, 松原 直紀, 西村 紳一郎 高分子 48 (5) 343 -348 1999年05月 [査読無し][通常論文]
  
• 不凍糖タンパク質の合成--不凍魚の秘密をさぐる

  西村 紳一郎 現代化学 (337) 56 -62 1999年04月 [査読無し][通常論文]
  
• 高分子担体を利用した糖鎖合成研究

  山田 久里子, 西村 紳一郎 繊維学会誌 54 (9) P318 -P322 1998年09月 [査読無し][通常論文]
  
• マンノース側鎖をもつキトサン, キチンの調製と性質

  栗田 恵輔, 小林 将之, 島田 久美, 森 知紀, 石井 茂, 西村 紳一郎, 西山 泰弘, 下條 学 キチン・キトサン研究 = Chitin and chitosan research 4 (2) 220 -221 1998年05月15日
• Polymer-assisted enzymatic synthesis of neosphingoglycolipids.

  K Yamada, SI Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 215 U117 -U117 1998年04月 [査読無し][通常論文]
  
• Chemo-enzymatic synthesis of new glycoconjugate polymers bearing sialyl Lewis(X) pendant chains.

  F Sallas, SI Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 215 U105 -U105 1998年04月 [査読無し][通常論文]
  
• Synthetic studies on specific glyco-ligands by molecular imprinting method

  N Nagahori, K Yamada, SI Nishimura ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 215 U117 -U117 1998年04月 [査読無し][通常論文]
  
• キチン・キトサンを基盤とする機能性超分子の設計

  西田 仁, 西村 紳一郎, 居城 邦治, OLAF Karthous, 下村 政嗣 キチン・キトサン研究 = Chitin and chitosan research 3 (2) 112 -113 1997年05月15日 [査読無し][通常論文]
  
• 糖鎖誘導体の精密合成

  西村 紳一郎 高分子 45 (8) 539 -543 1996年08月01日 [査読無し][通常論文]
  
• 糖転移酵素の新しい活性測定法

  富士 祥聡, 西村 紳一郎 日本分子生物学会年会プログラム・講演要旨集 19 393 -393 1996年08月01日 [査読無し][通常論文]
  
• RGDS含む人工タンパク質の合成とその生物活性

  松田 匡雄, 西村 紳一郎 日本分子生物学会年会プログラム・講演要旨集 19 393 -393 1996年08月01日 [査読無し][通常論文]
  
• 酵素反応を用いる高分子糖鎖の選択合成

  西村 紳一郎 高分子学会予稿集 45 (1) 16 -18 1996年05月10日 [査読無し][通常論文]
  
• 分子認識と糖鎖

  西村 紳一郎 キチン・キトサン研究 = Chitin and chitosan research 2 (2) 86 -87 1996年05月01日 [査読無し][通常論文]
  
• リン酸化キチンによる血液凝固現象

  辻 洋二, 吉田 孝, 西村 紳一郎, 坂入 信夫, 西 則雄, 戸倉 清一 キチン・キトサン研究 = Chitin and chitosan research 2 (2) 114 -115 1996年05月01日
• グリコシル化反応

  西村 紳一郎 高分子 45 (8) 563a -563a 1996年 [査読無し][通常論文]
  
• 糖供与体・糖受容体

  西村 紳一郎 高分子 45 (8) 563b -563b 1996年 [査読無し][通常論文]
  
• Biological activities of biodegradable polysaccharide

  S Tokura, SI Nishimura, N Sakairi, N Nishi MACROMOLECULAR SYMPOSIA 101 389 -396 1996年01月 [査読無し][通常論文]
   

  A low toxicity of chitin was demonstrated to be mostly due to biodegradability and the fast metabolization of hydrolysate in animal body. Chitosan, a deacetylated derivative of chitin, is also known to be a biocompatible polymer in spite of a slight immunoadjuvant activity in animal body. Chitosan is easily regenerated to fiber, film, beads and non woven fabrics owing to its high solubility toward diluted aqueous organic acids such as formic acid, acetic acid, glutamic acid and ascorbic acid. The regeneration of chitin was achieved into fibers, gels, porous foams and non woven fabrics following to dissolution of chitin with formic acid or calcium chloride dihydrate saturated methanol. Chitin and its derivatives have been applied as biomedical materials due to remarkable advantages such as antimicrobial activity, acceleration of epidermal cell assembly, low toxicity, and biodegradability.
• Synthesis of Antifreeze Glycoprotein

  TSUDA Tetsuro, NISHIMURA Shin-Ichiro Peptide chemistry : proceedings of the ... Symposium on Peptide Chemistry 1995 145 -148 1995年10月01日
• Structure-Function Relationship of Molecular Chaperone Protein -Nucleoplasmin-

  HOZUMI Kentaro, IWATA Kazumichi, FUKUE Keiji, ITOH Toru, SAKAIRI Nobuo, NISHIMURA Shin-Ichiro, TOKURA Seiichi, KATAGIRI Chiaki, NISHI Norio Peptide chemistry : proceedings of the ... Symposium on Peptide Chemistry 1995 341 -344 1995年10月01日
• Synthesis of Novel Clustered Glycopolymers Containing Triantennary Glycosides of N-Acetyllactosamine^1

  FURUIKE Tetsuya, NISHI Norio, TOKURA Seiichi, NISHIMURA Shin-ichiro Chemistry letters 1995 (9) 823 -824 1995年09月05日
• 再生キチンの薬物担持能

  戸倉 清一, 西 則雄, 坂入 信夫, 西村 紳一郎, 白井 昭宏, 高橋 清久 キチン・キトサン研究 = Chitin and chitosan research 1 (2) 72 -73 1995年04月15日
• 分枝型キチン誘導体の調製

  石井 茂, 小林 将之, 赤尾 裕史, 栗田 恵輔, 西村 紳一郎 キチン・キトサン研究 = Chitin and chitosan research 1 (2) 162 -163 1995年04月15日
• 精密多糖資源としてのキチンとキトサン

  西村 紳一郎 キチン・キトサン研究 = Chitin and chitosan research 1 (1) 15 -25 1995年01月30日 [査読無し][通常論文]
  
• SELECTIVE SULFATION OF CHITIN DERIVATIVES FOR BIOMEDICAL FUNCTIONS

  S TOKURA, K ITOYAMA, N NISHI, SI NISHIMURA, SAIKI, I, AZUMA, I JOURNAL OF MACROMOLECULAR SCIENCE-PURE AND APPLIED CHEMISTRY A31 (11) 1701 -1718 1994年 [査読無し][通常論文]
   

  The highest antithrombogenic activity was achieved by the N- and O-sulfation of partially N-deacetylated (70%) 6-O-carboxymethyl (0.60)-chitin (SCM-DAC-70) among various modified chitin derivatives. But a O-sulfated 6-O-carobxymethyl-chitin (SCM-chitin) was a quite inert heparinoid for the inhibition of blood clotting. It was also suggested that the distribution of N-sulfate and N-acetyl groups on the C-2 position of glucosamine residue might be essential to the selective adsorption of SCM-DAC-70 to antithrombin-III (AT-III) to inhibit thrombin activity. Kinetic evaluations demonstrated the noncompetitive inhibition of thrombin activity by SCM-DAC-70 and competitive inhibition of thrombin by SCM-DAC-70-AT-III complex. We were unable to find such a specific inhibition by the SCM-DAC-70 complex with blood clotting factors other than AT-III. SCM-chitin was found to inhibit the metastasis of cancer cells at about 80-85% without any side effect different from those of heparin. It was suggested that the inhibition of metastasis of B16-BL6 melanoma cells was achieved by bifunctional inhibitions for heparanase and type IV collagenase without growth inhibition of cancer cells.
• 糖鎖生物学と複合糖質モデル

  西村 紳一郎 繊維学会誌 48 (4) P158 -P164 1992年 [査読無し][通常論文]
  
• 朝鮮人参に含まれる生理活性多糖パナキサンAへの合成化学的アプローチ(有機化学・天然物化学-反応と合成(糖質)-)

  市川 幸孝, 西村 紳一郎, 坂入 信夫, 葛原 弘美, 瓜生 敏之 日本農藝化學會誌 62 (3) 1988年03月15日 [査読無し][通常論文]
  
• 各種脱アセチル化キチン繊維の調製とその性質

  戸倉 清一, 西村 紳一郎, 西 則雄, 中村 邦男, 指輪 仁之, 長谷川 長俊, 瀬尾 寛 繊維学会誌 43 (6) 288 -293 1987年 [査読無し][通常論文]
   

  キチン繊維より良好な糸質の繊維を得るため,種々の脱アセチル化キチン(DAC)を調製した。これらの紡糸ではCuSO₄-NH₄OH,アルコール製水酸化ナトリウム及びCuSO₄-H₂SO₄の3種の凝固系が見出され,いずれもよく似た糸質の繊維が得られた。しかし,繊維表面への蛋白質固定化では, CuSO₄-NH₄OH凝固系がより良好な結果を与えた。得られた種々のDAC繊維の糸質はキチンのそれより優れていたが特に湿潤時の引張り強度及び結節強度がキチン繊維やアルキルキチン繊維に比べ大巾に改善されていた。
• 非哺乳類多糖の新しい展開--新素材としてのキチンの医学領域での利用を中心に

  戸倉 清一, 西村 紳一郎 蛋白質核酸酵素 31 (15) p1621 -1631 1986年12月 [査読無し][通常論文]
  
• キチン及びその誘導体よりのリゾチーム受容性繊維

  戸倉 清一, 西 則雄, 西村 紳一郎, ソモリン オイン 繊維学会誌 39 (12) T507 -T511 1983年 [査読無し][通常論文]
   

  キチン及び化学修飾したキチン誘導体を繊維化し,それらのひっぱり糸質及びリゾチームに対する受容性を調べた。部分脱アセチル化キチンの繊維は特に湿強度の点で最もすぐれていた。一方,カルボキシルメチル(CM)基やジヒドロキシプロピル(DHP)基のような親水的なアルキル基を導入すると繊維強度は低下したが,リゾチーチの作用は受け易くなった。また, N-アセチルグルコサミン残基のC₆水酸基を修飾する限り,かさ高いアルキル基を導入してもリゾチームの作用を受け易くなる事がわかった。顕微鏡による観察で,リゾチーム受容性の増大は繊維表面の親水性増加によりおこる事が明らかとなった。

書籍等出版物

• Glycoscience 2nd Edition. 2115-2119

  Springer 2008年
• Glycoscience 2nd Edition. 1859-1871

  Springer 2008年
• 糖鎖のMALDI-TOFMSスペクトルデータブック (西村 紳一郎、福島信弘編)

  三共出版 2007年
• Comprehensive Glycoscience, 453-475

  Elsevier Ltd. 2007年
• 糖鎖のMALDI-TOFMSスペクトルデータブック

  黒河内 政樹, 広瀬 和子, 西村 紳一郎, 福島 信弘 
  三共出版 2007年 (ISBN: 9784782705278)
• Nanotechnology in Carbohydrate Chemistry (Yuasa H.監), 149-166

  Transworld Research Network 2006年
• Methods in Enzymology, 415, 202-212

  Academic Press 2006年
• 未来を拓く糖鎖科学 (永井克彦監), 419-420

  金芳堂 2005年
• Handbook of Carbohydrate Engineering (Yarema K. 編), 495-505

  Taylor & Francis 2005年
• 糖鎖生物学入門

  Taylor Maureen, Drickamer Kurt, 西村 紳一郎, 門出 健次 
  化学同人 2005年 (ISBN: 9784759810356)
• Methods in Enzymology, 362, 273-286

