Researcher Database

Shin-Ichiro Nishimura
Faculty of Advanced Life Science Advanced Transdisciplinary Science Drug Discovery Research

Researcher Profile and Settings


  • Faculty of Advanced Life Science Advanced Transdisciplinary Science Drug Discovery Research

Job Title

  • Professor


J-Global ID

Research Interests

  • Glycomedicine   Glycoconjugate   antibody   cancer specific epitope   organic chemistry   epitope   Medicinal chemistry   Chemical Biology   Glycotechnology   Bio-organic chemistry   

Research Areas

  • Nanotechnology/Materials / Chemical biology / Nanosome, exosome, DDS
  • Nanotechnology/Materials / Molecular biochemistry / Nanosome, exosome, DDS
  • Nanotechnology/Materials / Structural/physical organic chemistry / Nanosome, exosome, DDS
  • Nanotechnology/Materials / Nanobioscience / Nanosome, exosome, DDS
  • Life sciences / Genomics / glycotechnology, medicinal chemistry, epitope engineering
  • Life sciences / Structural biochemistry / glycotechnology, medicinal chemistry, epitope engineering
  • Life sciences / Bioorganic chemistry / glycotechnology, medicinal chemistry, epitope engineering
  • Life sciences / Applied biochemistry / glycotechnology, medicinal chemistry, epitope engineering

Educational Organization

Academic & Professional Experience

  • 2019/12 - Today 遠友ファーマ株式会社
  • 2006/04 - Today Hokkaido University
  • 2012/08 - 2022/03 医化学創薬株式会社
  • 2010/11 - 2012/08 医化学創薬合同会社
  • 2002/06 - 2010/03 AIST Hokkaido
  • 1995/04 - 2006/03 Hokkaido University
  • 1993/04 - 1995/03 Hokkaido University
  • 1993/07 - 1994/04 Johns Hopkins University 文部省在外研究員
  • 1992/04 - 1993/03 Hokkaido University
  • 1989/04 - 1992/03 Seikei University
  • 1988/04 - 1989/03 RIKEN
  • 1987/07 - 1988/03 RIKEN
  • 1986/04 - 1987/06 Hokkaido University


  • 1984/04 - 1987/06  Hokkaido University
  • 1982/04 - 1984/03  Hokkaido University
  • 1978/04 - 1982/03  Hokkaido University

Association Memberships

  • アメリカ化学会   日本化学会   高分子学会   日本臨床化学会   

Research Activities

Published Papers

  • Lareno L. Villones, Anna-Kristin Ludwig, Seiya Kikuchi, Rika Ochi, Shin-Ichiro Nishimura, Hans-Joachim Gabius, Herbert Kaltner, Hiroshi Hinou
    ChemBioChem 1439-4227 2023/03/09 [Refereed]
  • Hajime Wakui, Yasuhiro Yokoi, Chieko Horidome, Toyoyuki Ose, Min Yao, Yoshikazu Tanaka, Hiroshi Hinou, Shin-Ichiro Nishimura
    RSC Chemical Biology 2023 [Refereed][Not invited]
    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).
  • Michiru Otaki, Nozomi Hirane, Yayoi Natsume-Kitatani, Mari Nogami Itoh, Masanori Shindo, Yoichi Kurebayashi, Shin-Ichiro Nishimura
    Scientific Reports 12 (1) 17804  2022/10/24 [Refereed][Not invited]
    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.
  • 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  2022/10/23 [Refereed]
    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.
  • Takahiko Matsushita, Hiroshi Hinou, Shin-Ichiro Nishimura
    Chemistry Letters 51 1044 - 1048 0366-7022 2022/09/16 [Refereed]
  • 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 2022/07/21 [Refereed]
    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.
  • 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 [Refereed][Not invited]
    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.
  • Koidea R, Hirane N, Kambea D, Yoko Y, Otakia M, Nishimura S-I
    Biomaterials 280 121314  2022/01 [Refereed][Not invited]
  • 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 2041-6520 2020/12 [Refereed]
    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.
  • 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 1021-335X 2020/10/08 [Refereed][Not invited]
    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.
  • Yasuhiro Yokoi, Shin‐Ichiro Nishimura
    Chemistry – A European Journal 26 (54) 12363 - 12372 0947-6539 2020/09/25 [Refereed][Not invited]
  • 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 0022-2623 2020/08/13 [Refereed][Not invited]
    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.
  • 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 2020/03/31 [Refereed][Not invited]
    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.
  • 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 2020/02 [Refereed][Not invited]
    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.
  • 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 2041-6520 2020 [Refereed][Not invited]

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

  • 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) 2019/12 [Refereed][Not invited]
  • Ryosuke Koide, Shin‐Ichiro Nishimura
    Angewandte Chemie International Edition 58 (41) 14513 - 14518 1433-7851 2019/10/07 [Refereed][Not invited]
  • Sho Hideshima, Hiroki Hayashi, Hiroshi Hinou, Shunsuke Nambuya, Shigeki Kuroiwa, Takuya Nakanishi, Toshiyuki Momma, Shin-Ichiro Nishimura, Yoshihiro Sakoda, Tetsuya Osaka
    Scientific reports 9 (1) 11616 - 11616 2019/08/12 [Refereed][Not invited]
    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.
  • 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 1567-5769 2019/07 [Refereed][Not invited]
  • Hinou Hiroshi, Kikuchi Seiya, Ochi Rika, Igarashi Kota, Takada Wataru, Nishimura Shin-Ichiro
    BIOORGANIC & MEDICINAL CHEMISTRY 27 (13) 2822 - 2831 0968-0896 2019/07/01 [Refereed][Not invited]
  • Gebrehiwot AG, Melka DS, Kassaye YM, Gemechu T, Lako W, Hinou H, Nishimura SI
    BMC cancer 19 (1) 588  2019/06 [Refereed][Not invited]
  • Sanes JT, Hinou H, Lee YC, Nishimura SI
    Journal of agricultural and food chemistry 67 (1) 531 - 540 0021-8561 2019/01 [Refereed][Not invited]
    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.
  • 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 2018/09 [Refereed][Not invited]
  • Yogesh KV, Kamiyama T, Ohyama C, Yoneyama T, Nouso K, Kimura S, Hinou H, Nishimura SI
    MedChemComm 9 (8) 1351 - 1358 2040-2503 2018/08 [Refereed][Not invited]
    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 (一社)日本消化器外科学会 73回 685 - 685 2018/07
  • 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  1664-3224 2018/04/27 [Refereed][Not invited]
    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.
  • Gebrehiwot AG, Melka DS, Kassaye YM, Rehan IF, Rangappa S, Hinou H, Kamiyama T, Nishimura SI
    PloS one 13 (12) e0209515  2018 [Refereed][Not invited]
  • Ishihara S, Aoki K, Mizutani T, Amano M, Nishimura SI, Haga H
    Cell structure and function 43 (2) 177 - 185 0386-7196 2018 [Refereed][Not invited]
  • 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) 2017/12/06 [Refereed][Not invited]
    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.
  • 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 0002-7863 2017/12 [Refereed][Not invited]
    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.
  • Gerard Artigas, Joao T. Monteiro, Hiroshi Hinou, Shin-Ichiro Nishimura, Bernd Lepenies, Fayna Garcia-Martin
    JOURNAL OF MEDICINAL CHEMISTRY 60 (21) 9012 - 9021 0022-2623 2017/11 [Refereed][Not invited]
    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.
  • Takayuki Furukawa, Hiroshi Hinou, Seiji Takeda, Hitoshi Chiba, Shin-Ichiro Nishimura, Shu-Ping Hui
    CHEMBIOCHEM 18 (19) 1903 - 1909 1439-4227 2017/10 [Refereed][Not invited]
    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.
  • Shun Hayakawa, Yasuhiro Yokoi, Hiroshi Hinou, Shin-Ichiro Nishimura
    BIOCHEMISTRY 56 (33) 4379 - 4391 0006-2960 2017/08 [Refereed][Not invited]
    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.
  • 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) 1422-0067 2017/08 [Refereed][Not invited]
    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.
  • Nagaraju Brijesha, Shin-Ichiro Nishimura, Huligerepura Sosalegowda Aparna
    Journal of Agricultural and Food Chemistry 65 (8) 1496 - 1506 1520-5118 2017/03/01 [Refereed][Not invited]
    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.
  • 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 2470-1343 2017 [Refereed][Not invited]
    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.
  • Gerard Artigas, Hiroshi Hinou, Fayna Garcia-Martin, Hans-Joachim Gabius, Shin-Ichiro Nishimura
    CHEMISTRY-AN ASIAN JOURNAL 12 (1) 159 - 167 1861-4728 2017/01 [Refereed][Not invited]
    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.
  • Solomon T. Gizaw, Tetsu Ohashi, Masakazu Tanaka, Hiroshi Hinou, Shin-Ichiro Nishimura
    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1860 (8) 1716 - 1727 0304-4165 2016/08 [Refereed][Not invited]
    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.
  • 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 0032-0781 2016/08 [Refereed][Not invited]
    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.
  • Mamatha Bhanu L.S., S.-I. Nishimura, Aparna H.S.
    International Journal of Biological Macromolecules 88 138 - 145 0141-8130 2016/07 [Refereed][Not invited]
  • Naoki Ohyabu, Kiyoshi Kakiya, Yasuhiro Yokoi, Hiroshi Hinou, Shin-Ichiro Nishimura
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 138 (27) 8392 - 8395 0002-7863 2016/07 [Refereed][Not invited]
    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.
  • Inafuku S, Noda K, Amano M, Nishimura S, Ishida S
    Current eye research 41 (5) 721 - 724 0271-3683 2016/05 [Refereed][Not invited]
    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.
  • 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) 1536-5964 2016/04/01 [Refereed][Not invited]
    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.
  • Shun Hayakawa, Ryosuke Koide, Hiroshi Hinou, Shin-Ichiro Nishimura
    BIOCHEMISTRY 55 (5) 776 - 787 0006-2960 2016/02 [Refereed][Not invited]
    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.
  • Hiroshi Hinou, Yuya Abe, Shun Hayakawa, Kentaro Naruchi, Naoki Fujitani, Shin-Ichiro Nishimura
    TETRAHEDRON LETTERS 57 (7) 791 - 795 0040-4039 2016/02 [Refereed][Not invited]
    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.
  • R. S. Tan, H. Hinou, S.-I. Nishimura
    RSC Advances 6 (56) 50833 - 50836 2016 [Refereed][Not invited]

