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Hiroshi Hinou
Faculty of Advanced Life Science　Advanced Transdisciplinary Science　Drug Discovery Research
Professor
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Last Updated :2024/05/09

Researcher Profile and Settings

Affiliation

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

Job Title

  • Professor

Degree

  • PhD

URL

Researcher ID

  • A-4395-2012

J-Global ID

Research Interests

  • organic chemistry   生物有機化学   糖鎖工学   mass spectrometry   Analytical Chemistry   Surface chemistry   創薬化学   environmentally-controlled chemistry   cyclic compounds   

Research Areas

  • Nanotechnology/Materials / Chemical biology
  • Life sciences / Bioorganic chemistry
  • Nanotechnology/Materials / Synthetic organic chemistry
  • Nanotechnology/Materials / Analytical chemistry
  • Life sciences / Pharmaceuticals - chemistry and drug development
  • Life sciences / Bacteriology
  • Nanotechnology/Materials / Biochemistry
  • Manufacturing technology (mechanical, electrical/electronic, chemical engineering) / Applied biofunctional and bioprocess engineering
  • Nanotechnology/Materials / Nanobioscience
  • Nanotechnology/Materials / Green/sustainable/environmental chemistry
  • Environmental science/Agricultural science / Environmental effects of chemicals

Educational Organization

Academic & Professional Experience

  • 2019/04 - Today 北海道大学 先端生命科学研究院 次世代物質生命科学研究センター副センタ―長
  • 2019/01 - Today Hokkaido University Faculty of Advanced Life Science Professor
  • 2013/04 - 2018/12 Hokkaido University Faculty of Advanced Life Science Associate Professor
  • 2007/10 - 2013/03 Hokkaido University Faculty of Advanced Life Science Assistant Professor
  • 2010/02 - 2010/09 Institute for Research in Biomedicine (IRB barcelona) Visiting researcher
  • 2006 - 2009 産業技術総合研究所創薬シーズ探索研究ラボ 大学等非常勤研究員
  • 2005 - 2007 北海道大学理学研究科 助手
  • 2005 - 2006 産業技術総合研究所糖鎖工学研究センター 大学等非常勤研究員
  • 2003 - 2005 産業技術総合研究所糖鎖工学研究センター糖鎖自動合成チーム 研究員
  • 2002 - 2003 理化学研究所細胞制御化学研究室 共同研究員
  • 2002 - 2003 Nihon University School of Pharmacy
  • 2000 - 2002 東京理科大学理学部化学科 非常勤講師
  • 2000 - 2002 理化学研究所細胞制御化学研究室 協力研究員
  • 1999 - 2000 理化学研究所有機合成化学研究室 Junior Research Associate

Education

  • 1997/04 - 2000/03  Saitama University  Graduate School of Science and Engineering  Division of Biological and Environmental Sciences
  • 1995/04 - 1997/03  Saitama University  Graduate School of Science and Engineering  Division of Applied Chemistry
  •        - 1995  Saitama University  Faculty of Engineering

Association Memberships

  • The Mass Spectrometry Society of Japan   THE SOCIETY OF POLYMER SCIENCE, JAPAN   THE PHARMACEUTICAL SOCIETY OF JAPAN   THE JAPANESE SOCIETY OF CARBOHYDRATE RESEARCH   THE CHEMICAL SOCIETY OF JAPAN   GFRG Project   

Research Activities

Published Papers

  • Dereje G. Feleke, Bryan M. Montalban, Solomon T. Gizaw, Hiroshi Hinou
    2024/03/28
  • Bryan M. Montalban, Hiroshi Hinou
    ACS Infectious Diseases 2024/02/09 [Refereed][Not invited]
  • Shogo Urakami, Hiroshi Hinou
    International Journal of Molecular Sciences 2023/11/28 [Refereed][Invited]
  • Hiroshi Hinou
    Trends in Glycoscience and Glycotechnology 35 (204) E19 - E22 0915-7352 2023/03/25 [Refereed][Invited]
  • 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]
  • Bryan M. Montalban, Hiroshi Hinou
    Proteomics 1615-9853 2023 [Refereed][Not invited]
     