  Academic Press 2003年
• Carbohydrate-based Drug Discovery, 129-136

  Wiley-VCH, DEU 2003年
• 生命高分子科学入門 : 生物に学び生物を超える

  西村 紳一郎, 畑中 研一, 佐藤 智典, 和田 健彦 
  講談社 1999年 (ISBN: 4061397915)
• 糖質の科学と工学

  畑中 研一, 西村 紳一郎, 大内 辰郎, 小林 一清, 講談社サイエンティフィク 
  講談社 1997年 (ISBN: 4061397834)

講演・口頭発表等

• The dynamic epitope theory

  西村紳一郎

  The 13th International Polymer Conference (IPC2023) 2023年07月 口頭発表（基調）
• エクソソームのユニークな構造と機能に学ぶ新しい創薬研究への展開

  西村紳一郎

  官民研究開発投資拡大プログラム（PRISM） 「新薬創出を加速する人工知能の開発」 令4元年度成果報告会 2023年03月
• 実践的糖鎖工学プラットフォームの構築と次世代DDSの創成

  西村紳一郎

  第12回GFRGプロジェクトシンポジウム 2023年01月 口頭発表（一般）
• 前転移ニッチェを標的とする革新的薬物輸送システムの創製  [招待講演]

  西村紳一郎

  第9回日本細胞外小胞学会 2022年10月 口頭発表（招待・特別）
• 血中エクソソームの由来臓器を糖鎖解析により推定する基盤技術の開発

  西村紳一郎

  官民研究開発投資拡大プログラム（PRISM） 「新薬創出を加速する人工知能の開発」 令和3年度成果報告会 2022年05月
• Antibodies recognizing dynamic neoepitopes generated by cancer-specific truncated immature O-glycosylation  [招待講演]

  西村紳一郎

  環太平洋国際科学会議2021 2021年12月 口頭発表（招待・特別）
• 動的エピトープ理論－翻訳後糖鎖修飾が惹起するタンパク質の脆弱性  [招待講演]

  西村紳一郎

  第70回高分子討論会 2021年09月 口頭発表（招待・特別）
• 動的エピトープ理論による革新的創薬  [通常講演]

  西村紳一郎

  LIFE SCIENCE CONEECT 2021 2021年08月
• ヒト組織由来エクソソームの糖鎖プロファイリングによる新しいバイオマーカーの探索

  西村紳一郎

  官民研究開発投資拡大プログラム（PRISM） 「新薬創出を加速する人工知能の開発」 令和二年度成果報告会 2021年07月
• ローマは一日にして成らず。されど、すべての道は？  [招待講演]

  西村紳一郎

  GlycoTOKYO2019 2019年11月 口頭発表（招待・特別）
• Smart nanomedicine targeting of lysosomal glycohydrolases through activated endocytosis of cancer cells  [通常講演]

  西村紳一郎

  25th International Symposium on Glycoconjugates 2019年08月 ポスター発表
• Experience feedback from Hokkaido University researchers cooperating with European institutions, FP7 (Seventh Framework Programme, 2007-2013), “GlycoHIT: Glycomics by High-throughput Integrated Technologies” and “GLYCOPHARM: The sugar code from biochemical concept to clinics”  [招待講演]

  西村紳一郎

  EURAXESS Japan Tour 2019 2019年03月 シンポジウム・ワークショップパネル（指名）
• 独創的なシステム糖鎖工学プラットフォームからの創薬イノベーション  [招待講演]

  西村紳一郎

  医薬基盤研究所創薬教育セミナー 2018年03月 公開講演，セミナー，チュートリアル，講習，講義等
• 糖鎖研究の現状と将来展望  [通常講演]

  西村紳一郎

  第9回GFRG研究会シンポジウム 2017年12月 シンポジウム・ワークショップパネル（指名）
• 動的エピトープ理論：疾患特異抗原成立の新原理に基づく合理的創薬システムの構築  [招待講演]

  西村紳一郎

  第9回Neuroprotective Meeting for Young Researchers 2017年11月 公開講演，セミナー，チュートリアル，講習，講義等
• 動的エピトープ理論：タンパク質のダイナミックな翻訳後修飾が創出する疾患特異的抗原構造  [通常講演]

  西村 紳一郎

  第57回日本臨床化学会年次学術集会 2017年10月 口頭発表（招待・特別）
• Nanomedicine targeting intracellular glycoenzymes  [招待講演]

  西村 紳一郎

  2017 Gordon Research Conference (GRC) on Carbohydrate 2017年06月 口頭発表（招待・特別）
• New glycotechnology platform interfacing chemistry and biology  [招待講演]

  西村 紳一郎

  253rd ACS National Meeting 2017年04月 口頭発表（招待・特別）
• A theory of the dynamic epitope: Autoantibodies to the dynamic epitopes generated by aberrant glycosylation at consecutive threonine motifs in circulating carcinoma mucins  [通常講演]

  西村 紳一郎

  日本化学会第97春季年会 2017年03月 口頭発表（一般）
• Chemical Synthesis Uncovers Novel Regulation Mechanism in Protein Folding by Site-specific Glycosylation  [招待講演]

  西村 紳一郎

  A3 RONA and CPRH 2016年09月 口頭発表（一般）
• New trends in glycodrug discovery: Molecular targets unveiled by chemical synthesis and nanotechnology  [招待講演]

  西村 紳一郎

  GLYCOPHARM Final Conference 2016年07月 口頭発表（招待・特別）
• 動的な翻訳後修飾による疾患特異的抗原構造の獲得  [招待講演]

  西村 紳一郎

  北大・部局横断シンポジウム：研究ネットワーク促進プログラム「生体防御システムとその破綻」～免疫・感染・癌・炎症～ 2016年03月 口頭発表（招待・特別）
• Toward personalized medicine: Chemical and enzymatic synthesis of disease-relevant glycoproteins  [招待講演]

  西村 紳一郎

  103rd Indian Science Congress 2016 2016年01月 口頭発表（基調）
• Posttranslational glycosylation and cancer cell's life cycle: From discovery of disease-relevant epitope to development of new class anti-cancer drugs  [招待講演]

  西村 紳一郎

  第12回消化器外科Ⅰモーニングセミナー 2015年11月 公開講演，セミナー，チュートリアル，講習，講義等
• EPITOPE-DEFINED ANTIBODIES AS NEW CLASS THERAPEUTIC AND DIAGNOSTIC REAGENTS: TOWARD PERSONALIZED MEDICINE FROM CHEMICAL GLYCOBIOLOGY  [招待講演]

  西村紳一郎

  GLYCO23 2015年09月 口頭発表（基調）
• EPITOPE-DEFINED ANTIBODIES AS NEW CLASS THERAPEUTIC AND DIAGNOSTIC REAGENTS: TOWARD PERSONALIZED MEDICINE FROM CHEMICAL GLYCOBIOLOGY  [招待講演]

  西村 紳一郎

  EMBO Workshop 2015年06月 口頭発表（基調）
• 癌特異的糖ペプチドエピトープの探索と新しい抗体医薬の創出  [招待講演]

  西村 紳一郎

  基礎血液談話会 2015年04月 口頭発表（招待・特別）
• Intracellular traffic of cell surface mimetic quantum dots-anchored glycopeptides  [招待講演]

  西村 紳一郎

  249th ACS National Meeting & Exposition 2015年03月 口頭発表（招待・特別）
• Epitope-defined antibody discovered by new glycotechnology  [招待講演]

  西村 紳一郎

  2015 Queenstown Molecular Biology Meeting 2015年03月 口頭発表（招待・特別）
• Epitope-defined antibody as new class therapeutic and diagnostic reagents: Toward personalized medicine from chemical glycobiology  [招待講演]

  西村 紳一郎

  International Symposium on Chemical Biology Approach to Metabiomics, Chemical genomics and Epigenomics 2015年02月 口頭発表（基調）
• 糖鎖研究からの医薬品開発 - QuaDRAD™システムの構築と抗体医薬の創出  [招待講演]

  西村 紳一郎

  Clinical Oncology Research勉強会 2014年11月 公開講演，セミナー，チュートリアル，講習，講義等
• Toward personalized medicine from glycan biomarkers discovered by glycoblotting-assisted high throughput serum glycomics  [招待講演]

  西村 紳一郎

  Sino-Japan Workshop on Chemical Biology 2014年10月 口頭発表（招待・特別）
• GLYCOPHARM at work with glycopeptide epitopes: promising targets uncovered by comprehensive glycomics and robust synthetic compound library  [招待講演]

  西村 紳一郎

  GLYCOPHARM meeting 2014年06月 口頭発表（招待・特別）
• Antigenic glycopeptides: Conformational impact of protein glycosylation and disease-relevant antigenic structure  [招待講演]

  西村 紳一郎

  Special Lecture at Johns Hopkins School of Medicine 2014年03月 公開講演，セミナー，チュートリアル，講習，講義等
• High Performance Glycopeptide Microarray: Rapid Epitope Profiling of Monoclonal Antibodies and Serum Autoantibodies  [招待講演]

  西村 紳一郎

  27th International Carbohydrate Symposium (ICS2014) 2014年01月 口頭発表（招待・特別）
• Toward personalized medicine from glycan biomarkers discovered by glycoblotting-assisted high throughput serum glycomics  [招待講演]

  西村 紳一郎

  International Symposium on Chemical Biology – Drug Discovery 2014年01月 口頭発表（招待・特別）
• Autoantibodies to cancer-associated MUC1 fragments in healthy human sera discovered by high performance glycopeptide microarray platform  [招待講演]

  西村 紳一郎

  246th ACS National Meeting & Expositions 2013年09月 口頭発表（招待・特別）
• Drug discovery from chemical glycobiology  [招待講演]

  西村 紳一郎

  Special seminar at Wayne State University 2013年09月 公開講演，セミナー，チュートリアル，講習，講義等
• Toward personalized medicine from chemical glycobiology  [招待講演]

  西村 紳一郎

  GlycoMedicine Seminar 2013年08月 口頭発表（招待・特別）
• 糖鎖研究からの創薬－新しい抗体医薬品の開発を目指して  [招待講演]

  西村 紳一郎

  Conference for BioSignal and Medicine2013（CBSM2013） 2013年07月 口頭発表（招待・特別）
• Toward personalized medicine from glycan biomarkers discovered by glycoblotting-assisted high throughput serum glycomics  [招待講演]

  西村 紳一郎

  8th ISABS(International Society for Applied Biological Sciences) Conference 2013年06月 口頭発表（招待・特別）
• 学問の風景－Be ambitious!を胸に  [招待講演]

  西村 紳一郎

  学校法人高宮学園 代々木ゼミナール新校舎開校記念特別講演 2013年04月 公開講演，セミナー，チュートリアル，講習，講義等
• 人生に解なし―勇気を出してそれぞれの大志を抱け  [招待講演]

  西村 紳一郎

  北嶺中高等学校 進路講演会 2012年12月 公開講演，セミナー，チュートリアル，講習，講義等
• 生活習慣病・病気と糖鎖の関係  [招待講演]

  西村 紳一郎

  農林水産省委託プロジェクト研究「農林水産物・食品の機能性等を解析・評価するための基盤技術の開発」 第2回 医と食の市民公開講座 2012年11月 公開講演，セミナー，チュートリアル，講習，講義等
• 糖鎖生合成機構の制御と創薬  [招待講演]

  西村 紳一郎

  第17回 未来創薬・医療イノベーションセミナー 2012年09月 口頭発表（招待・特別）
• 全自動糖鎖解析システムの開発と臨床応用  [招待講演]

  西村 紳一郎

  JASIS2012 2012年09月 口頭発表（招待・特別）
• Glycomics and Drug Discovery  [招待講演]