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

  • Shobith Rangappa, Gerard Artigas, Risho Miyoshi, Yasuhiro Yokoi, Shun Hayakawa, Fayna Garcia-Martin, Hiroshi Hinou, Shin-Ichiro Nishimura
    MEDCHEMCOMM 7 (6) 1102 - 1122 2040-2503 2016 [Refereed][Not invited]
    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 [Refereed][Not invited]
  • 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 Nature Publishing Group 6 18703 - 18703 2045-2322 2016/01 [Refereed][Not invited]
    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.
  • Rehan IF, Ueda K, Mitani T, Amano M, Hinou H, Ohashi T, Kondo S, Nishimura SI
    J. Agricul. Food Chem. 63 (48) 10578 - 10590 0021-8561 2015/12 [Refereed][Not invited]
    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.
  • 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 2015/12 [Refereed][Not invited]
    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.
  • 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 0002-7863 2015/10 [Refereed][Not invited]
  • Gizaw ST, Koda T, Amano M, Kamimura K, Ohashi T, Hinou H, Nishimura S
    Biochimica et biophysica acta 1850 (9) 1704 - 1718 0006-3002 2015/09 [Refereed][Not invited]
  • Mamatha Bhanu L.S, Amano M, Nishimura S-I, Aparna H.S
    Glycoconj. J. 32 625 - 634 2015/08 [Refereed][Not invited]
  • 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 0146-0404 2015/08 [Refereed][Not invited]
    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.
  • 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 1424-3903 2015/07 [Refereed][Not invited]
  • Tan RS, Naruchi K, Amano M, Hinou H, Nishimura SI
    ACS Chemical Biology 10 (9) 2073 - 2086 1554-8929 2015/07 [Refereed][Not invited]
    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.
  • Koji Miyahara, Kazuhiro Nouso, Yuki Morimoto, Hideaki Kinugasa, Hironari Kato, Naoki Yamamoto, Koichiro Tsutsumi, Kenji Kuwaki, Hideki Onishi, Fusao Ikeda, Shinichiro Nakamura, Hidenori Shiraha, Akinobu Takaki, Taku Nakahara, Yoshiaki Miura, Hidehisa Asada, Maho Amano, Shin-Ichiro Nishimura, Kazuhide Yamamoto
    Pancreas 44 (4) 551 - 556 2015/05 [Refereed][Not invited]
    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.
  • 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 3 30 (3) 528 - 534 0815-9319 2015/03 [Refereed][Not invited]
    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.
  • Kimura Katsuki, Nishimura Shin-Ichiro, Miyoshi Risho, Hoque Asiful, Miyoshi Taro, Watanabe Yoshimasa
    BIORESOURCE TECHNOLOGY 179 180 - 186 0960-8524 2015/03 [Refereed][Not invited]
  • 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 2014/12 [Refereed][Not invited]
  • 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 0947-6539 2014/12 [Refereed][Not invited]
    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.
  • 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 1386-6346 2014/12 [Refereed][Not invited]
    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.
  • Fayna Garcia-Martin, Takahiko Matsushita, Hiroshi Hinou, Shin-Ichiro Nishimura
    CHEMISTRY-A EUROPEAN JOURNAL 20 (48) 15891 - 15902 0947-6539 2014/11 [Refereed][Not invited]
    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.
  • 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 0270-4137 2014/11 [Refereed][Not invited]
    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.
  • Michiyo Terashima, Maho Amano, Tomohiro Onodera, Shin-Ichiro Nishimura, Norimasa Iwasaki
    STEM CELL RESEARCH 13 (3) 454 - 464 1873-5061 2014/11 [Refereed][Not invited]
    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.
  • 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 0721-832X 2014/08 [Refereed][Not invited]
  • Shingo Hatakeyama, Maho Amano, Yuki Tobisawa, Tohru Yoneyama, Norihiko Tsuchiya, Tomonori Habuchi, Shin-Ichiro Nishimura, Chikara Ohyama
    JOURNAL OF UROLOGY 191 (3) 805 - 813 0022-5347 2014/03 [Refereed][Not invited]
    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.
  • 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 0304-4165 2014/03 [Refereed][Not invited]
    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.
  • Junya Ishida, Hiroshi Hinou, Kentaro Naruchi, Shin-Ichiro Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 24 (4) 1197 - 1200 0960-894X 2014/02 [Refereed][Not invited]
    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.
  • Koji Miyahara, Kazuhiro Nouso, Yasuhiro Miyake, Shinichiro Nakamura, Shuntaro Obi, Maho Amano, Kazuko Hirose, Shin-ichiro Nishimura, Kazuhide Yamamoto
    HEPATOLOGY 59 (1) 355 - 356 0270-9139 2014/01 [Refereed][Not invited]
  • 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 1751-7362 2014/01 [Refereed][Not invited]
    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.
  • Maho Amano, Hiroshi Hinou, Risho Miyoshi, Shin-Ichiro Nishimura
    Methods in Molecular Biology 1200 361 - 369 1064-3745 2014 [Refereed][Invited]
    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.
  • 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 0304-4165 2014/01 [Refereed][Not invited]
    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.
  • Ravi Kumar HV, Naruchi K, Miyoshi R, Hinou H, Nishimura S
    Organic letters 15 (24) 6278 - 6281 1523-7052 2013/12/20 [Refereed][Not invited]
  • 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  1932-6203 2013/10/07 [Refereed][Not invited]
    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.
  • 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 0944-1174 2013/10 [Refereed][Not invited]
    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.
  • MATSUSHITA Takahiko, HANDA Seiji, NARUCHI Kentaro, GARCIA-MARTIN Fayna, HINOU Hiroshi, NISHIMURA Shin-Ichiro
    Polymer journal Nature Publishing Group 45 (8) 854 - 862 0032-3896 2013/08 [Not refereed][Invited]
  • 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 (一社)日本消化器外科学会 68回 O - 5 2013/07
  • 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 0270-9139 2013/06 [Refereed][Not invited]
    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;)
  • Sho Hideshima, Hiroshi Hinou, Daisuke Ebihara, Ryosuke Sato, Shigeki Kuroiwa, Takuya Nakanishi, Shin-Ichiro Nishimura, Tetsuya Osaka
    ANALYTICAL CHEMISTRY 85 (12) 5641 - 5644 0003-2700 2013/06 [Refereed][Not invited]
    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 日本臨床分子医学会 50回 76 - 76 2013/04
  • 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 0947-6539 2013/03 [Refereed][Not invited]
    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.
  • Takayuki Furukawa, Misaki Arai, Fayna Garcia-Martin, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura
    GLYCOCONJUGATE JOURNAL 30 (2) 171 - 182 0282-0080 2013/02 [Refereed][Not invited]
    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 and Streptomyces hyalurolyticus (EC 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.
  • 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 0166-0934 2013/02 [Refereed][Not invited]
    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.
  • Feng F, Sakoda Y, Ohyanagi T, Nagahori N, Shibuya H, Okamastu M, Miura N, Kida H, Nishimura SI
    Antiviral therapy 1359-6535 2013/02 [Refereed][Not invited]
  • Noriko Nagahori, Tadashi Yamashita, Maho Amano, Shin-Ichiro Nishimura
    CHEMBIOCHEM 14 (1) 73 - 82 1439-4227 2013/01 [Refereed][Not invited]
    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.
  • Hirokazu Kai, Hiroshi Hinou, Kentaro Naruchi, Takahiko Matsushita, Shin-Ichiro Nishimura
    CHEMISTRY-A EUROPEAN JOURNAL 19 (4) 1364 - 1372 0947-6539 2013/01 [Refereed][Not invited]
    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.
  • Takahiko Matsushita, Naoki Ohyabu, Naoki Fujitani, Kentaro Naruchi, Hiroki Shimizu, Hiroshi Hinou, Shin-Ichiro Nishimura
    BIOCHEMISTRY 52 (2) 402 - 414 0006-2960 2013/01 [Refereed][Not invited]
    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.
  • 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 0956-3202 2013 [Refereed][Not invited]
    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.
  • 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 [Refereed][Not invited]
  • 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  2356-6140 2013 [Refereed][Not invited]
  • Katsuki Kimura, Ippei Tanaka, Shin-Ichiro Nishimura, Risho Miyoshi, Taro Miyoshi, Yoshimasa Watanabe
    WATER RESEARCH 46 (17) 5725 - 5734 0043-1354 2012/11 [Refereed][Not invited]
    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.
  • 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 1742-464X 2012/11 [Refereed][Not invited]
    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.
  • Hoshi H, Shimawaki K, Takegawa Y, Ohyanagi T, Amano M, Hinou H, Nishimura S
    Biochimica et biophysica acta 9 1820 1391 - 1398 0006-3002 2012/09 [Refereed][Not invited]
  • Naomi Manri, Yasuhiro Takegawa, Naoki Fujitani, Akihito Kaneko, Atsumu Hirabayashi, Shin-Ichiro Nishimura, Takeshi Sakamoto
    ANALYTICAL SCIENCES 28 (7) 723 - 727 0910-6340 2012/07 [Refereed][Not invited]
    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.
  • Hirokazu Kai, Hiroshi Hinou, Shin-Ichiro Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY 20 (8) 2739 - 2746 0968-0896 2012/04 [Refereed][Not invited]
    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.
  • Takayuki Furukawa, Hiroshi Hinou, Shin-Ichiro Nishimura
    ORGANIC LETTERS 8 14 (8) 2102 - 2105 1523-7060 2012/04 [Refereed][Not invited]
    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.
  • 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 0959-6658 2012/03 [Refereed][Not invited]
    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.
  • Shin-Ichiro Nishimura
    CLINICS IN LABORATORY MEDICINE 32 (1) 73 - + 0272-2712 2012/03 [Refereed][Not invited]
    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.
  • Midori Abe, Hideyuki Shimaoka, Masao Fukushima, Shin-Ichiro Nishimura
    POLYMER JOURNAL 44 (3) 269 - 277 0032-3896 2012/03 [Refereed][Not invited]
    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
  • Maho Amano, Ryo Hashimoto, Shin-Ichiro Nishimura
    CHEMBIOCHEM 13 (3) 451 - 464 1439-4227 2012/02 [Refereed][Not invited]
    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.
  • Fayna Garcia-Martin, Hiroshi Hinou, Takahiko Matsushita, Shun Hayakawa, Shin-Ichiro Nishimura
    ORGANIC & BIOMOLECULAR CHEMISTRY 10 (8) 1612 - 1617 1477-0520 2012 [Refereed][Not invited]
    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.
  • Shin-Ichiro Nishimura, Megumi Hato, Satoshi Hyugaji, Fei Feng, Maho Amano
    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 51 (14) 3386 - 3390 1433-7851 2012 [Refereed][Not invited]
  • Hiroshi Hinou, Kei Hyugaji, Fayna Garcia-Martin, Shin-Ichiro Nishimura, Fernando Albericio
    RSC ADVANCES 2 (7) 2729 - 2731 2046-2069 2012 [Refereed][Not invited]
    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.
  • 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 0004-3591 2011/11 [Refereed][Not invited]
    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.
  • 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 1742-464X 2011/10 [Refereed][Not invited]
    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.
  • Takayuki Furukawa, Hiroshi Hinou, Ken Shimawaki, Shin-Ichiro Nishimura
    Tetrahedron Letters 52 (43) 5567 - 5570 0040-4039 2011/10 [Refereed][Not invited]
  • Takeshi Ishihara, Isao Fukuda, Atsushi Morita, Yoshihiko Takinami, Hiroyuki Okamoto, Shin-Ichiro Nishimura, Yoshito Numata
    JOURNAL OF PROTEOMICS 74 (10) 2159 - 2168 1874-3919 2011/09 [Refereed][Not invited]
    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 (公社)日本生化学会 84回 4P - 0600 2011/09