    Sulfated N- and O-glycans exist in trace levels which are challenging to detect, especially when abundant neutral and sialylated glycans are present. Current matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based sulfoglycomics approaches effectively utilize permethylation to discriminate sulfated glycans from sialyl-glycans. And a charge-based separation to isolate the sulfated glycans from the rest of the permethylated neutral and sialyl-glycans. However, these approaches suffer from concomitant sample losses during cleanup steps. Herein, we describe Glycoblotting as a straightforward complementary method with seamless glycan purification, enrichment, methylation, and labeling on a single platform to address sulfated glycan enrichment, sialic acid methylation, and sample loss. Glycoblottings’ on-bead chemoselective ligation of reducing sugars with hydrazide showed excellent recovery of sulfated glycans, allowing the detection of more sulfated glycan species. On-bead methyl esterification of sialic acid using 3-methyl-1-p-tolyltriazene (MTT) effectively discriminates sulfated glycans from sialyl-glycans. Furthermore, we have shown that using MTT as a methylating agent allowed us to simultaneously detect and differentiate sulfate from phosphate groups in isobaric N-glycan species. We believe that Glycoblotting will contribute significantly to the MALDI-TOF MS-based Sulphoglycomics workflow.
  • 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).
  • Shogo Urakami, Hiroshi Hinou
    ACS Omega 7 (43) 39280 - 39286 2470-1343 2022/11/01 [Refereed]
  • Hiroshi Hinou
    Scientific Reports 12 (1) 2045-2322 2022/10/23 [Refereed][Not invited]
     
    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.
  • Erina Barada, Hiroshi Hinou
    International Journal of Molecular Sciences 23 (20) 12510  2022/10/22 [Refereed][Invited]
  • 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.
  • Shogo Urakami, Hiroshi Hinou
    Analysis & Sensing 2 (2) e20210004  2629-2742 2022/03 [Refereed]
  • Ana I Carbajo-Gordillo, José L Jiménez Blanco, Juan M Benito, Hugo Lana, Gema Marcelo, Christophe Di Giorgio, Cédric Przybylski, Hiroshi Hinou, Valentín Ceña, Carmen Ortiz Mellet, Francisco Mendicuti, Conchita Tros de Ilarduya, José M García Fernández
    Biomacromolecules 21 (12) 5173 - 5188 2020/12/14 [Refereed]
     
    The architectural perfection and multivalency of dendrimers have made them useful for biodelivery via peripheral functionalization and the adjustment of dendrimer generations. Modulation of the core-forming and internal matrix-forming structures offers virtually unlimited opportunities for further optimization, but only in a few cases this has been made compatible with strict diastereomeric purity over molecularly diverse series, low toxicity, and limited synthetic effort. Fully regular star polymers built on biocompatible macrocyclic platforms, such as hyperbranched cyclodextrins, offer advantages in terms of facile synthesis and flexible compositions, but core elaboration in terms of shape and function becomes problematic. Here we report the synthesis and characterization of star polymers consisting of functional trehalose-based macrocyclic cores (cyclotrehalans, CTs) and aminothiourea dendron arms, which can be efficiently synthesized from sequential click reactions of orthogonal monomers, display no cytotoxicity, and efficiently complex and deliver plasmid DNA in vitro and in vivo. When compared with some commercial cationic dendrimers or polymers, the new CT-scaffolded star polymers show better transfection efficiencies in several cell lines and structure-dependent cell selectivity patterns. Notably, the CT core could be predefined to exert Zn(II) complexing or molecular inclusion capabilities, which has been exploited to synergistically boost cell transfection by orders of magnitude and modulate the organ tropism in vivo.
  • 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.
  • Pablo A Guillen-Poza, Elena M Sánchez-Fernández, Gerard Artigas, José Manuel García Fernández, Hiroshi Hinou, Carmen Ortiz Mellet, Shin-Ichiro Nishimura, Fayna Garcia-Martin
    Journal of medicinal chemistry 63 (15) 8524 - 8533 2020/08/13 [Refereed]
     
    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.
  • 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 (19) 4999 - 5006 2041-6520 2020/05 [Refereed][Not invited]
     
    Aberrantly truncated immature O-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immature O-glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognises specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its solution NMR structure, an extended conformation induced by site-specific glycosylation. We demonstrate that this method accelerates dramatically the development of a new class of designated antibodies targeting a variety of "dynamic neoepitopes" elaborated by disease-specific O-glycosylation in the immunodominant mucin domains and mucin-like sequences found in intrinsically disordered regions of many proteins.
  • 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.
  • 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]
     

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

  • Hiroshi Hinou
    International Journal of Mass Spectrometry 1387-3806 2019/09 [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.
  • Hiroshi Hinou
    BMC cancer 19 (1) 588  2019/06/17 [Refereed][Not invited]
     