  西村 紳一郎

  26th International Carbohydrate Symposium 2012年07月 口頭発表（招待・特別）
• Toward Fully Automated Glycomics by Glycoblotting Method  [招待講演]

  西村 紳一郎

  Increasing the Impact of Glycoscience through New Tools and Technologies （Satellite Meeting of ICS2012）、、主催：Niels-Christian Reichardt group leader at CIC biomaGUNE 2012年07月 口頭発表（基調）
• グライコブロッティング法と糖鎖ナノテクノロジー  [招待講演]

  西村 紳一郎

  第16回腸内細菌学会 2012年06月 口頭発表（招待・特別）
• 各種質量分析システムを用いた糖タンパク質の特性解析 ～バイオ医薬品の開発における合理的アプローチ～  [招待講演]

  西村 紳一郎

  AB SCIEX LC/MS ユーザーズミーティング2012 2012年03月 公開講演，セミナー，チュートリアル，講習，講義等
• 医薬品開発のためのグライコミクス －高分枝型糖鎖マーカーを活用する前立腺癌治療薬の探索－  [招待講演]

  西村 紳一郎

  第7回臨床糖鎖研究会 2012年03月 口頭発表（招待・特別）
• 疾患グライコミクス  [招待講演]

  西村 紳一郎

  日本肝臓学会西部会 2011年12月 口頭発表（招待・特別）
• メタボロミクスターゲットとしての糖鎖修飾  [招待講演]

  西村 紳一郎

  第6回メタボロームシンポジウム 2011年10月 口頭発表（招待・特別）
• Nanocarries as an emerging platform for new molecular imaging and therapy.  [招待講演]

  西村 紳一郎

  神戸大学シグナル伝達医学GCOE学術講演会 2011年10月 公開講演，セミナー，チュートリアル，講習，講義等
• 糖鎖医薬品の研究開発を支援する新技術・デバイス  [通常講演]

  西村 紳一郎

  第5回ＧＦＲG研究会シンポジウム 2011年10月 口頭発表（一般）
• 全自動糖鎖解析法の実現と医療・創薬研究への展開  [通常講演]

  西村 紳一郎

  眼科学分野 医局リサーチカンファレンス 2011年09月 公開講演，セミナー，チュートリアル，講習，講義等
• 全自動糖鎖解析システムの開発と臨床応用  [招待講演]

  西村 紳一郎

  第51回日本臨床化学会年次学術集会 2011年08月 口頭発表（招待・特別）
• Identification of Disease Specific Glycopeptide Epitopes  [招待講演]

  西村 紳一郎

  European Young Investigators Workshop 2011年04月 口頭発表（招待・特別）
• 高速糖鎖解析技術による疾患検査マーカーの探索  [招待講演]

  西村 紳一郎

  第25回日本臨床検査自動化学会 2011年04月 口頭発表（招待・特別）
• Fully automated glycan analyzer: Importance of Indian glycan database both for early stage diagnosis of Indian and for discovery research of Indian-responsible therapeutic reagents  [招待講演]

  西村 紳一郎

  nternational Symposium on Challenges in Drug Discovery Programme 2011 2011年02月 口頭発表（基調）
• Glycoblotting-assisted high throughput glycomics and glycoproteomics  [招待講演]

  西村 紳一郎

  2010 International Chemical Congress of Pacific Basin Societies (PACIFICHEM2010） 2010年12月 口頭発表（基調）
• 疾患マーカー探索研究を加速する有機合成化学  [招待講演]

  西村 紳一郎

  平成22年度 秋季有機合成化学講習会 2010年11月 口頭発表（招待・特別）
• 高感度疾患マーカーとしての複合糖質の探索と医療への応用  [招待講演]

  西村 紳一郎

  神戸大学 先端医学シリーズ 2010年11月 公開講演，セミナー，チュートリアル，講習，講義等
• Glycoblotting method reveals new promising biomarkers during cell differentiation.  [招待講演]

  西村 紳一郎

  JAACT2010 2010年09月 口頭発表（基調）
• Cancer relevant epitopes uncovered by synthetic mucin glycopeptides  [招待講演]

  西村 紳一郎

  The 240th ACS National Meeting 2010年08月 口頭発表（招待・特別）
• Large-scale glycomics for the discovery of cancer markers：Diagnostic mass spectrometry based on glycoblotting technology  [招待講演]

  西村 紳一郎

  BIT's 3rd World Cancer Congress 2010 2010年06月 口頭発表（招待・特別）
• グライコブロッティング法と質量分析によるバイオマーカーの探索  [招待講演]

  西村 紳一郎

  第58回質量分析総合討論会、第1回アジア･オセアニア質量分析会議 2010年06月 口頭発表（招待・特別）
• Large-scale glycomics and glycoproteomics by glycoblotting method  [招待講演]

  西村 紳一郎

  ピッツバーグ分析化学・応用分光学会議（PITTCON) 2010年03月 口頭発表（招待・特別）
• 臨床グライコミクスと創薬研究  [招待講演]

  西村 紳一郎

  神戸大学医学研究科セミナー 2010年01月 公開講演，セミナー，チュートリアル，講習，講義等
• Chemistry and biological impact of complex carbohydrates  [招待講演]

  西村 紳一郎

  Special seminar on Mysore University 2009年12月 口頭発表（招待・特別）
• Drug discovery by integrating glycoblotting and automated glycan synthesis  [招待講演]

  西村 紳一郎

  International Conference on current Trends in Chemistry and Biochemistry (ICCTCB-2009) 2009年12月 口頭発表（基調）
• Comprehensive glycomics for basic and advanced studies in glycobiology  [招待講演]

  西村 紳一郎

  7th Annual Uppsala Conference 2009 2009年12月 口頭発表（招待・特別）
• 糖鎖版ＰＣＲ法の完成で加速する疾患マーカー探索研究  [招待講演]

  西村 紳一郎

  先端計測分析技術・機器開発事業 ５周年記念シンポジウム 2009年12月 口頭発表（招待・特別）
• 糖鎖の謎と魅力 －遺伝子だけでは語れない生命の不思議－  [招待講演]

  西村 紳一郎

  防衛大学特別講演会 2009年12月 公開講演，セミナー，チュートリアル，講習，講義等
• Large-scale serum glycomics of CFG KO mice based on the glycoblotting method  [通常講演]

  西村 紳一郎

  2009 Annual Meeting of the Society for Glycobiology 2009年11月 ポスター発表
• 大規模網羅的糖鎖解析と標的糖ペプチド解析  [招待講演]

  西村 紳一郎

  タンパク質研究・バイオマーカー研究セミナー 次世代プロテオミクス 最先端研究レポート 2009年11月 口頭発表（招待・特別）
• Automated glycan synthesis inspired from biosynthetic systems and their use in drug discovery research  [招待講演]

  西村 紳一郎

  Symposium on Bio-Inspired Engineering(ISBIE) 2009年10月 口頭発表（基調）
• 大規模グライコミクスによる癌マーカーの研究  [招待講演]

  西村 紳一郎

  第68回日本癌学会学術総会 2009年10月 口頭発表（招待・特別）
• 癌特異的糖ペプチド抗原の探索と疾患マーカーの開発 ―Anti-KL-6 抗体のエピトープマッピングから学んだこと―  [招待講演]

  西村 紳一郎

  第29回日本分子腫瘍マーカー研究会 2009年09月 口頭発表（招待・特別）
• Synthesis of highly complicated carbohydrates and their use in drug discovery research  [招待講演]

  西村 紳一郎

  Japanese-European Wiorkshop on Cellulose and Functional Polysaccharides 2009年09月 口頭発表（招待・特別）
• 原点に立ち返ってみよう－糖鎖構造解析のunmet needs－  [招待講演]

  西村 紳一郎

  第29回日本糖質学会年会 2009年09月 公開講演，セミナー，チュートリアル，講習，講義等
• 大規模糖鎖解析による新しい疾患マーカーの探索  [招待講演]

  西村 紳一郎

  消化器研究セミナー 2009年09月 公開講演，セミナー，チュートリアル，講習，講義等
• 糖鎖研究の新展開～遺伝子だけでは語れない生命の謎にせまる～  [招待講演]

  西村 紳一郎

  第49回 生命科学 夏の学校 2009年08月 口頭発表（招待・特別）
• 臨床グライコミクスによる新しい疾患マーカー分子の探索  [招待講演]

  西村 紳一郎

  第56回日本臨床検査医学会学術集会 2009年08月 口頭発表（招待・特別）
• 糖鎖研究と創薬～Unmet needs と defacto standards～  [招待講演]

  西村 紳一郎

  第25回創薬セミナー 2009年07月 口頭発表（招待・特別）
• Fully Automated Carbohydrate Synthesis by Combined Chemical and Enzymatic Protocol  [招待講演]

  西村 紳一郎

  15th European carbohydrate symposium 2009年07月 口頭発表（招待・特別）
• 北大リサーチ＆ビジネスパークにおける糖鎖研究の進捗状況  [招待講演]

  西村紳一郎

  北海道経済連合会 第185回常任理事会 2009年07月 口頭発表（招待・特別）
• 遺伝子だけでは語れない生命の謎に迫る化学研究  [招待講演]

  西村 紳一郎

  関西大学特別講演会 2009年06月 公開講演，セミナー，チュートリアル，講習，講義等
• New Trends in discovery research for cancer markers and glycosyltransferases inhibitors  [招待講演]

  西村 紳一郎

  NCI-Frederick Special Seminar 2009年03月 口頭発表（招待・特別）
• Glycoblotting: Potential "PCR" for Glycobiology  [招待講演]

  西村 紳一郎

  Johns Hopkins University Special Lecture 2009年03月 公開講演，セミナー，チュートリアル，講習，講義等
• 血液一滴から癌や生活習慣病を早期診断  [招待講演]

  西村 紳一郎

  OKINAWAライフサイエンスシンポジウム 生命科学の最前線と沖縄の可能性 2009年02月 口頭発表（招待・特別）
• 創薬ターゲットとしての複合糖質の魅力～革新的方法論の創出が探索研究を加速する  [招待講演]

  西村 紳一郎

  味の素医薬研究所講演会 2009年01月 口頭発表（招待・特別）
• 大規模グライコミクスの実現とその応用  [招待講演]

  西村 紳一郎

  オミックス医療研究会定期講演会 2009年01月 口頭発表（招待・特別）
• Chemistry and biological impact of comlpex carbohydrates  [招待講演]

  西村 紳一郎

  蓼科有機化学会議 2008年11月 口頭発表（招待・特別）
• 大規模糖鎖解析による癌マーカー分子の探索  [招待講演]

  西村 紳一郎

  第67回日本癌学会学術総会 2008年10月 口頭発表（招待・特別）
• Large-scale quantitative glycomics by glycoblotting method  [招待講演]

  西村 紳一郎

  BioJapan2008 World Business Forum 2008年10月 口頭発表（招待・特別）
• 大規模グライコミクスと疾患バイオマーカー探索研究  [招待講演]

  西村 紳一郎

  第28回日本糖質学会年会 2008年08月 口頭発表（招待・特別）
• 生命を探る－遺伝子だけでは語れない生命の謎がある－  [招待講演]

  西村 紳一郎

  平成20年度 日本学術会議第三部夏季部会 市民公開講演会 2008年08月 公開講演，セミナー，チュートリアル，講習，講義等
• Glycoblotting allows large-scale functional glycomics  [招待講演]

  西村 紳一郎

  International Carbohydrate Symposium 2008 2008年08月 口頭発表（基調）
• Toward carbohydrate-based drug discovery  [招待講演]