  • Hiroshi Hinou, Naohiro Saito, Takahiro Maeda, Masao Matsuda, Yuichi Kamiya, Shin-Ichiro Nishimura
    Journal of Carbohydrate Chemistry 30 (7-9) 575 - 586 0732-8303 2011/09/01 [Refereed][Not invited]
  • 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 0002-7863 2011/08 [Refereed][Not invited]
    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.
  • Toru Irie, Tokifumi Majima, Naohiro Sawaguchi, Tadanao Funakoshi, Shin-ichiro Nishimura, Akio Minami
    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A 97A (2) 111 - 117 1549-3296 2011/05 [Refereed][Not invited]
    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.
  • Kazuko Hirose, Maho Amano, Ryo Hashimoto, Yuan Chuan Lee, Shin-Ichiro Nishimura
    BIOCHEMISTRY 50 (21) 4757 - 4774 0006-2960 2011/05 [Refereed][Not invited]
    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.
  • 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 1861-4728 2011/04 [Refereed][Not invited]
    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.
  • 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 0947-6539 2011/02 [Refereed][Not invited]
    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.
  • Kentaro Naruchi, Shin-Ichiro Nishimura
    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 50 (6) 1328 - 1331 1433-7851 2011 [Refereed][Not invited]
  • Shin-Ichiro Nishimura
    ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL 65 65 219 - 271 0065-2318 2011 [Refereed][Not invited]
  • 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 0003-2700 2010/12 [Refereed][Not invited]
    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.
  • Masaki Kurogochi, Takahiko Matsushista, Maho Amano, Jun-ichi Furukawa, Yasuro Shinohara, Masato Aoshima, Shin-Ichiro Nishimura
    MOLECULAR & CELLULAR PROTEOMICS 9 (11) 2354 - 2368 1535-9476 2010/11 [Refereed][Not invited]
    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.
  • 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 0002-7863 2010/11 [Refereed][Not invited]
    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.
  • 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 0002-7863 2010/10 [Refereed][Not invited]
    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)).
  • 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 0959-6658 2010/08 [Refereed][Not invited]
    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.
  • 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 0960-894X 2010/08 [Refereed][Not invited]
    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.
  • 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 0022-2623 2010/08 [Refereed][Not invited]
    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).
  • 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 0006-2960 2010/07 [Refereed][Not invited]
    UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs, EC, 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.
  • Naohiro Sawaguchi, Tokifumi Majima, Tadanao Funakoshi, Kazumi Shimode, Kazuo Harada, Akio Minami, Shin-Ichiro Nishimura
    JOURNAL OF ORTHOPAEDIC SCIENCE 15 (4) 569 - 577 0949-2658 2010/07 [Refereed][Not invited]
    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.
  • Fei Feng, Nobuaki Miura, Norikazu Isoda, Yoshihiro Sakoda, Masatoshi Okamatsu, Hiroshi Kida, Shin-Ichiro Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 20 (12) 3772 - 3776 0960-894X 2010/06 [Refereed][Not invited]
    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.
  • Ryo Hashimoto, Kazuko Hirose, Taku Sato, Nobuhiro Fukushima, Nobuaki Miura, Shin-Ichiro Nishimura
    BMC SYSTEMS BIOLOGY 4 91  1752-0509 2010/06 [Refereed][Not invited]
    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, 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.
  • 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 1535-9476 2010/03 [Refereed][Not invited]
    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.
  • 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 0021-8561 2010/03 [Refereed][Not invited]
    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.
  • Takaomi Ito, Reiko Sadamoto, Kentaro Naruchi, Hiroko Togame, Hiroshi Takemoto, Hirosato Kondo, Shin-Ichiro Nishimura
    BIOCHEMISTRY 49 (11) 2604 - 2614 0006-2960 2010/03 [Refereed][Not invited]
    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.
  • Shingo Arioka, Masahiro Sakagami, Rie Uematsu, Hiroto Yamaguchi, Hiroko Togame, Hiroshi Takemoto, Hiroshi Hinou, Shin-ichiro Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY 18 (4) 1633 - 1640 0968-0896 2010/02 [Refereed][Not invited]
    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.
  • 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 0002-7863 2009/12 [Refereed][Not invited]
    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.
  • Hiroshi Hinou, Naohiro Saito, Masato Ogawa, Takahiko Maeda, Shin-Ichiro Nishimura
    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 10 (12) 5285 - 5295 1422-0067 2009/12 [Refereed][Not invited]
    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.
  • 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 0006-2960 2009/11 [Refereed][Not invited]
    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.
  • 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 0002-7863 2009/05 [Refereed][Not invited]
    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.
  • 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 1535-9476 2009/02 [Refereed][Not invited]
    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.
  • Noriyasu Kondo, Shin-Ichiro Nishimura
    CHEMISTRY-A EUROPEAN JOURNAL 15 (6) 1413 - 1421 0947-6539 2009 [Refereed][Not invited]
    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.
  • Noriko Nagahori, Midori Abe, Shin-Ichiro Nishimura
    BIOCHEMISTRY 48 (3) 583 - 594 0006-2960 2009/01 [Refereed][Not invited]
    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.
  • Masakazu Hachisu, Hiroshi Hinou, Manabu Takamichi, Sakae Tsuda, Shuhei Koshidaa, Shin-Ichiro Nishimura
    CHEMICAL COMMUNICATIONS (13) 1641 - 1643 1359-7345 2009 [Refereed][Not invited]
    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.
  • Xiao-Dong Gao, Satoru Moriyama, Nobuaki Miura, Neta Dean, Shin-Ichiro Nishimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 (47) 32534 - 32541 0021-9258 2008/11 [Refereed][Not invited]
    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.
  • 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 0022-1767 2008/11 [Refereed][Not invited]
    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.
  • 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 0304-3835 2008/11 [Refereed][Not invited]
  • 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 0006-2960 2008/10 [Refereed][Not invited]
    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.
  • 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 1549-3296 2008/09/15 [Refereed][Not invited]
  • 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 0022-1767 2008/09 [Refereed][Not invited]
    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.
  • Fengyu Su, Chunye Xu, Minoru Taya, Kimie Murayama, Yasuro Shinohara, Shin-Ichiro Nishimura
    Sensors 8 (7) 4282 - 4295 2008/07/18 [Refereed][Not invited]
  • 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 1549-3296 2008/07 [Refereed][Not invited]
  • Matsuhashi T, Iwasaki N, Nakagawa H, Hato M, Kurogochi M, Majima T, Minami A, Nishimura SI
    Osteoarthritis Cartilage. 16 (7) 772 - 778 2008/07 [Refereed][Not invited]
  • 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 0012-1797 2008/06 [Refereed][Not invited]
  • Hiroshi Ohira, Shinji Sakaue, Naofumi Itoh, Jun-Ichi Furukawa, Yoshiaki Miura, Ichizo Tsujino, Shin-Ichiro Nishimura, Masaharu Nishimura
    DIABETES 57 A704 - A704 0012-1797 2008/06 [Refereed][Not invited]
  • Kisaburo Deguchi, Takuro Keira, Kuriko Yamada, Hiroki Ito, Yasuhiro Takegawa, Hiroaki Nakagawa, Shin-Ichiro Nishimura
    JOURNAL OF CHROMATOGRAPHY A 1189 (1-2) 169 - 174 0021-9673 2008/05 [Refereed][Not invited]
    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.
  • Nicole Averbeck, Xiao-Dong Gao, Shin-Ichiro Nishimura, Neta Dean
    MOLECULAR BIOLOGY OF THE CELL 19 (5) 2169 - 2178 1059-1524 2008/05 [Refereed][Not invited]
    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.
  • Izuru Nagashima, Hiroki Shimizu, Takahiko Matsushita, Shin-Ichiro Nishimura
    Tetrahedron Letters 49 (21) 3413 - 3418 0040-4039 2008/05 [Refereed][Not invited]
  • Yasuhiro Takegawa, Hiroki Ito, Takuro Keira, Kisaburo Deguchi, Hiroaki Nakagawa, Shin-Ichiro Nishimura
    Journal of Separation Science 31 (9) 1585 - 1593 1615-9306 2008/05 [Refereed][Not invited]
  • Yasuhiro Takegawa, Megumi Hato, Kisaburo Deguchi, Hiroaki Nakagawa, Shin-Ichiro Nishimura
    Journal of Separation Science 31 (9) 1594 - 1597 1615-9306 2008/05 [Refereed][Not invited]
  • 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 (一社)日本外科学会 109 (臨増2) 144 - 144 0301-4894 2008/04
  • Jun-ichi Furukawa, Yasuro Shinohara, Hiromitsu Kuramoto, Yoshiaki Miura, Hideyuki Shimaoka, Masaki Kurogochi, Mika Nakano, Shin-Ichiro Nishimura
    Analytical Chemistry 80 (4) 1094 - 1101 0003-2700 2008/02 [Refereed][Not invited]
  • 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 1535-9476 2008/02 [Refereed][Not invited]
  • 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 0020-7136 2008/01 [Refereed][Not invited]
    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.
  • Takahiro Maeda, Shin-Ichiro Nishimuira
    CHEMISTRY-A EUROPEAN JOURNAL 14 (2) 478 - 487 0947-6539 2008 [Refereed][Not invited]
    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).
  • Taku Nakahara, Ryo Hashimoto, Hiroaki Nakagawa, Kenji Monde, Nobuaki Miura, Shin-Ichiro Nishimura
    NUCLEIC ACIDS RESEARCH 36 D368 - D371 0305-1048 2008/01 [Refereed][Not invited]
    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, 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.
  • Reiko Sadamoto, Takeshi Matsubayashi, Masataka Shimizu, Taichi Ueda, Shuhei Koshida, Toshiaki Koda, Shin-Ichiro Nishimura
    CHEMISTRY-A EUROPEAN JOURNAL 14 (33) 10192 - 10195 0947-6539 2008 [Refereed][Not invited]
  • 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 1019-6439 2008/01/01 [Refereed][Not invited]
  • 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 0193-1849 2007/10 [Refereed][Not invited]
    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.
  • 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 2007/09/21 
    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.
  • Naoki Fujitani, Hiroki Shimizu, Teruhiko Matsubara, Takashi Ohta, Yuuki Komata, Nobuaki Miura, Toshinori Sato, Shin-Ichiro Nishimura
    CARBOHYDRATE RESEARCH 342 (12-13) 1895 - 1903 0008-6215 2007/09 [Refereed][Not invited]
    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.
  • 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 1535-9476 2007/08 [Refereed][Not invited]
    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.
  • 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 1742-464X 2007/07 [Refereed][Not invited]
    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.
  • 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 1549-3296 2007/06 [Refereed][Not invited]
    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.
  • Satoshi Uemura, Fei Feng, Maya Kume, Kuriko Yamada, Kazuya Kabayama, Shin-Ichiro Nishimura, Yasuyuki Igarashi, Jin-Ichi Inokuchi
    GLYCOBIOLOGY 17 (6) 568 - 577 0959-6658 2007/06 [Refereed][Not invited]
    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.
  • Hiroaki Nakagawa, Megumi Hato, Yasuhiro Takegawa, Kisaburo Deguchi, Hiroki Ito, Masahiko Takahata, Norimasa Iwasaki, Akio Minami, Shin-Ichiro Nishimura