    BACKGROUND:Alterations in protein glycosylation patterns have potentially been targeted for biomarker discovery in a wide range of diseases including cancer. Although there have been improvements in patient diagnosis and survival for breast cancer (BC), there is no clinically validated serum biomarker for its early diagnosis. Here, we profiled whole serum and purified Immunoglobulin G (IgG) fraction N-glycome towards identification of non-invasive glycan markers of BC. METHODS:We employed a comprehensive glycomics approach by integrating glycoblotting-based glycan purification with MALDI-TOF/MS based quantitative analysis. Sera of BC patients belonging to stages I-IV and normal controls (NC) were collected from Ethiopian women during 2015-2016. IgG was purified by affinity chromatography using protein G spin plate and further subjected to glycoblotting for glycan release. Mass spectral data were further processed and evaluated rigorously, using various bioinformatics and statistical tools. RESULTS:Out of 35 N-glycans that were significantly up-regulated in the sera of all BC patients compared to the NC, 17 complex type N-glycans showed profound expression abundance and diagnostic potential (AUC = 0.8-1) for the early stage (I and II) BC patients. Most of these glycans were core-fucosylated, multiply branched and sialylated structures, whose abundance has been strongly associated with greater invasive and metastatic potential of cancer. N-glycans quantified form IgG confirmed their abundance in BC patients, of which two core-fucosylated and agalactosylated glycans (m/z 1591, 1794) could specifically distinguish (AUC = 0.944 and 0.921, p ≤ 0.001) stage II patients from NC. Abundance of such structural features in IgG is associated with a decrease in its immunosuppressive potential towards tumor cells, which in part may correlate with the aggressive nature of BC commonly noticed in black population. CONCLUSIONS:Our comprehensive study has addressed for the first time both whole serum and IgG N-glycosylation signatures of native black women suffering from BC and revealed novel glyco-biomarkers with marked overexpression and distinguishing ability at early stage patients. Further studies on direct identification of the intact glycoproteins using a glycoprteomics approach will provide a deeper understanding of specific biomarkers towards their clinical utility.
  • Hiroshi Hinou
    Bioorganic & medicinal chemistry 27 (13) 2822 - 2831 0968-0896 2019/05/06 [Refereed][Not invited]
     
    Structural and functional effects of core M1 type glycan modification catalyzed by protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) were investigated using a core M1 glycoform focused library of an α-dystroglycan fragment, 372TRGAIIQTPTLGPIQPTRV390. Evanescent-field fluorescence-assisted microarray system illuminated the specific binding pattern of plant lectins that can discriminate the glycan structure of core M1 glycan of the library. The comparative NMR analysis of synthetic glycopeptide having different length of the O-mannosylated glycans revealed a conformational change of the peptide backbone along with core M1 disaccharide formation. No long-range NOE signals of glycan-amino acid nor inter amino acid indicate the conformational change is induced by steric hindrance of core M1, the sole 1,2-O-modified form among protein binding sugar residue found in mammals.
  • Hiroshi Hinou
    Abstracts of Papers of the American Chemical Society 2019
  • Jurgen T. Sanes, Hiroshi Hinou, Yuan Chuan Lee, Shin-Ichiro Nishimura
    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.
  • Hiroshi Hinou
    PloS one 13 (12) e0209515  2018/12/28 [Refereed][Not invited]
     
    BACKGROUND:Most glycomics studies have focused on understanding disease mechanisms and proposing serum markers for various diseases, yet the influence of ethnic variation on the identified glyco-biomarker remains poorly addressed. This study aimed to investigate the inter-ethnic serum N-glycan variation among US origin control, Japanese, Indian, and Ethiopian healthy volunteers. METHODS:Human serum from 54 healthy subjects of various ethnicity and 11 Japanese hepatocellular carcinoma (HCC) patients were included in the study. We employed a comprehensive glycoblotting-assisted MALDI-TOF/MS-based quantitative analysis of serum N-glycome and fluorescence HPLC-based quantification of sialic acid species. Data representing serum N-glycan or sialic acid levels were compared among the ethnic groups using SPSS software. RESULTS:Total of 51 N-glycans released from whole serum glycoproteins could be reproducibly quantified within which 33 glycoforms were detected in all ethnicities. The remaining N-glycans were detected weakly but exclusively either in the Ethiopians (13 glycans) or in all the other ethnic groups (5 glycans). Highest abundance (p < 0.001) of high mannose, core-fucosylated, hyperbranched/hypersialylated N-glycans was demonstrated in Ethiopians. In contrast, only one glycan (m/z 2118) significantly differed among all ethnicities being highest in Indians and lowest in Ethiopians. Glycan abundance trend in Ethiopians was generally close to that of Japanese HCC patients. Glycotyping analysis further revealed ethnic-based disparities mainly in the branched and sialylated structures. Surprisingly, some of the glycoforms greatly elevated in the Ethiopian subjects have been identified as serum biomarkers of various cancers. Sialic acid level was significantly increased primarily in Ethiopians, compared to the other ethnicities. CONCLUSION:The study revealed ethnic-specific differences in healthy human serum N-glycome with highest abundance of most glycoforms in the Ethiopian ethnicity. The results strongly emphasized the need to consider ethnicity matching for accurate glyco-biomarker identification. Further large-scale study employing various ethnic compositions is needed to verify the current result.
  • Hiroshi Hinou
    ACS Medicinal Chemistry Letters 9 (9) 889 - 894 2018/09 [Refereed][Not invited]
  • K. Yogesh, Toshiya Kamiyama, Chikara Ohyama, Tohru Yoneyama, Kazuhiro Nouso, Satoshi Kimura, Hiroshi Hinou, Shin-Ichiro Nishimura
    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.
  • Risa Takayama, Shun Hayakawa, Hiroshi Hinou, Fernando Albericio, Fayna Garcia-Martin
    JOURNAL OF PEPTIDE SCIENCE 24 (8-9) e3111  1075-2617 2018/08 [Refereed][Not invited]
     