  西村 紳一郎

  BCN Biomed Seminar IRB 2008年07月 口頭発表（基調）
• 多糖鎖誘導体の整形外科領域での高次利用  [通常講演]

  西村 紳一郎

  繊維学会平成20年度年次大会 2008年06月 口頭発表（招待・特別）
• 糖鎖合成と創薬研究  [招待講演]

  西村 紳一郎

  天然高分子研究セミナー 2008年06月 口頭発表（招待・特別）
• 臨床グライコミクスによる疾患マーカー探索と創薬・医療への展開  [招待講演]

  西村 紳一郎

  第81回日本整形外科学会学術総会 2008年05月 口頭発表（招待・特別）
• Synthesis and characterization of MUC1-related glycopeptides  [招待講演]

  西村 紳一郎

  EPA Chemistry 3rd Flash Conference 2008年03月 口頭発表（招待・特別）
• 糖鎖修飾の大規模高速解析技術  [招待講演]

  西村 紳一郎

  大阪大学蛋白質研究所セミナー 2008年01月 公開講演，セミナー，チュートリアル，講習，講義等
• 糖鎖研究からバイオ産業育成への挑戦  [招待講演]

  西村 紳一郎

  日本応用糖質科学会平成19年度大会 2007年08月 口頭発表（招待・特別）
• Clinical Glycomics Based on glycoform-focused reverse genomics  [招待講演]

  西村 紳一郎

  Lecture at Genomics Research Center in Academia Sinca 2007年07月 口頭発表（招待・特別）
• Conformational and biological characterization of synthetic mucin glycopeptides  [招待講演]

  西村 紳一郎

  MICC-3 meeting 2007年07月 口頭発表（招待・特別）
• Mechanism-based drug design: Novel strategy toward highly selective therapeutic reagents  [招待講演]

  西村 紳一郎

  Gordon Research Conferences 2007 2007年06月 口頭発表（招待・特別）
• Glycoblotting-Based Clinical Glycomics  [招待講演]

  西村 紳一郎

  BENZON SYMPOSIUM No.54 2007年06月 口頭発表（招待・特別）
• 創薬研究開発を加速するChemical Biology  [招待講演]

  西村 紳一郎

  日本化学会第87春季年会（2007） 2007年03月 口頭発表（招待・特別）
• 臨床糖鎖情報科学へのアプローチ  [招待講演]

  西村 紳一郎

  日本バイオインフォマティクス学会北海道支部セミナー 2007年03月 口頭発表（招待・特別）
• 糖鎖の迅速解析法  [招待講演]

  西村 紳一郎

  JST-SORSTジョイントシンポジウム 2007年01月 口頭発表（招待・特別）
• 北大が開拓する未来創薬研究拠点  [招待講演]

  西村 紳一郎

  北海道大学21世紀COEプログラム 2007年01月 公開講演，セミナー，チュートリアル，講習，講義等
• Glycoform-focused reverse genomics (GFRG)  [招待講演]

  西村 紳一郎

  日本薬学会近畿支部 新春特別講演会 2007年01月 口頭発表（招待・特別）

所属学協会

• アメリカ化学会   日本化学会   高分子学会   日本臨床化学会   

共同研究・競争的資金等の研究課題

• 進化型MBRによる下水からの有機物回収：下水道の創エネルギーインフラへの転換

  日本学術振興会：科学研究費助成事業

  研究期間 : 2021年04月 -2025年03月 

  代表者 : 木村 克輝, 西村 紳一郎, 羽深 昭

   

  ベンチスケールのMBR実験システムを構築し、実下水処理場に実験装置を設置して連続ろ過実験を繰り返し実施した。本研究で提案・確立を行うエネルギー回収型MBRでは深刻な膜ファウリングの発生が確実に起こるため、これに対処するための担体を用いた機械的洗浄と薬品併用逆洗（CEB）を実施しながら連続ろ過運転を行った。HRT、SRT、DOを様々に変化させて実験を行い、有機物回収率、膜ファウリング発生度、処理水水質のそれぞれが高いレベルで調和するような運転条件の探索を試みた。HRTを極短の1.5-2時間、SRTも極短の0.5日とすることで、有機物回収率（初沈流入水に対して）は70％を超過する結果が得られた。実下水を用いた実験であったことからDOを一定範囲に制御することが難しく、MBR内のDO濃度は大きく変動した（1-7 mg/L）。処理水中のCOD濃度は20 mg/L程度となり、高い有機物回収率と処理水中の有機物濃度低減を同時に達成可能であることを実証した。膜ファウリングの発生については、CEBに用いる次亜塩素酸の濃度が洗浄効果に大きな影響を及ぼすことを観察した。本年度の研究では50-1000 ppmの範囲でCEBに用いる次亜塩素酸の濃度を変化させたが、最も安定したMBRの運転が可能になったのは次亜塩素酸の濃度を50 ppmに設定した時であった。過度に高い薬品濃度は微生物からの有機物放出を誘引し、MBR内の汚泥ろ過性を低下させるものと考えられた。CEBに用いる次亜塩素酸濃度を50 ppm とし、正味の膜透過水フラックスを20 LMHに設定した実験では５日間程度はほとんど膜ファウリングが発生しない状況を繰り返し観察した。
• 切除検体を基にした網羅的糖鎖解析による肝細胞癌悪性度解析と新規バイオマーカー開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2021年04月 -2024年03月 

  代表者 : 神山 俊哉, 西村 紳一郎, 旭 火華, 坂本 譲, 柿坂 達彦, 折茂 達也, 長津 明久

   

  これまでの糖鎖解析を行う癌に関する研究は、患者血清を用いるものが多かったが、患者背景：併存する疾患、人種などの影響があり、癌細胞上で行われる糖鎖修飾がどのように癌細胞の生物学的な悪性度を関連性があるかは未解決である。肝癌細胞株での実験で、これまで報告された患者血清から検出された糖鎖と異なるものが、浸潤能に関与していることが分かったため、実際の切除検体である癌細胞組織での検討が必要であるとの考えに至った。 今年度は、実際の切除検体である癌細胞組織での糖鎖解析による検討を行った。2001年から2006年までの肝細胞癌切除例48例の凍結検体の網羅的糖鎖解析による糖鎖発現を全自動血清糖鎖プロファイル解析で行った。1325.55m/zから3336.24m/zまでの39個の糖鎖を検出でき、肝癌組織のN型糖鎖プロファイルを解析できた。凍結保存された肝細胞癌切除標本の糖鎖解析が普遍的に可能であることが検証された。今後、同一患者さんで、非癌部の肝組織、血清での発現と、今回検出できた糖鎖との関連性を検証していく。さらにこれらの結果と臨床病理学的所見・組織病理学的所見、切除後の予後・再発との関連性を検討する予定であり、実際の肝細胞癌組織と癌悪性度、浸潤能などとの関連性がある特異の糖鎖が発見され、この糖鎖修飾を阻害することができる阻害剤が見つかれば、創薬の点からも癌治療に大きく貢献できると確信している。肝切除かあるいは分子標的薬による薬物治療かの治療選択基準となりうる新規バイオマーカーを検出できる可能性がある。
• タンパク質の翻訳後糖鎖修飾により生成する動的エピトープを標的とする抗体医薬の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2019年04月 -2023年03月 

  代表者 : 西村 紳一郎, 田中 良和, 比能 洋, 尾瀬 農之

   

  本研究課題は、申請者らの独創的な「動的エピトープを標的とする新薬開発」という発想と、既にその基盤が完成しており効率的でシームレスな抗体開発までを可能とする「糖鎖工学プラットフォームを活用する戦略」によって創出されたED抗体（epitope-defined antibody）を用いて、主としてがん領域におけるアンメットメディカルニーズに応え得る新たながん治療薬の開発を目的としている。 この研究課題においては多くのがん細胞表面に高発現してがんの増殖や転移の促進などに深く関与することが広く知られるムチンMUC1の細胞外タンデムリピートドメインに生成する多様な動的エピトープ（dynamic epitope）を特異的な標的とするED抗体である抗MUC1モノクローナル抗体の構造改変と低分子化によって高機能化することで新たな創薬モダリティーを創出することを具体的な目標として進めている。 2021年度（３年目）はMUC1-STを動的エピトープとするSN131のFab-MUC1-ST共結晶の構造解析に成功した。また、物理化学的・生化学的キャラクタリゼーションに加えて、すい臓がん細胞表面のMUC1との結合やSiglecなどとの相互作用などを多角的に解析した（論文投稿準備中）。SN131の改変型抗体であるSN132（動的エピトープとの結合領域を含むanti-parallel coiled-coil構造のFv-claspとリン脂質DPC膜結合性membrane-associated peptide領域を融合した分子量40K程度の全く新しいタイプの機能性抗体フラグメント）の作成法を確立した（特許出願準備中）。 また、SN131およびSN121のFv領域を、2種類のリンパ球を認識する抗体（OKT3およびL2K）のFv領域と融合させて新たに８種の二重特異性抗体の作成を進めており高活性を示す高性能な二重特異性抗体取得に向けて研究が進展している。
• 水中バイオポリマーの詳細な特性解明に基づく膜目詰まり問題の根本的解決

  日本学術振興会：科学研究費助成事業

  研究期間 : 2018年04月 -2021年03月 

  代表者 : 木村 克輝, 西村 紳一郎, 山村 寛

   

  浄水膜処理における膜ファウリングの発生において、水中天然有機物（NOM）の中のバイオポリマー（BP）と呼ばれる親水性高分子量画分の重要な関与が指摘されている。本研究では水道原水から高回収率・高純度でBPを回収・精製できる方法を新規に確立し、精製BPの物理化学特性および膜ファウリング発生ポテンシャルを評価した。BPの物理化学的性質は試料を採取する場所と季節により大きく変動することを明らかにした。BPのファウリングポテンシャルを評価する方法として水晶振動子マイクロバランス（QCM）分析を用いる方法を確立し、BPによるファウリングが起こりづらい膜材質を適切に選択できる可能性を提示した。
• 椎間板変性に起因した痛みと関連する血清中糖鎖の網羅的探索

  日本学術振興会：科学研究費助成事業

  研究期間 : 2018年06月 -2020年03月 

  代表者 : 須藤 英毅, 西村 紳一郎

   

  椎間板は、体幹を支える脊柱の主要構成要素であり、その代表的疾病である椎間板性腰痛や椎間板ヘルニア等では、激しい腰痛や下肢痛によって自立した生活が困難となるが、痛みを客観的に評価する標準的手法自体は確立されていない。そこで本研究の目的は、痛みを糖鎖医学の観点から分析し、椎間板変性に起因した痛みと血清中N型糖鎖のプロファイル変化との関連性を網羅的に検討することである。 方法としてウサギ椎間板に穿刺を行い、変性椎間板モデルを作製した。変性した椎間板組織と血清中糖鎖を網羅的に探索し正常組織及び血清と比較した。その結果、椎間板変性に伴う椎間板組織と血清のハイマンノース型N型糖鎖への糖鎖置換を明らかにした。
• 肝癌細胞株における糖鎖異常と浸潤能との関連性の解析

  日本学術振興会：科学研究費助成事業

  研究期間 : 2016年04月 -2020年03月 

  代表者 : 神山 俊哉, 西村 紳一郎, 島田 慎吾, 若山 顕治, 武冨 紹信, 横尾 英樹, 折茂 達也

   