    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.
  • 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  1476-4598 2007/05 [Refereed][Not invited]
    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.
  • Chihiro Kitatsuji, Masaki Kurogochi, Shin-Ichiro Nishimura, Koichiro Ishimori, Keisuke Wakasugi
    JOURNAL OF MOLECULAR BIOLOGY 368 (1) 150 - 160 0022-2836 2007/04 [Refereed][Not invited]
    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.
  • 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 1075-2617 2007/04 [Refereed][Not invited]
    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.
  • Akemi Kosaka, Daiko Wakita, Naoki Matsubara, Yuji Togashi, Shin-Ichiro Nishimura, Hidemitsu Kitamura, Takashi Nishimura
    INTERNATIONAL IMMUNOLOGY 19 (3) 249 - 256 0953-8178 2007/03 [Refereed][Not invited]
    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.
  • Hiroki Shimizu, Masahiro Sakamoto, Noriko Nagahori, Shin-Ichiro Nishimura
    Tetrahedron 63 (11) 2418 - 2425 0040-4020 2007/03 [Refereed][Not invited]
  • 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 0340-3696 2007/01 [Refereed][Not invited]
    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.
  • Hiroki Ito, Kuriko Yamada, Kisaburo Deguchi, Hiroaki Nakagawa, Shin-Ichiro Nishimura
    RAPID COMMUNICATIONS IN MASS SPECTROMETRY 21 (2) 212 - 218 0951-4198 2007 [Refereed][Not invited]
    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.
  • 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 0947-6539 2007 [Refereed][Not invited]
    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.
  • 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 0951-4198 2007 [Refereed][Not invited]
    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.
  • 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 1433-7851 2007 [Refereed][Not invited]
  • Yoshiaki Miura, Yasuro Shinohara, Jun-ichi Furukawa, Noriko Nagahori, Shin-ichiro Nishimurara
    CHEMISTRY-A EUROPEAN JOURNAL 13 (17) 4797 - 4804 0947-6539 2007 [Refereed][Not invited]
    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.
  • Masaki Kurogochi, Maho Amano, Masataka Fumoto, Akio Takimoto, Hirosato Kondo, Shin-ichiro Nishimura
    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 46 (46) 8808 - 8813 1433-7851 2007 [Refereed][Not invited]
  • Taku Nakahara, Ole Hindsgaul, Monica M. Palcic, Shin-Ichiro Nishimura
    PROTEIN ENGINEERING DESIGN & SELECTION 19 (12) 571 - 578 1741-0126 2006/12 [Refereed][Not invited]
    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.
  • 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 0022-3263 2006/12 [Refereed][Not invited]
    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.
  • Tohru Taniguchi, Kenji Monde, Shin-Ichiro Nishimura, Jun Yoshida, Hisako Sato, Akihiko Yamagishi
    Molecular Crystals and Liquid Crystals 460 (1) 107 - 116 1542-1406 2006/12/01
  • 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 1535-9476 2006/11 [Refereed][Not invited]
    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.
  • Akemi Kosaka, Ushaku Lee, Daiko Wakita, Naoki Matsubara, Yuji Togashi, Shin-Ichiro Nishimura, Hidemitsu Kitamura, Takashi Nishimura
    CANCER SCIENCE 97 (11) 1236 - 1241 1347-9032 2006/11 [Refereed][Not invited]
    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.
  • Tomohiro Onodera, Kenichi Niikura, Norimasa Iwasaki, Noriko Nagahori, Hideyuki Shimaoka, Ryusuke Kamitani, Tokifumi Majima, Akio Minami, Shin-Ichiro Nishimura
    BIOMACROMOLECULES 7 (11) 2949 - 2955 1525-7797 2006/11 [Refereed][Not invited]
    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.
  • Yasuhiro Takegawa, Kisaburo Deguchi, Hiroki Ito, Takuro Keira, Hiroaki Nakagawa, Shin-Ichiro Nishimura
    JOURNAL OF SEPARATION SCIENCE 29 (16) 2533 - 2540 1615-9306 2006/11 [Refereed][Not invited]
    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.
  • Ohyabu Naoki, Matsushita Takahiko, Hinou Hiroshi, Izumi Ryuko, Shimizu Hiroki, Kondo Hirosato, Nishimura Shin-Ichiro
    GLYCOBIOLOGY 16 (11) 1152 - 1152 0959-6658 2006/11 [Refereed][Not invited]
  • Ito Hiroki, Deguchi Kisaburo, Yamada Kuriko, Nagai Shinji, Fumoto Masataka, Hinou Hiroshi, Nakagawa Hiroaki, Shinohara Yasuro, Nishimura Shin-Ichiro
    GLYCOBIOLOGY 16 (11) 1136  0959-6658 2006/11 [Refereed][Not invited]
  • 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 日本癌学会 65回 100 - 100 0546-0476 2006/09
  • Noriko Nagahori, Shin-Ichiro Nishimura
    CHEMISTRY-A EUROPEAN JOURNAL 12 (25) 6478 - 6485 0947-6539 2006/08 [Refereed][Not invited]
    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.
  • 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 0027-8424 2006/06 [Refereed][Not invited]
    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.
  • Y Takegawa, K Deguchi, T Keira, H Ito, H Nakagawa, S Nishimura
    JOURNAL OF CHROMATOGRAPHY A 1113 (1-2) 177 - 181 0021-9673 2006/04 [Refereed][Not invited]
    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.
  • 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 1078-0432 2006/04 [Refereed][Not invited]
    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.
  • Takahiko Matsushita, Hiroshi Hinou, Masataka Fumoto, Masaki Kurogochi, Naoki Fujitani, Hiroki Shimizu, Shin-Ichiro Nishimura
    The Journal of Organic Chemistry 71 (8) 3051 - 3063 0022-3263 2006/04 [Refereed]
  • Yasuhiro Takegawa, Kisaburo Deguchi, Takuro Keira, Hiroki Ito, Hiroaki Nakagawa, Shin-Ichiro Nishimura
    Journal of Chromatography A 1113 (1-2) 177 - 181 0021-9673 2006/04 [Refereed][Not invited]
  • 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 0011-2240 2006/02 [Refereed][Not invited]
    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.
  • Nobuaki Miura, Tohru Taniguchi, Kenji Monde, Shin-Ichiro Nishimura
    Chemical Physics Letters 419 (4-6) 326 - 332 0009-2614 2006/02 [Refereed][Not invited]
  • 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 0951-4198 2006 [Refereed][Not invited]
    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.
  • K Deguchi, H Ito, Y Takegawa, N Shinji, H Nakagawa, SI Nishimura
    RAPID COMMUNICATIONS IN MASS SPECTROMETRY 20 (5) 741 - 746 0951-4198 2006 [Refereed][Not invited]
    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.
  • 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 1433-7851 2006 [Refereed][Not invited]
  • Hiroki Ito, Yasuhiro Takegawa, Kisaburo Deguchi, Shinji Nagai, Hiroaki Nakagawa, Yasuro Shinohara, Shin-Ichiro Nishimura
    RAPID COMMUNICATIONS IN MASS SPECTROMETRY 20 (23) 3557 - 3565 0951-4198 2006 [Refereed][Not invited]
    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.
  • Hiroshi Hinou, Masaki Kurogochi, Shin-Ichiro Nishimura
    GLYCOBIOLOGY 415 202 - + 0076-6879 2006 [Refereed][Not invited]
    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.
  • R Uematsu, J Furukawa, H Nakagawa, Y Shinohara, K Deguchi, K Monde, SI Nishimura
    MOLECULAR & CELLULAR PROTEOMICS 4 (12) 1977 - 1989 1535-9476 2005/12 [Refereed][Not invited]
    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.
  • 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 0947-6539 2005/11 [Refereed][Not invited]
    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.
  • T Masuko, N Iwasaki, S Yamane, T Funakoshi, T Majima, A Minami, N Ohsuga, T Ohta, SI Nishimura
    BIOMATERIALS 26 (26) 5339 - 5347 0142-9612 2005/09 [Refereed][Not invited]
    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.
  • H Hinou, M Kurogochi, H Shimizu, SI Nishimura
    BIOCHEMISTRY 44 (35) 11669 - 11675 0006-2960 2005/09 [Refereed][Not invited]
    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.
  • Y Takegawa, K Deguchi, H Nakagawa, SI Nishimura
    ANALYTICAL CHEMISTRY 77 (18) 6062 - 6068 0003-2700 2005/09 [Refereed][Not invited]
    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.
  • K Takaya, N Nagahori, M Kurogochi, T Furuike, N Miura, K Monde, YC Lee, SI Nishimura
    JOURNAL OF MEDICINAL CHEMISTRY 48 (19) 6054 - 6065 0022-2623 2005/09 [Refereed][Not invited]
    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+.
  • 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 1549-3296 2005/09/01 
    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.
  • 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 0021-9304 2005/09/01 [Refereed][Not invited]
    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.
  • 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 0363-5465 2005/08 [Refereed][Not invited]
    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.
  • 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 0002-7863 2005/08 [Refereed][Not invited]
    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.
  • Yayoi Yoshimura, Hiroki Shimizu, Hiroshi Hinou, Shin-Ichiro Nishimura
    Tetrahedron Letters 46 (28) 4701 - 4705 0040-4039 2005/07 [Refereed][Not invited]
  • 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 0947-6539 2005/06 [Refereed][Not invited]
    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.
  • T Majima, T Funakosi, N Iwasaki, ST Yamane, K Harada, S Nonaka, A Minami, SI Nishimura
    JOURNAL OF ORTHOPAEDIC SCIENCE 10 (3) 302 - 307 0949-2658 2005/05 [Refereed][Not invited]
    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.
  • T Masuko, A Minami, N Iwasaki, T Majima, SI Nishimura, YC Lee
    ANALYTICAL BIOCHEMISTRY 339 (1) 69 - 72 0003-2697 2005/04 [Refereed][Not invited]
    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.
  • Y Takegawa, K Deguchi, S Ito, S Yoshioka, H Nakagawa, S Nishimura
    ANALYTICAL CHEMISTRY 77 (7) 2097 - 2106 0003-2700 2005/04 [Refereed][Not invited]
    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.
  • H. Narita, I. Isshiki, N. Funamizu, T. Takakuwa, H. Nakagawa, S-I. Nishimura
    Environmental Technology 26 (4) 433 - 440 0959-3330 2005/04 [Refereed][Not invited]
  • T Matsushita, H Hinou, M Kurogochi, H Shimizu, SI Nishimura
    ORGANIC LETTERS 7 (5) 877 - 880 1523-7060 2005/03 [Refereed][Not invited]
    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.
  • T Masuko, A Minami, N Iwasaki, T Majima, SI Nishimura, YC Lee
    BIOMACROMOLECULES 6 (2) 880 - 884 1525-7797 2005/03 [Refereed][Not invited]
    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.
  • S Yamane, N Iwasaki, T Majima, T Funakoshi, T Masuko, K Harada, A Minami, K Monde, S Nishimura
    BIOMATERIALS 26 (6) 611 - 619 0142-9612 2005/02 [Refereed][Not invited]
    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.
  • Tetsuya Furuike, Reiko Sadamoto, Kenichi Niikura, Kenji Monde, Nobuo Sakairi, Shin-Ichiro Nishimura
    Tetrahedron 61 (7) 1737 - 1742 0040-4020 2005/02 [Refereed][Not invited]
  • SI Nishimura, N Nagahori, K Takaya, Y Tachibana, N Miura, K Monde
    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 44 (4) 571 - 575 1433-7851 2005 [Refereed][Not invited]
  • 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 1433-7851 2005 [Refereed][Not invited]
  • Y Takegawa, K Deguchi, S Ito, S Yoshioka, H Nakagawa, SI Nishimura
    RAPID COMMUNICATIONS IN MASS SPECTROMETRY 19 (7) 937 - 946 0951-4198 2005 [Refereed][Not invited]
    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.
  • 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 1433-7851 2005 [Refereed][Not invited]
  • N Kakegawa, N Hoshino, Y Matsuoka, N Wakabayashi, SI Nishimura, A Yamagishi
    CHEMICAL COMMUNICATIONS (18) 2375 - 2377 1359-7345 2005 [Refereed][Not invited]
    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.
  • K Deguchi, Y Takegawa, A Hirabayashi, H Nakagawa, SI Nishimura
    RAPID COMMUNICATIONS IN MASS SPECTROMETRY 19 (16) 2325 - 2330 0951-4198 2005 [Refereed][Not invited]