    Ester linkage (s) is a key chemical connector in organic chemistry, including natural products, peptides, and synthetic polymers. We herein describe a straightforward method for the efficient formation of ester linkage (s) on solid-phase. This method simply involves the use of amide coupling reagents under microwave irradiation. The robustness of this method relies on the use of classical solid-phase coupling reagents, heating by microwave irradiation, and a short time period, which results in high yields and the minimization of racemization.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • R. S. Tan, H. Hinou, S. -I. Nishimura
    RSC ADVANCES 6 (56) 50833 - 50836 2046-2069 2016 [Refereed][Not invited]
     
    A novel beta-galactosynthase-beta-mannosynthase dual-activity of beta-galactosidase from Aspergillus oryzae was uncovered, for the first time, using free monosaccharide substrates. The enzyme successfully converted galactose and mannose monomer sugars efficiently into Gal-beta(1-6)-Gal and Man-beta(1-6)-Man, respectively. The discovery could potentially revolutionize chemoenzymatic glycan and non-digestible oligosaccharide (NDO) 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.
  • Ibrahim F. Rehan, Koichiro Ueda, Tomohiro Mitani, Maho Amano, Hiroshi Hinou, Tetsu Ohashi, Seiji Kondo, Shin-Ichiro Nishimura
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 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.
  • Helena Coelho, Talcahiko Matsushita, Gerard Artigas, Hiroshi Hinou, F. Javier Canada, Richard Lo-Man, Claude Leclerc, Eurico J. Cabrita, Jesus Jimenez-Barbero, Shin-Ichiro Nishimura, Fayna Garcia-Martin, Filipa Marcelo
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 137 (39) 12438 - 12441 0002-7863 2015/10 [Refereed][Not invited]
     
    The identification of MUC1 tumor-associated Tn antigen (alpha GalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.
  • Solomon T. Gizaw, Toshiaki Koda, Maho Amano, Keiko Kamimura, Tetsu Ohashi, Hiroshi Hinou, Shin-Ichiro Nishimura
    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1850 (9) 1704 - 1718 0304-4165 2015/09 [Refereed][Not invited]
     
    Background: Huntington's disease (HD) is an autosomal, dominantly inherited and progressive neurodegenerative disease, nosologically classified as the presence of intranuclear inclusion bodies and the loss of GABA-containing neurons in the neostriatum and subsequently in the cerebellar cortex. Abnormal processing of neuronal proteins can result in the misfolding of proteins and altered post-translational modification of newly synthesized proteins. Total glycomics, namely, N-glycomics, O-glycomics, and glycosphingolipidomics (GSL-omics) of HD transgenic mice would be a hallmark for central nervous system disorders in order to discover disease specific biomarkers. Methods: Glycoblotting method, a high throughput glycomic protocol, and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) were used to study the total glycome expression levels in the brain tissue (3 mice of each sex) and sera (5 mice of each sex) of HD transgenic and control mice. All experiments were performed twice and differences in the expression levels of major glycoforms were compared between HD transgenic and control mice. Results: We estimated the structure and expression levels of 87 and 58 N-glycans in brain tissue and sera, respectively, of HD transgenic and control mice. The present results clearly indicated that the brain glycome and their expression levels are significantly gender specific when compared with those of other tissues and serum. Core-fucosylated and bisecting-GlcNAc types of N-glycans were found in increased levels in the brain tissue HD transgenic mice. Accordingly, core-fucosylated and sialic acid (particularly N-glycolylneuraminic acid, NeuGc) for biantennary type glycans were found in increased amounts in the sera of HD transgenic mice compared to that of control mice. Core 3 type O-glycans were found in increased levels in male and in decreased levels in both the striatum and cortexes of female HD transgenic mice. Furthermore, serum levels of core 1 type O-glycans decreased and were undetected for core 2 type O-glycans for HD transgenic mice. In glycosphingolipids, GD1a in brain tissue and GM2-NeuGc serum levels were found to have increased and decreased, respectively, in HD transgenic mice compared to those of the control group mice. Conclusion: Total glycome expression levels are significantly different between HD transgenic and control group mice. General significance: Glycoblotting combined with MALDI-TOF/MS total glycomics warrants a comprehensive, effective, novel and versatile technique for qualitative and quantitative analysis of total glycome expression levels. Furthermore, glycome-focused studies of both environmentally and genetically rooted neurodegenerative diseases are promising candidates for the discovery of potential disease glyco-biomarkers in the post-genome era. (C) 2015 Elsevier B.V. All rights reserved.
  • Roger S. Tan, Kentaro Naruchi, Maho Amano, Hiroshi Hinou, Shin-Ichiro Nishimura
    ACS CHEMICAL BIOLOGY 10 (9) 2073 - 2086 1554-8929 2015/09 [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.
  • Gizaw ST, Koda T, Amano M, Kamimura K, Ohashi T, Hinou H, Nishimura SI
    Biochimica et biophysica acta 1850 (9) 1704 - 1718 0006-3002 2015/04 [Refereed][Not invited]
  • 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.
  • Matsushita T, Takada W, Igarashi K, Naruchi K, Miyoshi R, Garcia-Martin F, Amano M, Hinou H, Nishimura S
    Biochimica et biophysica acta 3 1840 (3) 1105 - 1116 0006-3002 2014/03 [Refereed][Not invited]
  • 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.
  • 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.
  • HINO HIROSHI, NISHIMURA SHIN'ICHIRO
    化学 69 (1) 74 - 75 0451-1964 2014 [Not refereed][Invited]
  • Ravi H. Kumar, Kentaro Naruchi, Risho Miyoshi, Hiroshi Hinou, Shin-Ichiro Nishimura
    ORGANIC LETTERS 15 (24) 6278 - 6281 1523-7060 2013/12 [Refereed][Not invited]
     