  肝癌細胞株で、浸潤能と糖鎖の関連性を解析した。u-PA発現をWestern Blotで評価し、網羅的糖鎖解析を行った。HLEはu-PAの発現が高く、HepG2が最も低く、HLFはその中間であった。浸潤能はu-PA産生能順に、HLE、HLF、HepG2であった。HLE、HepG2で、E-Cadherin、N-Cadherinの発現をWestern Blotで評価した。HLEでE-Cadherin発現低下し、HepG2は高発現、N-Cadherinはともに発現していた。HLE、HLF、HepG2、u-PA発現制御下HLEでの糖鎖解析から1851m/z、2521m/zが浸潤能変化に共通糖鎖であった。
• 糖尿病網膜症における血管内皮増殖因子受容体の糖鎖変化

  日本学術振興会：科学研究費助成事業

  研究期間 : 2016年04月 -2018年03月 

  代表者 : 石田 晋, 西村 紳一郎, 野田 航介

   

  本研究では、糖尿病網膜症の病態責任分子である血管内皮増殖因子（VEGF）の受容体VEGFR-2上のN型糖鎖構造に関する検討をおこなった。既存データベースとの照合をおこなった結果、培養ヒト網膜血管内皮細胞におけるVEGFR-2由来のN型糖鎖16ピークについて、その糖鎖構造を推定することができた。しかしながら、糖尿病網膜症における病態変化の一つである低酸素条件に24時間曝露した培養したヒト網膜血管内皮細胞では、VEGFR-2上のN型糖鎖構造に変化を検出できなかった。
• グライコブロッティングを利用した多糖の選択的除去による膜ファウリングの効率的抑制

  日本学術振興会：科学研究費助成事業

  研究期間 : 2015年04月 -2018年03月 

  代表者 : 木村 克輝, 西村 紳一郎, 山村 寛

   

  本研究では、生体内試料から糖鎖のみを選択的に回収するグライコブロッティングに着目し、膜処理において膜を閉塞させるバイオポリマーを効率的に除去できる前処理方法の確立を試みた。常温下ではグライコブロッティングによるバイオポリマーの除去は限定的であったが、アニリンなどの触媒あるいは酸化剤の併用により常温下でもバイオポリマーの除去が可能となった。バイオポリマーにはグライコブロッティングにより除去しやすいものとしにくいものが混在していることが分かり、バイオポリマーの細分類が必要となった。バイオポリマーの分析法として国際標準となっている方法を改良し、バイオポリマーを細分類して分析することに成功した。
• 網羅的糖鎖解析による新規癌マーカーの探索と診断技術の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2013年05月 -2018年03月 

  代表者 : 西村 紳一郎, 能祖 一裕, 比能 洋, 大山 力, 神山 俊哉, 天野 麻穂

   

  世界初の「疾患糖鎖構造データベースの構築」を目標として、医師を含む臨床チームとの強力な連携により３５００件を超える患者検体を用いた大規模網羅的糖鎖解析による新規バイオマーカーの探索を進めた。特に消化器癌と泌尿器癌に焦点を絞りバイオマーカーとして有望な糖鎖構造情報を獲得するため、独創的な「全自動糖鎖解析装置」による大規模糖鎖解析からの疾患糖鎖データベースの構築に挑戦した。具体的には膵臓癌、膵炎、肝細胞癌、肝炎、潰瘍性大腸炎、大腸癌、自己免疫性膵炎等の消化器疾患領域、および腎細胞癌、腎炎、前立腺癌等の泌尿器疾患領域を中心に疾患糖鎖情報を収集して世界初の疾患関連糖鎖データベースの構築が実現した。
• グライコブロッティング法による糖尿病網膜症関連糖鎖の網羅的解析

  日本学術振興会：科学研究費助成事業

  研究期間 : 2014年04月 -2016年03月 

  代表者 : 石田 晋, 西村 紳一郎, 野田 航介

   

  糖尿病網膜症の有病率は糖尿病の罹病期間に関連するため、網膜症患者は増加の傾向にある。さまざまな基礎および臨床研究が行われてきたが、糖尿病網膜症のメカニズムにはいまだ不明な点が多い。しかし、近年N型糖鎖とよばれる蛋白上の構造変化が多くの疾患病態に関与することが明らかとなってきており、糖尿病網膜症眼内でのN型糖鎖変化も興味深い点である。 本検討では、1）糖尿病網膜症患者硝子体中においてN型糖鎖が増加していること、2）シアル酸付加型N型糖鎖が特に増加しており、全体のプロファイルも変化していること、3）そのシアル酸付加型N型糖鎖には複数のシアル酸転移酵素が関与することなどを明らかとした。
• 網羅的糖鎖解析グライコミクスによるぶどう膜炎炎症増悪マーカー探索

  日本学術振興会：科学研究費助成事業

  研究期間 : 2013年04月 -2016年03月 

  代表者 : 南塲 研一, 北市 伸義, 西村 紳一郎, 天野 麻穂

   

  今回我々はぶどう膜炎の発症時に特異的に上昇する糖鎖について検討をおこなった。インフリキシマブ導入後もぶどう膜炎を生じる患者、特に抗インフリキシマブ抗体が陽性患者、およびコントロールとしてインフリキシマブ導入後経過良好な患者の血清について、新しい糖鎖構造解析グライコブロッティング法をもちいて網羅的糖鎖解析をおこなった。2群間比較でm/z1680, m/z2337, m/z2379, m/z3195の４つの糖鎖において比較的明確な差異が認められ、いずれも無効例に多くみられるという結果であった。
• 非可逆的酵素阻害反応によるトリパノソーマ症治療薬の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2012年04月 -2014年03月 

  代表者 : 西村 紳一郎

   

  リパノソーマ症（シャーガス病）は中南米諸国で猛威を奮う深刻な感染症で既に2000万人以上が感染しており、毎年約２万人が本感染症により死亡している。現在、nifurtimoxとbenznidazoleのみが治療薬として承認されているが、その効果は満足できるものではないため新規な医薬品の開発が強く望まれている。 我々は新たなシアリダーゼの自殺基質型誘導体がシャーガス病に対する感染症治療薬の有望なリード化合物である事を発見した。そこで、本研究課題ではこの自殺基質型誘導体のナノ微粒子表面への集積法を確立することを第一の目的とし、ナノ医薬品としての治療薬候補の創製に繋げることを期間内の目標とした。
• 癌細胞表面に発現した分枝型Ｏ－グリカンによる宿主免疫逃避機構の解明

  日本学術振興会：科学研究費助成事業

  研究期間 : 2010年04月 -2014年03月 

  代表者 : 坪井 滋, 大山 力, 西村 紳一郎

   

  体内に生じた癌細胞を排除する免疫細胞の主要なものがNK細胞である。しかしながら、癌細胞がこのNK細胞の攻撃から逃避する能力を獲得すると、その癌細胞は体内で長く生存できるようになり、転移の原因になると考えられている。われわれは、ある種の癌細胞が、今までに知られていなかった新しい仕組みを使ってNK細胞の攻撃から逃避していることを発見した。これらの癌細胞では、細胞表面に付加している糖が、特殊なタイプの糖に変わっているため、NK細胞による攻撃から逃避できるようになったことを明らかにした。また、癌細胞が、転移先の臓器でどのようにして血管から脱出するのか、その機構の解明に貢献した。
• 新たな網羅的糖鎖解析グライコブロッティング法によるぶどう膜炎の病態解明

  日本学術振興会：科学研究費助成事業

  研究期間 : 2010年 -2012年 

  代表者 : 南場 研一, 西村 紳一郎, 北市 伸義, 天野 麻穂

   

  新しい網羅的糖鎖構造解析グライコブロッティング法を用いてぶどう膜炎の患者での糖鎖解析をおこなった。ぶどう膜炎患者では血清中の糖鎖量が増えていること、また、活動期に変動する糖鎖が3種類見られた。ベーチェット病ではHLA-B51との関連、眼外症状との関連性を示す糖鎖がみられたが、眼炎症発作との関連はみられなかった。マウス実験的自己免疫性ぶどう膜網膜炎モデルでの糖鎖変動を調べたところ、免疫により変動する糖鎖はみられたが、ぶどう膜炎特異的に変動していると思われる糖鎖はみられなかった。
• グライコミクスによる変形性関節症の早期診断に有効な血清糖鎖マーカー探索

  日本学術振興会：科学研究費助成事業

  研究期間 : 2011年 -2011年 

  代表者 : 西田 欽也, 岩崎 倫政, 西村 伸一郎, 天野 麻穂

   

  変形性関節症の早期診断のための血清マーカーの探索を目的に研究を進めた。変形性膝関節症(OA)患者および健常者の血清5-20μ1を用いた。血清中からグライコブロッティング法を用いて高速(5時間程度)かつ高効率に、糖蛋白質からN-結合型糖鎖の遊離精製を行った。得られたN-結合型糖鎖をMALDI-TOF/MSに供することで、各糖鎖構造を推定し、内部標準物質のピーク面積からの比例計算により定量化を行った。解析は現在進行中である。患者検体はX線所見にて膝OAと診断されたKellgren-Lawrence分類grade 2-4の患者より採取している。糖鎖解析は、本申請者が中心となり西村、天野らの協力のもと行っている。独自に開発したグライコブロッティング法は従来の解析法に比べ飛躍的に解析時間の高速化と高効率性を実現し、これまで困難とされていた大規模糖鎖解析を可能とし、本研究で予定している検体数の解析を早急に進めているところである。統計学的解析によりAUC(Area Under the Curve)が0.85以上の糖鎖およびその組み合わせを抽出する。先行研究の結果より、5から10程度の候補糖鎖および組み合わせが予想される。さらに、糖鎖関連分子のOAの病因における機能的役割を解明するためにスフィンゴ糖脂質を軟骨組織特異的にノックアウトしたマウス(UgcgKOマウス)を用いてOAモデルを作成し、その機能解析を行った。その結果、GSLsは軟骨の発生や分化には必須ではないが、正常な軟骨代謝を維持し、OA進行を抑制する機能を持つことが示唆された。
• 乳癌の網羅的糖鎖解析による新規バイオマーカーの開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2008年 -2010年 

  代表者 : 山下 啓子, 遠山 竜也, 杉浦 博士, 西村 紳一郎, 西村 紳一郎

   

  乳癌患者と健常人女性の血清を用いてグライコブロッティング法により網羅的糖鎖解析を行った結果、両者を区別できる単独の糖鎖マーカーは見出せなかった。次に、乳癌患者の血清を用いた網羅的糖鎖解析結果と臨床情報(乳癌のサブタイプ間での糖鎖プロファイル比較、予後解析など)の統計学的解析を行い、24種類の糖鎖を組み合わせることにより予後予測が可能となる可能性を見出した。
• 神経組織再生のための新規多機能生体内吸収性基材の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2006年 -2007年 

  代表者 : 三浪 明男, 西村 紳一郎, 眞島 任史, 岩崎 倫政

   

  本年度の研究によって、我々は以下のような成果結果を得ることができた。 1.基盤となる神経組織再建用材料の追加:平成18年度の報告書において我々はヒアルロン酸を導入したキトサン繊維を用いて神経再生の実験を行い、その結果を報告した。平成19年度ではこの素材の他に、新たな細胞外マトリックスが神経再生に有効であることを発見した。具体的にはこの細胞外マトリックスを使用することにより他の細胞外マトリックスを使用した場合と比較し、細胞(神経細胞のモデルとしてPC12細胞を使用)突起の伸長が有意に増加することを証明した。 2.神経組織再生用材料のための細胞接着分子・細胞増殖因子の導入:同様に平成18年度の報告書においてキトサンに細胞接着分子であるRGDモチーフを化学的に安定化した状態で導入したことを報告した。平成19年度ではRGDモチーフのほか、細胞外マトリックスの構成成分であるラミニンの細胞接着モチーフの2種類を化学的に合成(solid phase peptide synthesisにて)し、これらの生理活性の評価を行い、神経再生に対し望ましい効果を有することを証明した。 今後の予定: 前述した神経再生に有用な新しい素材に同じく前述の3つの細胞接着モチーフを化学的に導入することにより新たな神経再生用素材を作成する。これに前述のPC12細胞を播種し、神経突起伸長、神経細胞数の増加などをin vitroで評価をした後、臨床応用を見据え3次元のチューブを作成する予定である。、
• 金属微粒子を用いた選択的レーザー脱離イオン化TOF-MSによる複合糖質の機能探索