    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.
  • Shigeaki Abe, Hideki Moriyama, Kenichi Niikura, Fei Feng, Kenji Monde, Shin-Ichiro Nishimura
    Tetrahedron: Asymmetry 16 (1) 15 - 19 0957-4166 2005/01 [Refereed][Not invited]
  • Y Shinohara, J Furukawa, K Niikura, N Miura, SI Nishimura
    ANALYTICAL CHEMISTRY 76 (23) 6989 - 6997 0003-2700 2004/12 [Refereed][Not invited]
    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.
  • 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 0003-2700 2004/12 [Refereed][Not invited]
    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.
  • 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 0002-7863 2004/11 [Refereed][Not invited]
    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.
  • Tohru Taniguchi, Kenji Monde, Nobuaki Miura, Shin-Ichiro Nishimura
    Tetrahedron Letters 45 (46) 8451 - 8453 0040-4039 2004/11 [Refereed][Not invited]
  • M Kurogochi, SI Nishimura, YC Lee
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (43) 44704 - 44712 0021-9258 2004/10 [Refereed][Not invited]
    (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.
  • M Kurogochi, SI Nishimura
    ANALYTICAL CHEMISTRY 76 (20) 6097 - 6101 0003-2700 2004/10 [Refereed][Not invited]
    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.
  • Taniguchi Tohru, Miura Nobuaki, Uchida Naoto, Nishimura Shin-Ichiro, Monde Kenji
    Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会 (46) 401 - 406 2004/10/01 
    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.
  • T Taniguchi, N Miura, SI Nishimura, K Monde
    MOLECULAR NUTRITION & FOOD RESEARCH 48 (4) 246 - 254 1613-4125 2004/09 [Refereed][Not invited]
  • Yuki Tachibana, Kenji Monde, Shin-Ichiro Nishimura
    Macromolecules 37 (18) 6771 - 6779 0024-9297 2004/09 [Refereed][Not invited]
  • Kenji Monde, Tohru Taniguchi, Nobuaki Miura, Shin-Ichiro Nishimura
    Journal of the American Chemical Society 126 (31) 9496 - 9497 0002-7863 2004/08 [Refereed][Not invited]
  • 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 1525-7797 2004/05 [Refereed][Not invited]
    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.
  • T Ueda, F Feng, R Sadamoto, K Niikura, K Monde, SI Nishimura
    ORGANIC LETTERS 6 (11) 1753 - 1756 1523-7060 2004/05 [Refereed][Not invited]
    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.
  • H Moriyama, T Tsukida, Y Inoue, K Yokota, K Yoshino, H Kondo, N Miura, S Nishimura
    JOURNAL OF MEDICINAL CHEMISTRY 47 (8) 1930 - 1938 0022-2623 2004/04 [Refereed][Not invited]
    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.
  • F Sallas, K Niikura, SI Nishimura
    CHEMICAL COMMUNICATIONS (5) 596 - 597 1359-7345 2004/03 [Refereed][Not invited]
    New amphiphilic cyclodextrins fully substituted with sugar residues on the primary face have been synthesised and enzymatically modified.
  • T Tsukida, H Moriyama, Y Inoue, H Kondo, K Yoshino, SI Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 14 (6) 1569 - 1572 0960-894X 2004/03 [Refereed][Not invited]
    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.
  • R Sadamoto, K Niikura, T Ueda, K Monde, N Fukuhara, SI Nishimura
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 126 (12) 3755 - 3761 0002-7863 2004/03 [Refereed][Not invited]
    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.
  • 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 0032-3896 2004/03 [Refereed][Not invited]
  • 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 0006-291X 2004/02 [Refereed][Not invited]
    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.
  • H Hoshi, H Nakagawa, S Nishiguchi, K Iwata, K Niikura, K Monde, SI Nishimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (4) 2341 - 2349 0021-9258 2004/01 [Refereed][Not invited]
    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.
  • S Andre, H Kaltner, T Furuike, SI Nishimura, HJ Gabius
    BIOCONJUGATE CHEMISTRY 15 (1) 87 - 98 1043-1802 2004/01 [Refereed][Not invited]
    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.
  • Y Tachibana, GL Fletcher, N Fujitani, S Tsuda, K Monde, SI Nishimura
    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 43 (7) 856 - 862 1433-7851 2004 [Refereed][Not invited]
  • Y Takegawa, S Ito, S Yoshioka, K Deguchi, H Nakagawa, K Monde, SI Nishimura
    RAPID COMMUNICATIONS IN MASS SPECTROMETRY 18 (4) 385 - 391 0951-4198 2004 [Refereed][Not invited]
    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.
  • M Sato, R Sadamoto, K Niikura, K Monde, H Kondo, SI Nishimura
    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 43 (12) 1516 - 1520 1433-7851 2004 [Refereed][Not invited]
  • F Feng, K Okuyama, K Niikura, T Ohta, R Sadamoto, K Monde, T Noguchi, SI Nishimura
    ORGANIC & BIOMOLECULAR CHEMISTRY 2 (11) 1617 - 1623 1477-0520 2004 [Refereed][Not invited]
    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).
  • M Kurogochi, T Matsushita, SI Nishimura
    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 43 (31) 4071 - 4075 1433-7851 2004 [Refereed][Not invited]
  • 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 0165-2478 2003/09 [Refereed][Not invited]
  • H Moriyama, T Tsukida, Y Inoue, H Kondo, K Yoshino, SI Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 13 (16) 2737 - 2740 0960-894X 2003/08 [Refereed][Not invited]
    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.
  • H Moriyama, T Tsukida, Y Inoue, H Kondo, K Yoshino, SI Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 13 (16) 2741 - 2744 0960-894X 2003/08 [Refereed][Not invited]
    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.
  • Kenji Monde, Tohru Taniguchi, Nobuaki Miura, Shin-Ichiro Nishimura, Nobuyuki Harada, Rina K Dukor, Laurence A Nafie
    Tetrahedron Letters 44 (32) 6017 - 6020 0040-4039 2003/08 [Refereed][Not invited]
  • Tetsuya Furuike, Kuriko Yamada, Takashi Ohta, Kenji Monde, Shin-Ichiro Nishimura
    Tetrahedron 59 (27) 5105 - 5113 0040-4020 2003/06 [Refereed][Not invited]
  • Noriko Nagahori, Kenichi Niikura, Reiko Sadamoto, Masahiro Taniguchi, Akihiko Yamagishi, Kenji Monde, Shin-Ichiro Nishimura
    Advanced Synthesis & Catalysis 345 (67) 729 - 734 1615-4150 2003/06 [Refereed][Not invited]
  • Koji Matsuoka, Takumi Ohtawa, Hiroshi Hinou, Tetsuo Koyama, Yasuaki Esumi, Shin-Ichiro Nishimura, Ken Hatano, Daiyo Terunuma
    Tetrahedron Letters 44 (18) 3617 - 3620 0040-4039 2003/04 [Refereed][Not invited]
  • 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 0960-894X 2003/03 [Refereed][Not invited]
  • Kadota K, Nishimura S, Bono H, Nakamura S, Hayashizaki Y, Okazaki Y, Takahashi K
    Physiological genomics 12 (3) 251 - 259 1094-8341 2003/02 [Refereed][Not invited]
  • 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 0006-291X 2003/01 [Refereed][Not invited]
    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.
  • 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 1433-7851 2003 [Refereed][Not invited]
  • Sadamoto R, Niikura K, Monde K, Nishimura S
    Methods in enzymology 362 273 - 286 0076-6879 2003 [Refereed][Not invited]
  • Noriko Nagahori, Kenichi Niikura, Reiko Sadamoto, Kenji Monde, Shin-Ichiro Nishimura
    Australian Journal of Chemistry 56 (6) 567 - 567 0004-9425 2003 [Refereed][Not invited]
    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.
  • Yuki Tachibana, Naoki Matsubara, Fumio Nakajima, Tetsuro Tsuda, Sakae Tsuda, Kenji Monde, Shin-Ichiro Nishimura
    Tetrahedron 58 (51) 10213 - 10224 0040-4020 2002/12 [Refereed][Not invited]
  • N Nagahori, RT Lee, S Nishimura, D Page, R Roy, YC Lee
    CHEMBIOCHEM 3 (9) 836 - 844 1439-4227 2002/09 [Refereed][Not invited]
    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.
  • 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 0002-7863 2002/08 [Refereed][Not invited]
  • Jin Nishida, Kazutaka Nishikawa, Shin-Ichiro Nishimura, Shigeo Wada, Takeshi Karino, Takehiro Nishikawa, Kuniharu Ijiro, Masatsugu Shimomura
    Polymer Journal 34 (3) 166 - 174 0032-3896 2002/03 [Refereed][Not invited]
  • M Sawa, H Kondo, S Nishimura
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 12 (4) 581 - 584 0960-894X 2002/02 [Refereed][Not invited]
    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.
  • 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 0036-8075 2002/01/04 [Refereed][Not invited]
    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.
  • T Sukegawa, T Furuike, K Niikura, A Yamagishi, K Monde, SI Nishimura
    CHEMICAL COMMUNICATIONS (5) 430 - 431 1359-7345 2002 [Refereed][Not invited]
    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.
  • Takehiko Saito, Wilina Lim, Takashi Suzuki, Yasuo Suzuki, Hiroshi Kida, Shin-Ichiro Nishimura, Masato Tashiro
    Vaccine 20 (1-2) 125 - 133 0264-410X 2001/10 [Refereed][Not invited]
  • S Nishiguchi, K Yamada, Y Fuji, S Shibatani, A Toda, SI Nishimura, SI Nishimura
    CHEMICAL COMMUNICATIONS (19) 1944 - 1945 1359-7345 2001/10 [Refereed][Not invited]
    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.
  • Shin-Ichiro Nishimura
    Current Opinion in Chemical Biology 5 (3) 325 - 335 1367-5931 2001/06 [Refereed][Not invited]
  • Daijyu Kumagai, Masaki Miyazaki, Shin-Ichiro Nishimura
    Tetrahedron Letters 42 (10) 1953 - 1956 0040-4039 2001/03/04 [Refereed][Not invited]
    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.
  • Masao Matsuda, Shin-Ichiro Nishimura, Fumio Nakajima, Takashi Nishimura
    Journal of Medicinal Chemistry 44 (5) 715 - 724 0022-2623 2001/03 [Refereed][Not invited]
  • 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 1389-1723 2001 [Refereed][Not invited]
    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.
  • Nagahori N, Nishimura S
    Biomacromolecules 2 (1) 22 - 24 1525-7797 2001 [Refereed][Not invited]
  • T Furuike, S Aiba, SI Nishimura
    TETRAHEDRON 56 (51) 9909 - 9915 0040-4020 2000/12 [Refereed][Not invited]
    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.
  • 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 0344-5704 2000/07 [Refereed][Not invited]
  • K Washiya, T Furuike, F Nakajima, YC Lee, SI Nishimura
    ANALYTICAL BIOCHEMISTRY 283 (1) 39 - 48 0003-2697 2000/07 [Refereed][Not invited]
    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.
  • Ohta A, Sekimoto M, Sato M, Koda T, Nishimura S.-I, Iwakura Y, Sekikawa K, Nishimura T
    Journal of Immunology 165 (2) 956 - 961 2000/07 [Refereed][Not invited]
  • 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 0006-291X 2000/05/27 [Refereed][Not invited]
    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.
  • Takehiro Nishikawa, Jin Nishida, Ryusuke Ookura, Shin-Ichiro Nishimura, Volker Scheumann, Manfred Zizlsperger, Reinald Lawall, Wolfgang Knoll, Masatsugu Shimomura
    Langmuir 16 (3) 1337 - 1342 0743-7463 2000/02 [Refereed][Not invited]
  • 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 0008-8749 2000/01/10 [Refereed][Not invited]
    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.
  • Tachibana, Y, Tsuda, T, Matsubara, N, Tsuda, S, Nishimura, SI
    Abstracts of Papers of the American Chemical Society 219 U244 - U244 0065-7727 2000 [Refereed][Not invited]
  • S. I. Nishimura, N. Kimura, K. Matsuoka, Yuan Chuan Lee
    Carbohydrate Letters 4 (2) 77 - 84 1073-5070 2000 [Not refereed][Not invited]
    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 [Refereed][Not invited]
  • F Sallas, SI Nishimura
    JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 1 (13) 2091 - 2103 1470-4358 2000 [Refereed][Not invited]
    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.
  • 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 1470-4358 2000 [Refereed][Not invited]
    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.