    A novel protocol for the synthesis of general N-glycan core structures was established by means of Man beta(1 -> 4)Man peracetate derived from a naturally abundant locust bean gum as a key starting material. Phenyl (2-O-benzy1-4,6-O-benzylidine-beta-D-mannopyranosyl)-(1 -> 4)-3,6-di-O-benzyl-2-azido-2-deoxy-1-thio-beta-D-glucopyranoside facilitated the synthesis of key intermediates leading to hyperbranched N-glycan core structures.
  • Takahiko Matsushita, Seiji Handa, Kentaro Naruchi, Fayna Garcia-Martin, Hiroshi Hinou, Shin-Ichiro Nishimura
    Polymer Journal 45 (8) 854 - 862 0032-3896 2013/08 [Not refereed][Invited]
     
    Functionalized, high-generation (G7) polyamidoamine (PAMAM) dendrimers are a convenient scaffold for the fully automated enzymatic synthesis of oligosaccharides such as biologically important sialyl Lewis X tetrasaccharide derivatives. In this study, we expanded this strategy to the synthesis of more complicated glycopeptides by assessing the feasibility of G7 PAMAM dendrimer-based polymer supports for attaching glycopeptide intermediates during the subsequent enzymatic modification steps. A monosaccharide-attached glycopeptide containing an N-terminal heterobifunctional linker was prepared by microwave-assisted solid-phase synthesis and was coupled with an aminooxy-functionalized G7 PAMAM dendrimer through oxime bond formation. This reaction proceeded smoothly at pH 4 and afforded the conjugates in 98% yield when 0.2 equivalents of the glycopeptide were combined with 1 equivalent of the aminooxy group of dendrimer. Although modifications using recombinant human β1,4-galactosyltransferase/uridine-5'-diphospho-α-D-galactose disodium salt and recombinant rat α2,3-sialyltransferase/cytidine-5'- monophospho-β-D-N-acetylneuraminic acid disodium salt gave the trisaccharide Neu5Acα2,3Galβ1,4GlcNAc in quantitative yields, treatment with recombinant human α1,3-fucosyltransferase in the presence of excess guanosine 5'-diphospho-β-L-fucose disodium salt did not convert this trisaccharide into the tetrasaccharide sialyl Lewis X tetrasaccharide on the dendrimer. Further optimization studies are required to improve the efficiency of branched-type sugar elongations and product release from polymers by selective peptidases for constructing a high-throughput glycopeptide synthetic system. © 2013 The Society of Polymer Science, Japan (SPSJ) All rights reserved.
  • 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.
  • 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 3.2.1.35) and Streptomyces hyalurolyticus (EC 4.2.2.1). It was demonstrated that glycoblotting method allows for high throughput and quantitative analysis of hyaluronan fragments within a wide dynamic range (1 similar to 1,000 pmole) when 5 mu g of hyaluronan digests were applied for this enrichment protocol. Molecular size and distribution of hyaluronan fragments were proved to be influenced strongly by conditions and hyaluronidases employed while source of hyaluronan did not affect the degradation profiles. Strikingly, the present method uncovered the existence of the smallest and unusual hyaluronan degradation fragments such as a disaccharide GlcA beta 1-3GlcNAc during the digestion by bovine hyaluronidase and a trisaccharide GlcA beta 1-3GlcNAc beta 1-4GlcA derivative by Streptomyces hyaluronidase. Bovine testis hyaluronidases afforded hyaluronan tetra- and hexasaccharides as major products. On the other hand, it was demonstrated that Streptomyces hyaluronidase can produce odd number fragments from three to nine sugar residues while even number fragments from four to fourteen sugar residues were major products.
  • 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.
  • Hiroshi Hinou
    Biochimica et Biophysica Acta (BBA) - General Subjects 1820 (9) 1391 - 1398 0006-3002 2012/09 [Refereed][Not invited]
  • 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.
  • 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.
  • 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.
  • Takayuki Furukawa, Hiroshi Hinou, Ken Shimawaki, Shin-Ichiro Nishimura
    TETRAHEDRON LETTERS 52 (43) 5567 - 5570 0040-4039 2011/10 [Refereed][Not invited]
     