  日本学術振興会：科学研究費助成事業

  研究期間 : 2005年 -2007年 

  代表者 : 西村 紳一郎, 長堀 紀子, 比能 洋, 篠原 康郎

   

  本研究では"糖鎖提示ナノ微粒子および質量分析計"を用いることで、複合糖質の分子レベでの機能解明すなわち「相互作用相手分子を効率的に探索し、同定する新規方法論」を開発することを目的とした。 〈非特異吸着抑制型水溶性糖鎖提示ナノ微粒子の作製〉 本研究では、金属表面とチオール基との親和性を利用して金属ナノ微粒子表面修飾を行った。金属ナノ微粒子表面には糖鎖を〜数百分子程度提示できるため、相互作用相手分子との強い結合力が得られる(糖鎖クラスター効果)。また、アミノオキシ基と糖鎖還元末端との化学選択的反応を利用することであらゆる糖鎖を提示可能なシステムを構築した。これを利用し、糖鎖提示ナノ懲粒子のライブラリー化を行った。標的タンパク質を共有結合で捕捉可能な自殺基質型の糖鎖誘導体を微粒子表面に簡便に提示する方法の開発も合わせて行った。金属-チオール結合がレーザーによって切断されることを利用し、ナノ微粒子表面への糖鎖の提示をMALDI-TOF MSによって評価した。ナノ微粒子への非特異吸着を低減するために、双性イオン型チオール化合物を合成し、これを用いた。作製したすべてのナノ微粒子は高い水溶性および安定性を示した。 〈糖鎖相互作用分子の探索および同定方法の確立〉 本研究で作製したナノ微粒子は水溶性であるため、相互作用相手分子を捕捉後そのまま各種生化学的アッセイおよびMALDI-TOF MS測定に供することができる。生体試料からの相互作用相手分子(タンパク質)の捕捉においては、ナノ微粒子と試料溶液をインキュベートし洗浄後そのままSDS-PAGEにアプライし、目的のバンドを切り出してプロテオミクスの常法に従ってタンパク質の同定が可能であった。このように、競合糖鎖による溶出が必要なアフィニティークロマトグラフィー等の従来法と比較して、サンプルロスが少ないことによる高感度化、および作業工程の簡素化による大幅な效率化を達成した。
• 人工細胞外マトリックスの精蜜分子設計と靭帯および関節軟骨細胞再生への応用

  日本学術振興会：科学研究費助成事業

  研究期間 : 2003年 -2005年 

  代表者 : 眞島 任史, 岩崎 倫政, 門出 健次, 西村 紳一郎, 三浪 明男

   

  フィブロネクチン、コラーゲン、ヒアルロン酸レセプタータンパク質のモルフォロジー制御に応用して軟骨・腱・靱帯等の細胞接着・伸展制御実験に供した。またヒアルロン酸、アルギン酸、キトサンなどのグルコサミノグリカン(プロテオグリカン)のモデルとなる多糖とコラーゲン、フィブロネクチンなどの高分子ハイブリッドの簡易複合化法を考案した。具体的にはキトサンに細胞接着分子であるRGD(Arg-Gly-Asp)配列をキトサンに導入することに成功した。RGD配列をキトサンに導入することにより線維芽細胞および軟骨細胞の接着性を有意に改善することができた。 キトサンとヒアルロンサン、またはキトサンとアルギンサンを用いて新規な人工細胞外マトリックス(繊維成分)をデザインし作成した。これらの高分子材料をウサギ線維芽細胞および軟骨細胞培養系に加え細胞の増殖およびコラーゲンやヒアルロン酸などの細胞外マトリックス成分産性能をin vitroで評価し、本研究により合成された新規な人工細胞外マトリックス材料の構造・組成・物性と細胞接着・伸展との間の関係を議論する上で重要な基礎データベースを構築した。靭帯および関節軟骨再生にはキトサンとヒアルロンサンのハイブリッド細胞培養基材すぐれていた。キトサンにヒアルロンサンをハイブリッド化することによりその軟骨細胞および線維芽細胞に対する接着性が有意に改善し、さらに運動器再生用scaffoldとしての強度も上げることができ、生体内にある組成と同じ細胞外基質の産生が確認された。本効果は生体内でも認められ、従来軟骨は繊維軟骨、靭帯は瘢痕でしか修復しなかったが、本人工細胞外マトリックスを用いることで、正常組織として生体内で再生させることが可能となった。さらにキトサンとヒアルロンサンのハイブリッド細胞培養基材を用いてin vivoで生体内毒性試験でその安全性を確認した。
• 神経組織再生のための新規多機能生体内吸収性基材の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2003年 -2004年 

  代表者 : 三浪 明男, 西村 紳一郎, 眞島 任史, 岩崎 倫政

   

  昨年度は、神経組織再生用チューブの作成のため、基盤材料にRGDモチーフおよび細胞増殖因子(タンパク)を化学的に安定化した状態で導入する技術を確立した。本年度は、基盤材料に導入したタンパクが基盤材料の分解と共に放出され細胞に対し、有効な生理活性を持つことを証明する実験を行った。RGDモチーフおよび細胞増殖因子を導入した基盤材料は、コントロールに比べ明らかに高い細胞接着性および増殖性を示した。さらに、基盤材料の分解と共に導入されたタンパクは基盤から放出され、長期にわたりタンパク活性を維持することも明らかとなった。 しかし、導入基盤の持つこれらの細胞に対する作用は神経細胞に特異的ではないため、本年度はさらに、神経細胞に対しより特異的作用を持つと考えられる糖鎖を基盤に導入し、神経細胞に対する特異的生理活性の有無を確認した。基盤材料に、糖鎖をより安定化した状態で化学的に導入する方法については確立した。糖鎖を導入した基盤の神経細胞へ対する接着性および増殖性評価においては、コントロールに比べ両評価共に有意な向上を認めた。 これら新規に開発した基盤材料へのタンパクおよび糖鎖の化学的導入技術の確立およびin vitro評価におけるその有用性については証明された。今後は、基盤材料の生体内親和性および生分解性のコントロール技術をより確実なものにし、生体内における神経組織再生用チューブの完成と神経組織再生手法の確立を目指す。
• 乳酸菌表層への化学提示法を用いた経口ワクチンの開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 2003年 -2004年 

  代表者 : 西村 紳一郎

   

  バクテリア細胞壁へのムラミン酸誘導体導入による表面修飾という本手法の発展として、ムラミン酸誘導体をもちいた新しい抗菌剤について検討した。 ムラミン酸の4位をフッ素化した細胞壁前駆体誘導体(フッ素化パークヌクレオチド)を、N-アセチルガラクトサミンを出発原料として合成するルートを新たに確立した。このフッ素化パークヌクレオチドは、バクテリア細胞壁合成経路においてLipidIIの合成を阻害することにより、抗菌作用を示すことが期待される。この抗菌作用について乳酸菌を用いて調べた。菌株には、ラクトバシルス属のL.plantarum JCM1149を用いた。この細菌をフッ素化パークヌクレオチドを1mg/ml(0.87mMの,0.01mg/ml(0.0087mM)で含む培地(市販のラクトバチリMRSブロスを22.5g/lで蒸留水に溶かしたもの)に添加し、嫌気性条件下37℃で培養した。比較として、化合物(Ia)を含まない培地でも培養した。1、2、3、5、7、10時間後の細菌の個体数を、寒天培地上で形成されたコロニーを数えることにより算出した。各時間での細菌の個体数を算出した結果、フッ素化パークヌクレオチドを含む培地では含まない培地にくらべ、増殖率が低下することが確認された。これらの成果はOrg.Lett.2004,6,1753-1756に掲載された。
• 糖ペプチド医薬の分子設計

  日本学術振興会：科学研究費助成事業

  研究期間 : 2000年 -2002年 

  代表者 : 西村 紳一郎, 前川 宜彦, 近藤 裕郷, 西村 孝司, 前川 宣彦

   

  本研究課題は近年急速に研究が進展しつつある糖鎖工学分野における基盤技術の一つである機能分子設計法開拓の一環として位置づけられる。特に本課題においては糖ペプチドの分子設計を主たる目的としており、いずれの化合物も将来の創薬研究開発において鍵となるものであり、また合成・設計の標準的な指針を広く提供するものである。 以下の3つのタイプの異なる糖ペプチドについての合成法を確立し、各々の生理活性と構造に関する有効な知見を得た。 (1)不凍糖タンパク質(AFGP)の構造と機能に関する研究 (2)環状糖ペプチドからなるインフルエンザウイルス感染阻止剤の分子設計 (3)免疫応答制御作用を持つ新規2官能性糖ペプチドの分子設計
• 複合的バイオ新素材の開発に関する共同研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 1999年 -2001年 

  代表者 : 西 則雄, 覺知 豊次, 坂入 信夫, 西村 紳一郎, 野水 基義

   

  多くの生体関連高分子の中で、蛋白質、核酸、糖質等に関する研究は、極めて大きく進展している。ただ、それぞれの分野がそれぞれ独自の歴史と世界を持ち、各分野間の交流や共同研究は少ない。生命体を構成する成分は、多くの生体関連高分子やその周辺化合物よりなる複合体である事を考えると各分野が融合した分野にこそ多くの超機能性物質創出の可能性がある。 本研究では、核酸(DNA及びRNA)、ペプチド、蛋白質、糖質、脂質等、ほぼすべての生体関連有機化合物をカバーできる日独の研究者グループが、お互いの情報を密度濃く交換し、意見交換や議論を深く行うことにより研究の進展をはかり、生医学、環境化学等の多くの分野で、極めて新しいタイプのバイオ新素材を創出することを目的とする。 当該研究期間に、DNA-キトサン系二層式膜よりなる外科手術用接着材の調製、糖ペプチド及び糖タンパク質合成の基礎研究、紫外線照射によるDNAの固定化法の検討等を行った。例えば、紫外線照射法を利用して水不溶化DNAを含む各種素材、たとえば、DNAを固定化したガラスビーズ、不織布等を調製し、これらによるダイオキシン等環境ホルモン除去作用を検討した。特に母乳中のダイオキシン除去、タバコ煙中のベンツピレン、ダイオキシン除去等、具体的なプロジェクトを進めた。 さらに、核酸-タンパク質複合体であるDNA-コラーゲン複合体の構造や形成メカニズムにつき検討を進めた。 研究期間中、日独の研究者が相互訪問し、特に札幌ではProf.Kunz を迎えて"Sapporo Bioconjugate Colloquium"を開催し、研究討論と交流を行った。
• 糖鎖自動合成システムを用いる複合糖質の効率的合成研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 1999年 -2001年 

  代表者 : 西村 紳一郎

   