  • Takehiro Nishikawa, Jin Nishida, Ryusuke Ookura, Shin-Ichiro Nishimura, Shigeo Wada, Takeshi Karino, Masatsugu Shimomura
    Materials Science and Engineering: C 8-9 495 - 500 0928-4931 1999/12 [Refereed][Not invited]
    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.
  • 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 0928-4931 1999/12 [Refereed][Not invited]
    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.
  • 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 11 9 (11) 1213 - 1224 0959-6658 1999/11 [Refereed][Not invited]
    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).
  • 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 0022-1007 1999/04/05 [Refereed][Not invited]
    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.
  • K Yamada, S Matsumoto, SI Nishimura
    CHEMICAL COMMUNICATIONS (6) 507 - 508 1359-7345 1999/03 [Refereed][Not invited]
    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.
  • SI Nishimura, M Matsuda, H Kitamura, T Nishimura
    CHEMICAL COMMUNICATIONS (15) 1435 - 1436 1359-7345 1999 [Not refereed][Not invited]
    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.
  • Keisuke Kurita, Kumi Shimada, Yasuhiro Nishiyama, Manabu Shimojoh, Shin-Ichiro Nishimura
    Macromolecules 31 (15) 4764 - 4769 0024-9297 1998/07/28 [Refereed][Not invited]
    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.
  • 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 0968-5677 1998/07 [Refereed][Not invited]
  • SI Nishimura, S Nomura, K Yamada
    CHEMICAL COMMUNICATIONS (5) 617 - 618 1359-7345 1998/03 [Refereed][Not invited]
    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.
  • T Sukegawa, M Matsuda, SI Nishimura, M Shimomura, K Ijiro, O Karthaus
    MOLECULAR RECOGNITION AND INCLUSION 519 - 522 1998 [Not refereed][Not invited]
    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.
  • 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 0008-6215 1998/01 [Refereed][Not invited]
  • Convenient fluorescence probes for the characterisation of glycosyltransferases related to the acrosome reaction
    Nishimura S. -I, Washiya K
    Zygote 6 35 - 36 1998 [Refereed][Not invited]
  • 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 [Not refereed][Not invited]
  • K Yamada, E Fujita, SI Nishimura
    CARBOHYDRATE RESEARCH 305 (3-4) 443 - 461 0008-6215 1997/12 [Refereed][Not invited]
    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.
  • S Nishimura, K Yamada
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 119 (43) 10555 - 10556 0002-7863 1997/10 [Refereed][Not invited]
  • 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 0014-5793 1997/03 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Hitoshi Yoshino, Shin-Ichiro Nishimura, Shigeru Ishii, Tomonori Mori, Yasuhiro Nishiyama
    Carbohydrate Polymers 32 (3-4) 171 - 175 0144-8617 1997 [Refereed][Not invited]
    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.
  • Matsuoka K, Nishimura S. I, Lee Y. C
    Fluorescence Spectroscopy 278 519 - 528 1997 [Refereed][Not invited]
  • Keisuke Kurita, Soichiro Hashimoto, Hitoshi Yoshino, Shigeru Ishii, Shin-Ichiro Nishimura
    Macromolecules 29 (6) 1939 - 1942 0024-9297 1996/03/11 [Refereed][Not invited]
    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.
  • T Tsuda, T Furuike, SI Nishimura
    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 69 (2) 411 - 416 0009-2673 1996/02 [Refereed][Not invited]
    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.
  • M Ohmae, S Suzuki, S Tokura, N Nishi, SI Nishimura
    JOURNAL OF BIOCHEMISTRY 119 (2) 367 - 371 0021-924X 1996/02 [Refereed][Not invited]
    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).
  • Keisuke Kurita, Shin-Ichi Watabe, Shin-Ichiro Nishimura, Shigeru Ishii
    Journal of Polymer Science, Part A: Polymer Chemistry 34 (3) 429 - 438 0887-624X 1996 [Refereed][Not invited]
    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.
  • Tetsuro Tsuda, Shin-Ichiro Nishimura
    Chemical Communications (24) 2779 - 2780 1359-7345 1996 [Refereed][Not invited]
    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.
  • Kayo Shimoda, Kohji Nakajima, Yoshiharu Hiratsuka, Shin-Ichiro Nishimura, Keisuke Kurita
    Carbohydrate Polymers 29 (2) 149 - 154 0144-8617 1996 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Soichiro Hashimoto, Shigeru Ishii, Tomonori Mori, Shin-Ichiro Nishimura
    Polymer Journal 28 (8) 686 - 689 0032-3896 1996 [Refereed][Not invited]
    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.
  • K Yamada, SI Nishimura
    TETRAHEDRON LETTERS 36 (52) 9493 - 9496 0040-4039 1995/12 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Yukiko Suzuki, Takahito Enari, Shigeru Ishii, Shin-Ichiro Nishimura
    Macromolecules 28 (6) 1801 - 1806 1520-5835 1995/11/01 [Refereed][Not invited]
    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.
    MACROMOLECULES 28 (21) 7241 - 7247 0024-9297 1995/10 [Refereed][Not invited]
    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.
    JOURNAL OF BIOCHEMISTRY 118 (2) 278 - 284 0021-924X 1995/08 [Refereed][Not invited]
    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.
  • Kyung Bok Lee, Koji Matsuoka, Shin-Ichiro Nishimura, Yuan Chuan Lee
    Analytical Biochemistry 230 (1) 31 - 36 0003-2697 1995 [Refereed][Not invited]
    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.
    MACROMOLECULES 28 (8) 2961 - 2968 1995 [Refereed][Not invited]
    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 68 (6) 1715 - 1720 0009-2673 1995 [Refereed][Not invited]
    CARBOHYDRATE RESEARCH 276 (1) 31 - 42 1995 [Refereed][Not invited]
  • Keisuke Kurita, Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, Shin-Ichiro Nishimura
    Macromolecules 27 (26) 7544 - 7549 1520-5835 1994/12/01 [Refereed][Not invited]
    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.
    NEOGLYCOCONJUGATES, PT A 242 235 - 246 0076-6879 1994 [Refereed][Not invited]
  • Akihiro Shirai, Mikio Takahashi, Hiroaki Kaneko, Shin-Ichiro Nishimura, Masato Ogawa, Norio Nishi, Seiichi Tokura
    International Journal of Biological Macromolecules 16 (6) 297 - 300 0141-8130 1994 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Shigeru Ishii, Koji Tomita, Shin‐Ichiro Nishimura, Kayo Shimoda
    Journal of Polymer Science Part A: Polymer Chemistry 32 (6) 1027 - 1032 1099-0518 1994 [Refereed][Not invited]
    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.
  • Shigeru Ishii, Shin‐Ichi Watabe, Shin‐Ichiro Nishimura, Keisuke Kurita
    Journal of Polymer Science Part A: Polymer Chemistry 32 (3) 575 - 577 1099-0518 1994 [Refereed][Not invited]
  • Tokura Seiichi, Miura Yoshiaki, Johmen Masayoshi, Nishi Norio, Nishimura Shin-Ichiro
    Journal of Controlled Release 28 (1-3) 235 - 241 0168-3659 1994 [Refereed][Not invited]
    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.
  • 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 1099-0518 1994 [Refereed][Not invited]
    MACROMOLECULES 27 (18) 4876 - 4880 1994 [Refereed][Not invited]
    TETRAHEDRON-ASYMMETRY 5 (12) 2335 - 2338 1994 [Refereed][Not invited]
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 199 (1) 249 - 254 1994 [Refereed][Not invited]
    TETRAHEDRON LETTERS 35 (31) 5657 - 5660 1994 [Refereed][Not invited]
  • NISHIMURA Shin-ichiro, MIURA Yoshiaki, REN Longdi, SATO Manabu, YAMAGISHI Akihiko, NISHI Norio, TOKURA Seiichi, KURITA Keisuke, ISHII Shigeru
    Chemistry Letters The Chemical Society of Japan 1993 (9) 1623 - 1626 0366-7022 1993/09/05 
    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.
  • Keisuke Kurita, Koji Tomita, Tomoyoshi Tada, Shin-Ichiro Nishimura, Shigeru Ishii
    Polymer Bulletin 30 (4) 429 - 433 0170-0839 1993/04 [Refereed][Not invited]
    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.
  • Itoyama Koki, Nishimura Shin-ichiro, Kawamura Yoshihide, Seo Hiroshi, Sato Kaoru, Dosako Shun-ichi, Nishi Norio, Tokura Seiichi
    Sen-ito Kogyo The Society of Fiber Science and Technology, Japan 49 (11) 580 - 585 0037-9875 1993 
    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.
  • Keisuke Kurita, Koji Tomita, Shigeru Ishii, Shin‐Ichiro Nishimura, Kayo Shimoda
    Journal of Polymer Science Part A: Polymer Chemistry 31 (9) 2393 - 2395 1099-0518 1993 [Refereed][Not invited]
  • 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 1099-0518 1993 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Hitoshi Yoshino, Shin-Ichiro Nishimura, Shigeru Ishii
    Carbohydrate Polymers 20 (4) 239 - 245 0144-8617 1993 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Hitoshi Yoshino, Koji Yokota, Motonari Ando, Satoshi Inoue, Shigeru Ishii, Shin-Ichiro Nishimura
    Macromolecules 25 (14) 3786 - 3790 1520-5835 1992/07/01 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Satoshi Inoue, Kiichi Yamamura, Hitoshi Yoshino, Shigeru Ishii, Shin-Ichiro Nishimura
    Macromolecules 25 (14) 3791 - 3794 1520-5835 1992/07/01 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Seiji Iwawaki, Shigeru Ishii, Shin‐Ichiro Nishimura
    Journal of Polymer Science Part A: Polymer Chemistry 30 (4) 685 - 688 1099-0518 1992 [Refereed][Not invited]
  • Keisuke Kurita, Naohiro Mikawa, Shigeru Ishii, Shin-Ichiro Nishimura
    Macromolecules 24 (26) 6853 - 6855 1520-5835 1991/12/01 [Refereed][Not invited]
  • Shin-Ichiro Nishimura, Osamu Kohgo, Keisuke Kurita, Hiroyoshi Kuzuhara
    Macromolecules 24 (17) 4745 - 4748 1520-5835 1991/08/01 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Mitsuo Kawata, Yoshiyuki Koyama, Shin‐Ichiro Nishimura
    Journal of Applied Polymer Science 42 (11) 2885 - 2891 1097-4628 1991 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Satoshi Inoue, Shin‐Ichiro Nishimura
    Journal of Polymer Science Part A: Polymer Chemistry 29 (6) 937 - 939 1099-0518 1991 [Refereed][Not invited]
  • Keisuke Kurita, Mami Kamiya, Shin-Ichiro Nishimura
    Carbohydrate Polymers 16 (1) 83 - 92 0144-8617 1991 [Refereed][Not invited]
    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.
    MACROMOLECULES 24 (15) 4236 - 4241 1991 [Refereed][Not invited]
  • Shin-Ichiro Nishimura, Hiroyoshi Kuzuhara
    Carbohydrate Research 206 (2) 207 - 217 0008-6215 1990/10/10 [Refereed][Not invited]
    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.
  • Shin-Ichiro Nishimura, Koji Matsuoka, Keisuke Kurita
    Macromolecules 23 (18) 4182 - 4184 1520-5835 1990 [Refereed][Not invited]
  • Keisuke Kurita, Yoshiyuki Koyama, Satoshi Inoue, Shin-Ichiro Nishimura
    Macromolecules 23 (11) 2865 - 2869 1520-5835 1990 [Refereed][Not invited]
    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.
  • Keisuke Kurita, Naohiro Mikawa, Yoshiyuki Koyama, Shin-Ichiro Nishimura
    Macromolecules 23 (10) 2605 - 2609 1520-5835 1990 [Refereed][Not invited]
    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.
  • Kurita Keisuke, Takanobu Takeda, Shinichiro Nishimura
    Polymer Journal 22 (5) 429 - 434 1349-0540 1990 [Refereed][Not invited]
    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.
  • Shin-Ichiro Nishimura, Hiroyoshi Kuzuhara, Yasuyuki Takiguchi, Kenzo Shimahara
    Carbohydrate Research 194 (C) 223 - 231 0008-6215 1989/12/01 [Refereed][Not invited]
    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.
    JAPANESE JOURNAL OF CANCER RESEARCH 80 (3) 200 - 203 0910-5050 1989/03 [Refereed][Not invited]
    INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 9 (4) 225 - 232 0141-8130 1987/08 [Refereed][Not invited]
    CARBOHYDRATE RESEARCH 134 (2) 305 - 312 0008-6215 1984 [Refereed][Not invited]
  • Norio Nishi, Hiroyuki Ohnuma, Shin-Ichiro Nishimura, Oyin Somorin, Seiichi Tokura
    Polymer Journal 14 (11) 913 - 919 1349-0540 1982 [Refereed][Not invited]