    Development of beta-selective glucuronylation reaction using phenyl 2,4-di-O-acetyl-1-thio-beta-D-glucopyranosidurono-6,3-lactone was described. Glycosylations of this glycosyl donor with hexosamine derivatives proceeded with excellent yield and beta-stereoselectivity to afford glycosaminoglycan-type disaccharides. (C) 2011 Elsevier Ltd. All rights reserved.
  • 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.
  • 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.
  • 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 [Refereed][Not invited]
     
    The simple doping of calcium chloride allowed highly improved yields in the glycosylation promoted by sulfated zirconia (SZ) without calcination. It was revealed by means of per-O-benzylated galactose trichloroacetimidate as a model glycosyl donor that this effect depends greatly on the cationic ion radius of used metal chlorides. Temperature-programmed desorption of ammonia (NH3 TPD) and the pyridine IR method showed clearly that coordination of calcium ion provides the SZ surface with newly formed Lewis acidic sites while the Bronsted acid site disappeared, indicating that the enhanced catalytic potency of SZ is due to the increased Lewis acid sites by doping the optimal calcium ions. The present findings might give insight into the relationship between a catalytic mechanism and superacidity of SZ, which is crucial for the design of novel superacid-based catalysts and environmentally benign glycosylation processes.
  • 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.
  • 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.
  • 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 2.4.1.41), a family of key enzymes that initiate posttranslational modification with O-glycans in mucin synthesis by introduction of alpha-GalNAc residues, are structurally composed of a catalytic domain and a lectin domain. It has been known that multiple Ser/Thr residues are assigned in common mucin glycoproteins as potential O-glycosylation sites and more than 20 distinct isoforms of this enzyme family contribute to produce densely O-glycosylated mucin glycoproteins. However, it seems that the functional role of the lectin domain of ppGalNAcTs remains unclear. We considered that electron capture dissociation mass spectrometry (ECD-MS), a promising method for highly selective fragmentation at peptide linkages of glycopeptides to generate unique c and z series of ions, should allow for precise structural characterization to uncover the mechanism in O-glycosylation of mucin peptides by ppGalNAcTs. In the present study, it was demonstrated that a system composed of an electrospray source, a linear RFQ ion trap that isolates precursor ions, the ECD device, and a TOF mass spectrometer is a nice tool to identify the preferential O-glycosylation sites without any decomposition of the carbohydrate moiety. It should be noted that electrons used for ECD are accelerated within a range from 1.75 to 9.75 eV depending on the structures of glycopeptides of interest. We revealed for the first time that additional installation of a alpha-GalNAc residue at potential glycosylation sites by ppGalNAcT2 proceeds smoothly in various unnatural glycopeptides having alpha-Man, alpha-Fuc, and beta-Gal residues as well as alpha-GalNAc residues. The results may suggest that ppGalNAcT2 did not differentiate totally presubstituted sugar residues in terms of configuration of functional groups, D-, L-configuration, and even alpha-, beta-stereochemistry at an anomeric carbon atom when relatively short synthetic peptides were employed for the acceptor substrates. Unexpected characteristics of ppGalNAcT2 motivated us to challenge site-directed installation of alpha-GalNAc residues at desired position(s) by protecting some hydroxyl groups of Thr/Ser residues with selectively removable sugars, notably a novel concept as "carbohydrate as protective groups", toward a goal of the systematic chemical and enzymatic synthesis of biologically important mucin glycopeptides.
  • 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.
  • Hiroshi Hinou
    Molecular Imaging for Integrated Medical Therapy and Drug Development 2010
  • 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][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.
  • 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.
  • An Efficient Strategy for the Explortion of Specific Inhibitors of Sialyltransferases
    Kensku Hosoghchi, Hiroshi Hinou, Shin-Ichiro Nishimura
    Proceeding of Molecular Imaging for Integrated Medical Therapy and Drug Development 294 - 301 2009 [Not refereed][Invited]
  • Hiroki Shimizu, Yayoyi Yoshimura, Hiroshi Hinou, Shin-Ichiro Nishimura
    TETRAHEDRON 64 (43) 10091 - 10096 0040-4020 2008/10 [Refereed][Not invited]
     