  初年度は糖転移酵素群による効果的な糖鎖伸長反応とその迅速な評価法を確立させた。次年度目は糖鎖自動合成装置の開発において鍵となる「糖転移酵素」をくり返し利用するために固定化の検討を行った。まず既存の固定化法を用いて酵素を固定化し利用することが可能であることを、β1,4-ガラクトシルトランスフェラーゼやα2,3-シアリルトランスフェラーゼで確認した。更に、糖転移酵素をマルトース結合蛋白質との融合蛋白質の状態で大腸菌に生産させ、側鎖にマルトースを持つ高分子に結合させる固定化法を開発した。 最終年度である平成13年度は、最終目標であった「糖鎖自動合成装置」を開発した。生合成を真似た方法論(バイオミメティック法)は実用化に耐えられる酵素活性の実現等に向けてさまざまな改良を行う必要はあるが、自動合成システムの構築にとって重要なブレークスルーとなることは間違いない。このようにして必須の2つのアイテム(酵素と基質)がそろったので図1に示したような自動合成装置の第一号機(試作機)が完成した。 このような一連の糖転移酵素と高分子型のプラーマー基質を用いる免疫制御薬の候補の一つとして期待されている細胞接着糖ペプチドやセレクチンのリガンドとして知られている複雑なスフィンゴ糖脂質なども合成できるようになった。 今後は実用化に向けた研究開発と共に、試作した糖鎖を用いて生理活性等の研究の発展に期待ができる。
• サケ白子DNAを含む機能性複合素材の開発とその利用

  日本学術振興会：科学研究費助成事業

  研究期間 : 1998年 -2001年 

  代表者 : 西 則雄, 覺知 豊次, 坂入 信夫, 西村 紳一郎, 平 敏夫, 山田 秀明, 長田 篤雄

   

  ほとんど全ての生物の遺伝子であるDNAは超高分子物質で、様々の物質と極めて特異性、選択性の高い相互作用をする機能性物質とも言える。我々はDNAをアルギン酸とのコンプレックスにする方法により、DNAをその天然構造を保ったまま効率的に不溶化する方法を見出し、この方法を用いて、多くの利用の道を開発した。一方、上記の研究の過程でDNAはコラーゲンとの複合体にもでき、特に医用高分子の分野で極めて有望な複合素材ができること、また、紫外線照射によりDNAをセルロース不織布、ガラスビーズ、シリカゲル等、様々な材料に結合させることができることなどが明らかになった。 本研究では上記の成果をさらに発展させ、特に北海道で多量に廃棄されているサケ白子DNAを含む各種の機能性複合素材を開発し、例えば環境ホルモン除去素材、生医学材料などとしての実用化を目指すものである。 研究期間中にコラーゲン水溶液をDNA水溶液中にノズルを通して押し出すと、DNA-コラーゲン複合体が形成され、この方法によりDNA-コラーゲン複合体のフィルム、繊維、ゲル等を調製できることがわかった。また、紫外線照射により架橋構造をもつ水不溶性DNAフィルムを調製できる事、この方法を使ってDNAをセルロース不織布、多孔質ガラスビーズ等、様々な材料に結合させ得ることも明らかになった。主としてこれらの知見をもとにして、DNAを含むポリイオンコンプレックスを利用した外科手術用補助素材及び皮膚に優しい創傷被覆材の開発、環境ホルモン集積、除去用担体の開発等の研究を行った。 また、紫外線照射法でDNAを固定化した素材の化学構造を明らかにした。またこの方法を応用して、母乳中に含まれるダイオキシンの選択的除去等のプロジェクトを推進した。さらに紫外線照射法に続くDNA固定化の一方法としてゾルーゲル法の応用を検討した。
• スーパーバイオシステムの高次認識糖鎖分子による構築

  日本学術振興会：科学研究費助成事業

  研究期間 : 1997年 -2000年 

  代表者 : 瓜生 敏之, 古川 清, 小林 一清, 西村 紳一郎, 畑中 研一, 秋吉 一成, 山形 達也

   

  (1)平成9年度〜平成11年度の3年間に6回の公開シンポジウムを開催した。また、本特定領域研究で得られた研究成果を広く社会に公表するための公開シンポジウムを平成13年1月23日に東京国際フォーラムにて開催した。本特定領域研究の研究成果発表の他に、他の領域の研究者による招待講演、次の領域への発展を含めた講演などを催し、特定領域研究に携わった研究者以外にも多数の参加者(特に企業からの参加者)があった。 (2)計画研究ならびに公募研究における各自の研究成果をまとめた。本特定領域研究において平成9年度〜平成11年度の3年間(公募研究に関しては1〜3年間)に行われた各研究者の研究成果をまとめた。 (3)各班ごとの研究成果をまとめた。4つの班すなわちA01班(糖鎖の超精密合成に関する基礎研究)、A02班(糖分子鎖集合体)、A03班(糖鎖による生命分子制御)、A04班(高次認識糖鎖による細胞機能制御)ごとに研究成果を集約し、本特定領域研究で得られた知見について考察し、当初の目標がどこまで達成されたかについて検討した。その結果、各班とも目標を上回る成果が得られたことを確認した。 (4)班間の共同研究成果も含めた領域全体としての成果について、総括班会議を開催して取りまとめを行った。その際に、各班間の連携が生み出した成果についても検討し、領域全体としての3年間の成果報告書を英文で作成した(総ページ数は316ページ)。 (5)研究成果は、946報の原著論文、187報の総説、1000件以上の口頭発表、121編の著書として公表した。 (6)A01班からA04班の研究者の中から各班5〜7名を選んで、本特定領域研究で得られた成果を中心にして、学会出版センターから『季刊化学総説:糖鎖分子の設計と生理機能』を出版予定である(印刷中)。
• 糖鎖の超精密合成に関する基礎研

  日本学術振興会：科学研究費助成事業

  研究期間 : 1997年 -1999年 

  代表者 : 西村 紳一郎, 碓氷 泰市, 木曽 真, 葛原 弘美, 坂入 信夫, 伊藤 幸成

   

  昨年度はGM3をはじめそのアナログの合成に有効なプライマーが選定できたので、平成11年度はこの手法を拡張してシアリルルイスX構造を含む複雑なガングリオシドならびにラクトサミノグリカン鎖の構築などに応用することに成功した(西村)。 グロボ3糖を12個、6個、4個結合させた、各種カルボシランデンドリマーを調整して、病原性大腸菌やその毒素への接着活性を比較検討した。このうち、12個のグロボ3糖を結合させた第一世代デンドリマーの合成を達成した。同様の手法を用いて、アレー型第1世代デンドリマーに6個の、第0世代デンドリマーに4個のグロボ3糖を結合させた化合物を新たに合成した(葛原)。 新しいシアリルルイスX関連化合物の精密合成と糖鎖抗体の作成により、白血球交通、炎症、がん転移、血液凝固などの生命現象に深く関わるセレクチンの特異的な生体内リガンド構造を検索し、その分子機構を明らかにした。特に、6-sulfode-N-acetyl-sialyl LeXとその関連ガングリオシドの系統的精密合成によりL-セレクチンの特異的リガンド構造を解明し、白血球交通に関わる全く新しい動的制御システムの存在を分子レベルで明らかにした。さらに、P-セレクチンのカウンター・レセプターとしてムチン型コア構造を含むシアリルルイスX誘導体を系統的に構築し、特異抗体の反応性と分子構造を明らかにする。他方、脳神経機能に重要な働きをするガングリオ系ガングリオシドの系統的精密合成の一貫として、ハイブリッド型ガングリオシド及び新規ポリシアロガングリオシドの合成を行って、コリン作動性ニユーロンならびにミエリン形成における分子機能を解明した(木曾)。 量産化に成功したラクト-N-テトラオースやネオテトラオースを出発原料にフコシル5糖LexやH型オリゴ糖の酵素合成法を確立した。これらのオリゴ糖を高分子に組み込んだ細胞表層糖鎖モデルやムチン型糖鎖高分子を構築し、バイオミメティック材料としての有効化を促進した(碓氷)。 高次の糖鎖構造の合成に関する基礎研究を継続的に行う。特に以下の3点に重点をおいて検討を行った。 1)新規糖タンパク糖鎖構造の構築 2)糖タンパク化学合成をめざした方法論の開拓 3)立体選択的グリコシル化反応の固相合成への展開(伊藤)。 これまでの研究で見い出されたラミナリビオースやコンドロシンなど多糖の限定分解で得られる二糖類の位置選択的な化学変換法を基に植物の生長を制御する環状糖脂質及びその類縁体の短工程合成法を確立した。また、構造と生理活性の相関を検討した(坂入)。
• 新しい蛍光性糖質化合物の合成と生化学的利用

  日本学術振興会：科学研究費助成事業

  研究期間 : 1998年 -1998年 

  代表者 : 西村 紳一郎, 西 則雄, LEE Yuan C.

   

  生体内で重要な働きをしている糖転移酵素はその分布活性を調べるのが困難である。このことが糖鎖機能や生合成システムの謎を解明する妨げとなっている。たとえば、受精や老化等の様々な生命現象の解明を遅らせている。 そのため、この反応を容易に測定できる可能性の高いエネルギートランスファーの手法を用いることを試みた。これは酵素の二つの基質間の蛍光強度の変化を測定するだけで酵素活性を測定することができる方法で、非常に簡単で放射能を使わないので安全である。このために蛍光ラベルをした種々の糖転移酵素の基質を合成した。 現在までターゲットにした糖転移酵素はβ-1,4-ガラクトシルトランスフェラーゼとシアリルトランスフェラーゼである。 β-1,4-ガラクトシルトランスフェラーゼの基質についてはガラクトースの6位をナフチルアミンで蛍光ラベルしたUDP-Galとナフチルメチルアミンで蛍光ラベルしたUDP-Galの合成に成功した(蛍光ドナー)。また、モノマー型またはポリマー型でグルコサミンの1位にスペーサーを挟んで(β型)ダンシル基で蛍光ラベルした化合物を合成した(蛍光アクセプター)。前者の蛍光ドナーを用いた場合は、見事に酵素活性を測定することができたが、エネルギートランスファーの効率が悪く、後者はナフチルメチル基が立体障害となったためか、糖転移反応が起こらなかった。このため2位にナフチルメチル基を導入したUDP-Galを合成した。 シアリルトランスフェラーゼの基質については、9位にナフチルメチル基を導入したCMP-NANAの合成に成功した(蛍光ドナー)。また、1位にスペーサーを挟んで(β型)ダンシル基を導入したガラクトースを合成した(蛍光アクセプター)。これらの基質を使って酵素活性を測定した。
• 生体基盤材料の開発

  日本学術振興会：科学研究費助成事業

  研究期間 : 1997年 -1997年 

  代表者 : 前田 瑞夫, 山下 俊, 相田 卓三, 長崎 幸夫, 西村 紳一郎, 高橋 保

   

  近年、世界における材料研究の大きな流れとして、より高次な生体機能が注目されるようになってきた。一方、物性の優れた高分子材料をどのようにして生体内で働かせるのかということも重要な課題となりつつある。この様な趨勢を踏まえ、本企画調査研究では、高分子材料の機能発言と生体親和性、あるいは生体内輸送を軸に、生体分子と親和性のある新しい高分子(高分子複合体)を合成し、生体におけるその作用機構を高分子化学に基づく分子レベルで研究することを目的とした。 このため、異なる分野の専門的知見に基づく研究討論を促進し、異なる高分子および生体分子を扱う研究者間の情報交換を円滑に進めるべく、以下の活動を行った。 (1)本企画調査研究の公開シンポジウムを開催して、生体基盤材料の今後の研究の方向性について議論を行い、お互いの理解を深めた。 (2)インターネット上にメイリンググループを結成し、電子メイルを用いて研究分担者間の遠隔討諭を繰り返し行うことにより、分子レベルで生体系を理解するための方法論について研究した。 (3)特定領域研究の申講のための準備委員会を11回行った。 (4)研究成果報告書を作成し、分担者それぞれの研究方法と成果を公表した。 これらの活動を通じて研究方法に関するコンセンサスを構築し、これを取りまとめて平成10年度発足・科学研究費「特定領域研究(B)」に領域申請を行った。
• DNAの高度機能性素材としての利用