Books etc

  • Glycoscience 2nd Edition. 2115-2119
    Springer 2008
  • Glycoscience 2nd Edition. 1859-1871
    Springer 2008
  • 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
  • Comprehensive Glycoscience, 453-475
    Elsevier Ltd. 2007
  • 黒河内 政樹, 広瀬 和子, 西村 紳一郎, 福島 信弘 
    三共出版 2007 (ISBN: 9784782705278)
  • Nanotechnology in Carbohydrate Chemistry (Yuasa H.監), 149-166
    Transworld Research Network 2006
  • Methods in Enzymology, 415, 202-212
    Academic Press 2006
  • 未来を拓く糖鎖科学 (永井克彦監), 419-420
    金芳堂 2005
  • Handbook of Carbohydrate Engineering (Yarema K. 編), 495-505
    Taylor & Francis 2005
  • Taylor Maureen, Drickamer Kurt, 西村 紳一郎, 門出 健次 
    化学同人 2005 (ISBN: 9784759810356)
  • Methods in Enzymology, 362, 273-286
    Academic Press 2003
  • Carbohydrate-based Drug Discovery, 129-136
    Wiley-VCH, DEU 2003
  • 西村 紳一郎, 畑中 研一, 佐藤 智典, 和田 健彦 
    講談社 1999 (ISBN: 4061397915)
  • 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 (ISBN: 4061397834)