    The efficiency of microwave irradiation at low temperature for glycosylations is described. Although oligosaccharide synthesis usually requires reactive donors for glycosylations, which have leaving groups on the another positions, i.e., trichloroacetoimidates, halogenates, thioalkyl glycosides, etc., the suitable donors in our microwave supported synthesis of Lewis X oligosaccharide were very stable acetate derivatives. Regarding glycosylation with a fucosyl acetate donor and a glucosamine acceptor, microwave irradiation with simultaneous cooling improved yields. Moreover, further synthesis to Lewis X derivatives was achieved only with microwave irradiation at low temperatures. Without microwave irradiation, we could only obtain byproducts and none of the designed product at any reaction temperature. (C) 2008 Elsevier Ltd. All rights reserved.
  • 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]
  • 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.
  • Naoki Ohyabu, Takahiko Matsushita, Hiroshi Hinou, Ryuko Izumi, Hiroki Shimizu, Hirosato Kondo, Shin-Ichiro Nishimura
    GLYCOBIOLOGY 16 (11) 1152 - 1152 0959-6658 2006/11 [Refereed][Not invited]
  • Hiroki Ito, Kisaburo Deguchi, Kuriko Yamada, Shinji Nagai, Masataka Fumoto, Hiroshi Hinou, Hiroaki Nakagawa, Yasuro Shinohara, Shin-Ichiro Nishimura
    GLYCOBIOLOGY 16 (11) 1136 - 1136 0959-6658 2006/11 [Refereed][Not invited]
  • T Matsushita, H Hinou, M Fumoto, M Kurogochi, N Fujitani, H Shimizu, SI Nishimura
    JOURNAL OF ORGANIC CHEMISTRY 71 (8) 3051 - 3063 0022-3263 2006/04 [Refereed][Not invited]
     
    A MUCI-related glycopeptide having five core-2 hexasaccharide branches C330H527N46O207, MW = 8450.9) was synthesized by a new strategy using a combination of microwave-assisted solid-phase synthesis (MA-SPGS) and enzymatic sugar elongation. Synthesis of a key glycopeptide intermediate was best achieved in a combination of PEGA [poly(ethylene glycol)-poly-(NN-dimethylacrylamide) copolymer] resin and MA-SPGS using glycosylated amino acid building blocks with high speed and high purity. Deprotection of the glycopeptide intermediate and subsequent glycosyltransferase-catalyzed sugar elongations were performed for generation of the additional diversities with the sugar moieties of glycopeptides using beta 1,4-galactosyltransferase (beta 1,4-GaIT) and two kinds of alpha 2,3-sialyltransferases [ST3Gal III; alpha 2,3-(N)-SiaT and ST3Gal II; alpha 2,3-(O)-SiaT]. These reactions proceeded successfully in the presence of 0.2% Triton X-100 to convert the chemically synthesized trisaccharide glycans to disialylated hexasaccharide.
  • 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]
  • 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.
  • 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.
  • 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.
  • Y Yoshimura, H Shimizu, H Hinou, SI Nishimura
    TETRAHEDRON LETTERS 46 (28) 4701 - 4705 0040-4039 2005/07 [Refereed][Not invited]
     
    Efficient microwave-assisted glycosylations from methyl glucopyranosides are described. We have discussed the effects of microwave irradiation on this unique glycoside exchanging reaction from view points such as amount of Lewis acid promoters and acceptors, hydroxyl protecting groups of methyl glucopyranosides donors for reactivity, and neighboring effect. (c) 2005 Published by Elsevier Ltd.
  • 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.
  • 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]
  • 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]
  • H Hinou, XL Sun, Y Ito
    JOURNAL OF ORGANIC CHEMISTRY 68 (14) 5602 - 5613 0022-3263 2003/07 [Refereed][Not invited]
     
    Bisubstrate-type sialyltransferase inhibitors 1/2a-e, having CMP-NeuAc and N-acetyllactosamine (or lactose) moieties connected by an alkanedithiol linker, were synthesized systematically. A uniform synthetic strategy was adopted that consists of consecutive couplings of three components (N-acetyllactosamine or lactose, sialic acid, and CMP), followed by oxidation. Due to the sensitivity of the compounds under alkaline conditions, final deprotection required careful monitoring by H-1 NMR. The inhibitory activities of 1/2a-e toward ST6N and ST3N indicated that both the structure of the acceptor moiety and the distance between donor and acceptor moieties were important.
  • K Kawamura, H Hinou, G Matsuo, T Nakata
    TETRAHEDRON LETTERS 44 (28) 5259 - 5261 0040-4039 2003/07 [Refereed][Not invited]
     
    An efficient convergent strategy for the construction of a trans-fused 6-6-6-6-membered tetracyclic ether ring system was developed. The key steps involve coupling of two cyclic ethers by esterification, SmI2-promoted intramolecular reductive cyclization of iodo ester to hemiacetal, dehydration to dihydropyran, hydroboration, oxidation, intramolecular acetalization, and Lewis-acid catalyzed silane reduction. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • K Matsuoka, T Ohtawa, H Hinou, T Koyama, Y Esumi, SI Nishimura, K Hatano, D Terunuma
    TETRAHEDRON LETTERS 44 (18) 3617 - 3620 0040-4039 2003/04 [Refereed][Not invited]
     