  日本学術振興会：科学研究費助成事業

  研究期間 : 1995年 -1997年 

  代表者 : 西 則雄, 長田 篤雄, 平 敏夫, 西村 紳一郎, 久保木 芳徳, 戸倉 清一

   

  DNAを遺伝子としてではなく高度機能性素材として、たとえば細胞培養用バイオマトリックス、抗DNA抗体検出用素材、発癌性物質検出用フィルターなどの各種生医学材料、抗菌性物質徐放性材料、さらには高選択性クロマトグラフィーの担体などとして実用化を目指す研究を行った。 たとえば、酸性多糖のアルギン酸でDNAを不溶化する方法を見出し、DNA-アルギン酸フィルム、ゲル、繊維等を調整することができた。この方法でDNAをミリポア-フィルターに固定化し、発癌性の環境汚染物質を特異的に集積、あるいは除去するフィルターを開発した。 さらにDNA-アルギン酸を用いたELISA法により抗DNA抗体の検出が可能となったことから、臨床的なデータとあわせてこの系の有用性を追求し,診断法として確立する基礎を築いた。 また、不溶化DNA-金属錯体をアルギン酸を用いてフィルム化し抗菌性を付与した徐放性機能性物質を作製し、その抗菌活性について細胞培養法より検討した。その結果、銀イオンを配位させたDNA-アルギン酸フィルムが抗菌性フィルムとして極めて有用であることを見出した。すなわち、DNAを用いる事によりアルギン酸フィルム中に銀をより多く固定化できることがわかり、また、そのように調整されたフィルムは大腸菌および黄色ブドウ球菌の生育を阻害することが示された。
• スーパーバイオシステムの高次認識糖鎖分子による構築

  日本学術振興会：科学研究費助成事業

  研究期間 : 1996年 -1996年 

  代表者 : 瓜生 敏之, 重政 好弘, 葛原 弘美, 西村 紳一郎, 小林 一清, 山形 達也

   

  重点領域研究「スーパーバイオシステムの高次認識糖鎖分子による構築」、略称「スーパー糖鎖分子」の採用決定に伴い配布された基盤研究(B)で、研究代表者と22名の研究分担者が個々に研究を行った。研究分野は4つに大別される。第一は新しい糖鎖合成を研究したもので、「糖鎖自動合成の可能性」、「複合糖鎖の効率的合成を目指して」、「オリゴ糖鎖ライブラリー作成のための実践的合成法の開発」などが研究され、講演会で発表された。第二は糖分子鎖集合体の合成と機能に関するもので、「スーパーバイオリガンド機能を持つ人工複合糖質ポリマー」、「高分子ロタキサンの合成」、「硫酸化ポリ(グリコシルオキシエチルメタクリレート)の細胞および生体分子との相互作用」などが研究された。第三は生理活性を有する多糖・オリゴ糖の合成と物性に関するもので、「多糖・オリゴ糖エイズ薬」、「キチン、キトサンの生体内分分解性」、「溶液中におけるヒアルロン酸の構造と物性」などが研究され成果を挙げた。第四は細胞機能制御ができる糖鎖分子に関して研究され、「神経突起伸長に必須のガングリオシドGMl」、「神経回路形成と脳特異プロテオグリカン」、「活性化に伴うヒト末梢白血球表面の生化学的変化の解析」などに研究成果を挙げた。平成9年度から3年間「スーパー糖鎖分子」の重点領域研究が実施されるに当り、現段階の研究水準や進行状況を計画班員が討議しあったのが、平成9年1月28日に開かれた講演会においてであった。本重点領域研究が行われると、本研究分野が世界をリ-ドする優れた研究領域へ導かれるような予感が感じられた。
• キトサン医用材料の生体適合性に関する研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 1993年 -1995年 

  代表者 : 西村 紳一郎, ラッキエビッチ B, ポロビンスキー S, リップシモノビッチ B, ウルバンチェク G, ショスランド L, ツーラ M, クチャルスカ M, ダトキエビッチ J, 高井 光男, 西 則雄, 戸倉 清一, 東 市郎, リップシモノビッチ B., ラッキエビッチ B., ウルバンチェク G., ボロビンスキー S., ショスランド L., ツーラー M., クチャルスカ M., ダトキエビッチ J., ツーラ M.

   

  平成5年度および6年度に得られた基礎的データをもとに応用・実用化に向けた以下の各項目を詳しく検討した。 (1)これまでに合成された各種硫酸化キチン誘導体・キトサン誘導体の抗血栓性ならびに抗エイズウイルス活性等を精査する。これらのデータをもとに合成高分子表面のコーティング素材としての適性を確認する。 (2)人工血管素材となり得るキチン中空系を製作するための新手法を開発する。とくに,これまでに見られない新しい安全な溶媒系の発見に努力する。また,この繊維の力学物性をはじめ生体適応性・抗ウイルス性などの基本物性を評価する。 (3)小動物・大動物の体内にこれらキチン・キトサン由来の医用材料を埋めこんだ際の免疫反応・炎症反応に関するデータを蓄積する。 【方法・研究経過】 (1)西村らにより始めて開発されたN-フタロイル化法にもとずく,キトサンの有機溶媒への可溶化法を利用して,有用な鍵中間体である6-0-ナリチルキトサンと6-0-トリチルキチンを硫酸化後,脱トリチル化して,3位のみ,あるいは2,3位の硫酸化された新しい硫酸化多糖を位置選択的に合成した。これらの構造はNMR,とくに^<13>C-NMRにおける環炭素シグナルの硫酸化による顕著な低磁場シフトにより確認できた。 (2)上記の新しい硫酸化多糖の抗血栓性を主としてアメリカ薬局方に記載された実験方法に従って評価した。その結果,6位への硫酸化による6S体で92.6unit/mg,3位への置換体である3S体で2.8unit/mg,さらに,2位と3位への置換体である23S体で3.4unit/mgの抗血栓性を示した。これは,この活性が硫酸基の量ではなく,6位の第1級水酸基側への置換が有効であり,以然に発見した一連のヘパリノイドの性質に矛盾しない。一方,2位,あるいは3位の面(Face)への硫酸化では全くこの活性が見られない事から,少なくともトロンビンへの結合については6位硫酸基の存在が必須である事が明確となった。 (3)これらの抗エイズ活性をin vitroでチェックしたところ,逆に23S体および3S体が各々0.14μg/mL,4.83μg/mLという低濃度で抗エイズウイルス活性を発現したのに対して,6S体では28.5μg/mLを必要とする事から,エイズウイルスの主要なエンベロープタンパク質であるGP-120の塩基性配列部分への効果的な結合にとってはC-2位とC-3位の側の硫酸基が重要な因子となっている事が初めて確かめられた。この活性は,これまで高いエイズウイルス感染阻害活性が知られているカードラン硫酸に匹敵する程の高い活性であった。現在,in vivoにおける実験および安全性・毒性試験等を検討中であるが,今後,極めて有望な医薬・人工臓器表面被覆剤に応用される可能性が高い。 (4)ポーランド側ウッジ工科大学グループではキチン系化合物の成型・加工法についての改良・大巾機能向上を目的とした紡糸装置および膜物生評価装置等が開発された。これらの手法により,キチン等の多糖系繊維の大量かつコンスタントな物性を持った調製が可能となった。一方,戸倉はキチンの新しい溶媒として塩化カルシウム-メタノール系を発見した。この系は,中性であるため,主鎖の分解は全く越らず,温度を60℃程度で数日混合・撹拌するだけで均一で粘稠な溶液を得る事を見出した。この発見により,キチンの紡糸は飛躍的に簡素化できた。これらの手法を駆使して現在,中空糸開発の研究が進行中であり,今後,人工血管材料への応用が急速に実現化するものと考えられる。 以上,新規キチン硫酸の開発と繊維科学のハイブリッド化により,生体適合性医用多糖の研究指針となる大きな研究成果が得られた。
• 自己組織形成能を有する新しい両親媒性多糖の分子設計

  日本学術振興会：科学研究費助成事業

  研究期間 : 1994年 -1994年 

  代表者 : 西村 紳一郎

   

  上記研究課題に関し,次の各実験データ・実績を得た。 (1)鍵中間体となる種々の新規誘導体の位置選択的合成に成功した。これらの化合物はいづれも良好な溶解性を示し,両親媒性の付与・脱保護により容易に新しい両親媒性キトサン誘導体に変換できることを見出した。 (2)正電荷・負電荷を有する化合物について単分子膜形成能およびリポソーム形成能について検討したところ,いづれも安定で再現性良く分子集合体を形成できる事がわかった。 (3)リポソームに取り込まれた糖鎖はWGAなどのレクチンによって特異的に認識される事を見出し,薬物を特異的臓器に運搬するいわゆるターゲッティング機能を有する事も発見した。
• 多糖の分子設計による分離および不斉認識機能の発現

  日本学術振興会：科学研究費助成事業

  研究期間 : 1989年 -1989年 

  代表者 : 栗田 恵輔, 西村 紳一郎

   

  キチンの構造特異性を生かした機能化研究を進め、以下の結果を得た。 1)キチンに対するアラニンNCAのグラフト共重合により、ポリペプチド側鎖を導入する方法を確立した。次に、その側鎖末端に補酵素NADHの活性部位を固定化して高分子不斉還元試薬とし、還元反応におけるスペ-サ-の有効性を明らかにした。また、より効果的なスペ-サ-として、アラニン1分子を介してNADHモデルを固定化した。 2)新しい化学修飾法の開発の一環としてラクトンを用いたヒドロキシアシル化を検討した。生成物の高い吸湿性を示す。キトサンのアミノ基の約50%を特殊な条件下でアセチル化すると水溶性キチンが生成することを見いだした。この調製法は従来法に比べてきわめて簡便であり、多くの利点をもつ。また、キチンを効率的にDEAE化する方法を提示し、さらに、その誘導体が分離用の充填剤として有効であることを報告した。また、アミノ基およびメルカプト基を導入する方法を開発した。 3)トシル化およびヨウ素化キチンを用いてグラフト共重合を行った。ルイス酸の存在下、およびエキシマ-レ-ザ-照射下での反応により、それぞれカチオン的、およびラジカル的な共重合が可能であることを明らかにした。また、オキサゾリンのグラフト共重合、およびセリウム塩による共重合も報告した。共重合体は親水性材料として有用である。 4)キトサンに嵩高い基を導入すると溶媒に可溶になるため、多くの化学修飾を均一溶液中で進めることができるようになった。これにより位置特異的に糖を側鎖として導入する方法が開発され、構造的に明確な分枝キチンの調製を可能にした。また、N-アセチルグルコサミンを不飽和アルコ-ルでグリコシル化したものはラジカル重合性を示し、新しいタイプの複合糖質モデルを調製するのに有用である。これらの誘導体は生体活性成分に対して特異性の高い認識機能を発現すると期待される。

産業財産権

• 特許5682767:糖鎖捕捉分子を用いた糖鎖精製濃縮法および糖鎖構造解析法  

  西村 紳一郎, 新倉 謙一, 中川 裕章, 岡山 峰伸  塩野義製薬株式会社  201303087879113624
• PCT/JP/018094:細胞内物質移送システムおよびその利用  
• 特許5958911:糖ペプチドアレイ  
• 特許5916017:新規なMUC1抗体  
• 特許5978204:がん関連糖ペプチドエピトープ、抗体および使用方法  
• 特許5773352:抗MUC1抗体  
• 特許5669081:生体分子固定化担体および生体分子担体固定化方法