Conference Activities & Talks

  • The dynamic epitope theory
    Shi-Ichiro Nishimura
    The 13th International Polymer Conference (IPC2023)  2023/07
  • 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
    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  2023/03
  • 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
    第12回GFRGプロジェクトシンポジウム  2023/01
  • Creation of an innovative drug delivery system targeting pre-metastatic niche  [Invited]
    第9回日本細胞外小胞学会  2022/10
  • 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
    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  2022/05
  • Antibodies recognizing dynamic neoepitopes generated by cancer-specific truncated immature O-glycosylation  [Invited]
    Shin-Ichiro Nishimura
    The 2021 International Chemical Congress of Pacific Basin Societies / Pacifichem2021  2021/12
  • The dynamic epitope theory-Vulnerability of proteins induced by posttranslational glycosylation  [Invited]
    Shin-Ichiro Nishimura
    70th Symposium on Macromolecules  2021/09
  • 動的エピトープ理論による革新的創薬  [Not invited]
    LIFE SCIENCE CONEECT 2021  2021/08
  • 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
    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  2021/07
  • ローマは一日にして成らず。されど、すべての道は?  [Invited]
    GlycoTOKYO2019  2019/11
  • Smart nanomedicine targeting of lysosomal glycohydrolases through activated endocytosis of cancer cells  [Not invited]
    Shin-Ichiro Nishimura
    25th International Symposium on Glycoconjugates  2019/08
  • 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”  [Invited]
    Shin-Ichiro Nishimura
    EURAXESS Japan Tour 2019  2019/03
  • 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  [Invited]
    医薬基盤研究所創薬教育セミナー  2018/03
  • 糖鎖研究の現状と将来展望  [Not invited]
    第9回GFRG研究会シンポジウム  2017/12
  • 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  [Invited]
    第9回Neuroprotective Meeting for Young Researchers  2017/11
  • 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  [Not invited]
    西村 紳一郎
    第57回日本臨床化学会年次学術集会  2017/10
  • Nanomedicine targeting intracellular glycoenzymes  [Invited]
    Shin-Ichiro Nishimura
    2017 Gordon Research Conference (GRC) on Carbohydrate  2017/06
  • New glycotechnology platform interfacing chemistry and biology  [Invited]
    Shin-Ichiro Nishimura
    253rd ACS National Meeting  2017/04
  • A theory of the dynamic epitope: Autoantibodies to the dynamic epitopes generated by aberrant glycosylation at consecutive threonine motifs in circulating carcinoma mucins  [Not invited]
    Shin-Ichiro Nishimura
    日本化学会第97春季年会  2017/03
  • Chemical Synthesis Uncovers Novel Regulation Mechanism in Protein Folding by Site-specific Glycosylation  [Invited]
    Shin-Ichiro Nishimura
    A3 RONA and CPRH  2016/09
  • New trends in glycodrug discovery: Molecular targets unveiled by chemical synthesis and nanotechnology  [Invited]
    Shin-Ichiro Nishimura
    GLYCOPHARM Final Conference  2016/07
  • 動的な翻訳後修飾による疾患特異的抗原構造の獲得  [Invited]
    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~  2016/03
  • Toward personalized medicine: Chemical and enzymatic synthesis of disease-relevant glycoproteins  [Invited]
    Shin-Ichiro Nishimura
    103rd Indian Science Congress 2016  2016/01
  • Posttranslational glycosylation and cancer cell's life cycle: From discovery of disease-relevant epitope to development of new class anti-cancer drugs  [Invited]
    Shin-Ichiro Nishimura
    第12回消化器外科Ⅰモーニングセミナー  2015/11
    Shin-Ichiro Nishimura
    GLYCO23  2015/09
    Shin-Ichiro Nishimura
    EMBO Workshop  2015/06
  • Epitope-defined antibody drugs discovered by novel glycotechnology  [Invited]
    Shin-Ichiro Nishimura
    基礎血液談話会  2015/04
  • Intracellular traffic of cell surface mimetic quantum dots-anchored glycopeptides  [Invited]
    Shin-Ichiro Nishimura
    249th ACS National Meeting & Exposition  2015/03
  • Epitope-defined antibody discovered by new glycotechnology  [Invited]
    Shin-Ichiro Nishimura
    2015 Queenstown Molecular Biology Meeting  2015/03
  • Epitope-defined antibody as new class therapeutic and diagnostic reagents: Toward personalized medicine from chemical glycobiology  [Invited]
    Shin-Ichiro Nishimura
    International Symposium on Chemical Biology Approach to Metabiomics, Chemical genomics and Epigenomics  2015/02
  • 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  [Invited]
    Shin-Ichiro Nishimura
    Clinical Oncology Research勉強会  2014/11
  • Toward personalized medicine from glycan biomarkers discovered by glycoblotting-assisted high throughput serum glycomics  [Invited]
    Shin-Ichiro Nishimura
    Sino-Japan Workshop on Chemical Biology  2014/10
  • GLYCOPHARM at work with glycopeptide epitopes: promising targets uncovered by comprehensive glycomics and robust synthetic compound library  [Invited]
    Shin-Ichiro Nishimura
    GLYCOPHARM meeting  2014/06
  • Antigenic glycopeptides: Conformational impact of protein glycosylation and disease-relevant antigenic structure  [Invited]
    Shin-Ichiro Nishimura
    Special Lecture at Johns Hopkins School of Medicine  2014/03
  • High Performance Glycopeptide Microarray: Rapid Epitope Profiling of Monoclonal Antibodies and Serum Autoantibodies  [Invited]
    Shin-Ichiro Nishimura
    27th International Carbohydrate Symposium (ICS2014)  2014/01
  • Toward personalized medicine from glycan biomarkers discovered by glycoblotting-assisted high throughput serum glycomics  [Invited]
    Shin-Ichiro Nishimura
    International Symposium on Chemical Biology – Drug Discovery  2014/01
  • Autoantibodies to cancer-associated MUC1 fragments in healthy human sera discovered by high performance glycopeptide microarray platform  [Invited]
    Shin-Ichiro Nishimura
    246th ACS National Meeting & Expositions  2013/09
  • Drug discovery from chemical glycobiology  [Invited]
    Shin-Ichiro Nishimura
    Special seminar at Wayne State University  2013/09
  • Toward personalized medicine from chemical glycobiology  [Invited]
    Shin-Ichiro Nishimura
    GlycoMedicine Seminar  2013/08
  • 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  [Invited]
    西村 紳一郎
    Conference for BioSignal and Medicine2013(CBSM2013)  2013/07
  • Toward personalized medicine from glycan biomarkers discovered by glycoblotting-assisted high throughput serum glycomics  [Invited]
    Shin-Ichiro Nishimura
    8th ISABS(International Society for Applied Biological Sciences) Conference  2013/06
  • 学問の風景-Be ambitious!を胸に  [Invited]
    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  2013/04
  • 人生に解なし―勇気を出してそれぞれの大志を抱け  [Invited]
    西村 紳一郎
    北嶺中高等学校 進路講演会  2012/12
  • 生活習慣病・病気と糖鎖の関係  [Invited]
    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  2012/11
  • Regulation of glycan biosynthetic mechanism and drug discovery  [Invited]
    Shin-Ichiro Nishimura
    第17回 未来創薬・医療イノベーションセミナー  2012/09
  • Development and clinical application of fully automated glycan analytical system  [Invited]
    Shin-Ichiro Nishimura
    JASIS2012  2012/09
  • Glycomics and Drug Discovery  [Invited]
    Shin-Ichiro Nishimura
    26th International Carbohydrate Symposium  2012/07
  • Toward Fully Automated Glycomics by Glycoblotting Method  [Invited]
    Shin-Ichiro Nishimura
    Increasing the Impact of Glycoscience through New Tools and Technologies (Satellite Meeting of ICS2012)、、主催:Niels-Christian Reichardt group leader at CIC biomaGUNE  2012/07
  • グライコブロッティング法と糖鎖ナノテクノロジー  [Invited]
    西村 紳一郎
    第16回腸内細菌学会  2012/06
  • 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~  [Invited]
    西村 紳一郎
    AB SCIEX LC/MS ユーザーズミーティング2012  2012/03
  • 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-  [Invited]
    西村 紳一郎
    第7回臨床糖鎖研究会  2012/03
  • 疾患グライコミクス  [Invited]
    西村 紳一郎
    日本肝臓学会西部会  2011/12
  • Posttranslational glycosylation as a potential target for clinical metabolomics  [Invited]
    Shin-Ichiro Nishimura
    第6回メタボロームシンポジウム  2011/10
  • Nanocarries as an emerging platform for new molecular imaging and therapy.  [Invited]
    西村 紳一郎
    神戸大学シグナル伝達医学GCOE学術講演会  2011/10
  • 糖鎖医薬品の研究開発を支援する新技術・デバイス  [Not invited]
    西村 紳一郎
    第5回GFRG研究会シンポジウム  2011/10
  • 全自動糖鎖解析法の実現と医療・創薬研究への展開  [Not invited]
    西村 紳一郎
    眼科学分野 医局リサーチカンファレンス  2011/09
  • 全自動糖鎖解析システムの開発と臨床応用  [Invited]
    西村 紳一郎
    第51回日本臨床化学会年次学術集会  2011/08
  • Identification of Disease Specific Glycopeptide Epitopes  [Invited]
    Shin-Ichiro Nishimura
    European Young Investigators Workshop  2011/04
  • 高速糖鎖解析技術による疾患検査マーカーの探索  [Invited]
    西村 紳一郎
    第25回日本臨床検査自動化学会  2011/04
  • 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  [Invited]
    Shin-Ichiro Nishimura
    nternational Symposium on Challenges in Drug Discovery Programme 2011  2011/02
  • Glycoblotting-assisted high throughput glycomics and glycoproteomics  [Invited]
    Shin-Ichiro Nishimura
    2010 International Chemical Congress of Pacific Basin Societies(PACIFICHEM2010)  2010/12
  • 疾患マーカー探索研究を加速する有機合成化学  [Invited]
    西村 紳一郎
    平成22年度 秋季有機合成化学講習会  2010/11
  • 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  [Invited]
    西村 紳一郎
    神戸大学 先端医学シリーズ  2010/11
  • Glycoblotting method reveals new promising biomarkers during cell differentiation.  [Invited]
    Shin-Ichiro Nishimura
    JAACT2010  2010/09
  • Cancer relevant epitopes uncovered by synthetic mucin glycopeptides  [Invited]
    Shin-Ichiro Nishimura
    The 240th ACS National Meeting  2010/08
  • Large-scale glycomics for the discovery of cancer markers:Diagnostic mass spectrometry based on glycoblotting technology  [Invited]
    Shin-Ichiro Nishimura
    BIT's 3rd World Cancer Congress 2010  2010/06
  • Glycoblotting and mass spectrometry for the discovery of new biomaekers  [Invited]
    Shin-Ichiro Nishimura
    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  2010/06
  • Large-scale glycomics and glycoproteomics by glycoblotting method  [Invited]
    Shin-Ichiro Nishimura
    PITTCON  2010/03
  • 臨床グライコミクスと創薬研究  [Invited]
    西村 紳一郎
    神戸大学医学研究科セミナー  2010/01
  • Chemistry and biological impact of complex carbohydrates  [Invited]
    Shin-Ichiro Nishimura
    Special seminar on Mysore University  2009/12
  • Drug discovery by integrating glycoblotting and automated glycan synthesis  [Invited]
    Shin-Ichiro Nishimura
    International Conference on current Trends in Chemistry and Biochemistry (ICCTCB-2009)  2009/12
  • Comprehensive glycomics for basic and advanced studies in glycobiology  [Invited]
    Shin-Ichiro Nishimura
    7th Annual Uppsala Conference 2009  2009/12
  • 糖鎖版PCR法の完成で加速する疾患マーカー探索研究  [Invited]
    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  2009/12
  • 糖鎖の謎と魅力 -遺伝子だけでは語れない生命の不思議-  [Invited]
    西村 紳一郎
    防衛大学特別講演会  2009/12
  • Large-scale serum glycomics of CFG KO mice based on the glycoblotting method  [Not invited]
    Shin-Ichiro Nishimura
    2009 Annual Meeting of the Society for Glycobiology  2009/11
  • 大規模網羅的糖鎖解析と標的糖ペプチド解析  [Invited]
    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  2009/11
  • Automated glycan synthesis inspired from biosynthetic systems and their use in drug discovery research  [Invited]
    Shin-Ichiro Nishimura
    Symposium on Bio-Inspired Engineering(ISBIE)  2009/10
  • Cancer-related markers discovered by large-scale glycomics  [Invited]
    Shin-Ichiro Nishimura
    第68回日本癌学会学術総会  2009/10
  • 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―  [Invited]
    西村 紳一郎
    第29回日本分子腫瘍マーカー研究会  2009/09
  • Synthesis of highly complicated carbohydrates and their use in drug discovery research  [Invited]
    Shin-Ichiro Nishimura
    Japanese-European Wiorkshop on Cellulose and Functional Polysaccharides  2009/09
  • 原点に立ち返ってみよう-糖鎖構造解析のunmet needs-  [Invited]
    西村 紳一郎
    第29回日本糖質学会年会  2009/09
  • 大規模糖鎖解析による新しい疾患マーカーの探索  [Invited]
    西村 紳一郎
    消化器研究セミナー  2009/09
  • 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~  [Invited]
    西村 紳一郎
    第49回 生命科学 夏の学校  2009/08
  • 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  [Invited]
    西村 紳一郎
    第56回日本臨床検査医学会学術集会  2009/08
  • 糖鎖研究と創薬~Unmet needs と defacto standards~  [Invited]
    西村 紳一郎
    第25回創薬セミナー  2009/07
  • Fully Automated Carbohydrate Synthesis by Combined Chemical and Enzymatic Protocol  [Invited]
    Shin-Ichiro Nishimura
    15th European carbohydrate symposium  2009/07
  • 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  [Invited]
    北海道経済連合会 第185回常任理事会  2009/07
  • 遺伝子だけでは語れない生命の謎に迫る化学研究  [Invited]
    西村 紳一郎
    関西大学特別講演会  2009/06
  • New Trends in discovery research for cancer markers and glycosyltransferases inhibitors  [Invited]
    Shin-Ichiro Nishimura
    NCI-Frederick Special Seminar  2009/03
  • Glycoblotting: Potential "PCR" for Glycobiology  [Invited]
    Shin-Ichiro Nishimura
    Johns Hopkins University Special Lecture  2009/03
  • 血液一滴から癌や生活習慣病を早期診断  [Invited]
    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  2009/02
  • 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  [Invited]
    西村 紳一郎
    味の素医薬研究所講演会  2009/01
  • 大規模グライコミクスの実現とその応用  [Invited]
    西村 紳一郎
    オミックス医療研究会定期講演会  2009/01
  • Chemistry and biological impact of comlpex carbohydrates  [Invited]
    Shin-Ichiro Nishimura
    Tateshina Conference on Organic Chemistry  2008/11
  • 大規模糖鎖解析による癌マーカー分子の探索  [Invited]
    西村 紳一郎
    第67回日本癌学会学術総会  2008/10
  • Large-scale quantitative glycomics by glycoblotting method  [Invited]
    Shin-Ichiro Nishimura
    BioJapan2008 World Business Forum  2008/10
  • 大規模グライコミクスと疾患バイオマーカー探索研究  [Invited]
    西村 紳一郎
    第28回日本糖質学会年会  2008/08
  • 生命を探る-遺伝子だけでは語れない生命の謎がある-  [Invited]
    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  2008/08
  • Glycoblotting allows large-scale functional glycomics  [Invited]
    Shin-Ichiro Nishimura
    International Carbohydrate Symposium 2008  2008/08
  • Toward carbohydrate-based drug discovery  [Invited]
    Shin-Ichiro Nishimura
    BCN Biomed Seminar IRB  2008/07
  • 多糖鎖誘導体の整形外科領域での高次利用  [Not invited]
    西村 紳一郎
    繊維学会平成20年度年次大会  2008/06
  • 糖鎖合成と創薬研究  [Invited]
    西村 紳一郎
    天然高分子研究セミナー  2008/06
  • 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  [Invited]
    西村 紳一郎
    第81回日本整形外科学会学術総会  2008/05
  • Synthesis and characterization of MUC1-related glycopeptides  [Invited]
    Shin-Ichiro Nishimura
    EPA Chemistry 3rd Flash Conference  2008/03
  • 糖鎖修飾の大規模高速解析技術  [Invited]
    西村 紳一郎
    大阪大学蛋白質研究所セミナー  2008/01
  • 糖鎖研究からバイオ産業育成への挑戦  [Invited]
    西村 紳一郎
    日本応用糖質科学会平成19年度大会  2007/08
  • Clinical Glycomics Based on glycoform-focused reverse genomics  [Invited]
    Shin-Ichiro Nishimura
    Lecture at Genomics Research Center in Academia Sinca  2007/07
  • Conformational and biological characterization of synthetic mucin glycopeptides  [Invited]
    Shin-Ichiro Nishimura
    MICC-3 meeting  2007/07
  • Mechanism-based drug design: Novel strategy toward highly selective therapeutic reagents  [Invited]
    Shin-Ichiro Nishimura
    Gordon Research Conferences 2007  2007/06
  • Glycoblotting-Based Clinical Glycomics  [Invited]
    Shin-Ichiro Nishimura
    BENZON SYMPOSIUM No.54  2007/06
  • 創薬研究開発を加速するChemical Biology  [Invited]
    西村 紳一郎
    日本化学会第87春季年会(2007)  2007/03
  • 臨床糖鎖情報科学へのアプローチ  [Invited]
    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  2007/03
  • 糖鎖の迅速解析法  [Invited]
    西村 紳一郎
    JST-SORSTジョイントシンポジウム  2007/01
  • 北大が開拓する未来創薬研究拠点  [Invited]
    西村 紳一郎
    北海道大学21世紀COEプログラム  2007/01
  • Glycoform-focused reverse genomics (GFRG)  [Invited]
    西村 紳一郎
    日本薬学会近畿支部 新春特別講演会  2007/01


Industrial Property Rights

  • 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  

Awards & Honors

  • 2022/02 Hokkaido University Hokkaido University President's Award for Excellence in Research and Teaching for AY2022
    受賞者: Shin-Ichiro Nishimura
  • 2020/04 Ministry of Education, Culture, Sports, Science and Technology-Japan MEXT (Minister of Education, Culture, Sports, Science and Technology) Minister’s, Science and Technology for AY2020
    受賞者: Shin-Ichiro Nishimura
  • 2009/06 Cabinet Office 7th Industry-Academia-Government Collaboration Contribution Award
    受賞者: Shin-Ichiro Nishimura
  • 2008/11 The Hokkaido Shimbun Press The Hokkaido Shimbun Culture Prize 2008
    受賞者: Shin-Ichiro Nishimura
  • 2006/03 Japan Association for Chemical Innovation METI (Ministry of Economy, Trade and Industry) Minister’s Award at the GSC Awards for 2006
     Automated Glycoconjugate Synthesis by Artificial Golgi Apparatus 
    受賞者: Shin-Ichiro Nishimura;Shionogi;Co., Ltd;TOYOBO;HITACHI
  • 2005/09 JST, NEDO Outstanding Performance Awards of UBS Special Award Innovation Japan 2005
     Glycan Purification Kit S-Bio BlotGlyco 
    受賞者: Shin-Ichiro Nishimura
  • 2004/05 The Society of Polymer Science, Japan The Award of the Society of Polymer Science, Japan
     Studies on Synthesis of Glycoconjugate 
    受賞者: Shin-Ichiro Nishimura
  • 1999/03 The Chemical Society of Japan The Chemical Society of Japan Award for Creative Work for 1998
     Development of Efficient Synthesis of Glycoconjugate and Application to Its Functional Analysis 
    受賞者: Shin-Ichiro Nishimura
  • 1998/07 The Japanese Society of Carbohydrate Research JSCR (The Japanese Society of Carbohydrate Research) Incentive Awards
     Functional Analysis of Glycoconjugate and Its Application 
    受賞者: Shin-Ichiro Nishimura
  • 1997/05 Johns Hopkins University Membership of the Johns Hopkins Society of Scholars
     Shin-Ichiro Nishimura

Educational Activities

Teaching Experience

  • Functional and Regulatory Life Science
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 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
  • Soft Matter Science
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : (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
  • Transdisciplinary Life Science
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : (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
  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : Drug discovery, design, screening, developing and sales
  • Applied Biological Organic Chemistry
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 理学部
    キーワード : 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

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