    A novel anomeric beta-thioacetate of an N-acetyllactosamine derivative was efficiently synthesized in high yield from the known 2-azido glycosyl chloride using thioacetic acid as a convenient reagent. The synthesis involved not only an S,2 replacement of the chloride by a carbothiolate anion but also a reductive acetamidation of the azide group. Applications of the thioacetate for glycosidation were demonstrated to provide both O- and S-glycosides in high yields. Furthermore, both intermediates gave a new class of glycoclusters that included thioglycosidic linkages. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • H Hinou, XL Sun, Y Ito
    TETRAHEDRON LETTERS 43 (50) 9147 - 9150 0040-4039 2002/12 [Refereed][Not invited]
     
    A convergent strategy for the construction of bisubstrate-type sialyltransferase inhibitor (1) was developed. It consists of consecutive coupling of three components (N-acetyllactosamine, sialic acid, and CMP), followed by oxidation and deprotection. As expected, compound I showed potent inhibitory activities toward both 2,3-(N)- and 2,6-(N)-sialyltransferase. (C) 2002 Elsevier Science Ltd. All rights reserved.
  • H Hinou
    TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY 12 (65) 185 - 190 0915-7352 2000/05 [Not refereed][Invited]
     
    Hinou, H, 2000, '二糖類を出発物とした有用化合物の合成研究', vol. 12, no. 65, pp. 185-190.
  • G Matsuo, H Hinou, H Koshino, T Suenaga, T Nakata
    TETRAHEDRON LETTERS 41 (6) 903 - 906 0040-4039 2000/02 [Refereed][Not invited]
     
    A very efficient convergent strategy for the construction of the trans-fused 6-6-6-6-membered tetracyclic ether ring system was developed based on the acetylide-triflate coupling of two tetrahydropyrans, oxidation of the alkyne group to an alpha-diketone, double cyclization to 6,6,6,6-membered tetracyclic diacetal, and stereoselective reduction of the diacetal with Et3SiH-TMSOTf. (C) 2000 Elsevier Science Ltd. All rights reserved.
  • H Hinou, A Umino, K Matsuoka, D Terunuma, S Takahashi, Y Esumi, H Kuzuhara
    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 73 (1) 163 - 171 0009-2673 2000/01 [Refereed][Not invited]
     
    With interest in clustering bioactive chitooligosaccharides, a pair of amphiphilic derivatives of around DP (degree of polymerization) 6, which has been considered to be the minimum molecular length for some kinds of bioactivity, was synthesized. Homologous derivatives of chitopentaose and chitoheptaose carrying tetradecanoyl and tetradecyloxy groups at the NH and C-1 of the reducing ends, respectively, were actually obtained using a 1,6-anhydro-2-azido-2-deoxy-beta-D-glucopyranose derivative as the terminal precursor and a chitobiose derivative as the elongation unit. Micelle formation of both derivatives was assumed from the results obtained by dye solubilization tests.
  • Hiroshi Hinou, Hidehiro Kurosawa, Koji Matsuoka, Daiyo Terunuma, Hiroyoshi Kuzuhara
    Tetrahedron Letters 40 (8) 1501 - 1504 0040-4039 1999/02/19 [Refereed][Not invited]
     
    For the preparation of L-iduronic acid, trehalose was converted into a derivative of a novel disaccharide, β-L-idopyranosyl β-L-idopyranoside, through diastereoselective hydroboration of the 5,5'-di-eno intermediate. The 6- and 6'-hydroxy groups were then oxidized in two steps to give a disaccharide composed of 2 units of L-iduronate moieties, which underwent acidic cleavage of the glycosidic bond to give the target compound.
  • Y Ogawa, H Hinou, K Matsuoka, D Terunuma, H Kuzuhara
    TETRAHEDRON LETTERS 39 (32) 5789 - 5792 0040-4039 1998/08 [Refereed][Not invited]
     
    A pair of disaccharidic glycals thoroughly O-benzylated except the 4'- or the 6'-hydroxyl groups were prepared as the monomers for iodonium ion-promoted polymerization, which proceeded under dark conditions to give polysaccharides of more than DP 12 (24 saccharide). Reductive removal of the iodine atom and subsequent deprotection gave polysaccharides alternatively composed of beta-D-glucopyranosyl and 2-deoxy-alpha, beta-D-glucopyranosyl residues. (C) 1998 Elsevier Science Ltd. All rights reserved.
  • A Umino, H Hinou, K Matsuoka, D Terunuma, S Takahashi, H Kuzuhara
    JOURNAL OF CARBOHYDRATE CHEMISTRY 17 (2) 231 - 239 0732-8303 1998 [Refereed][Not invited]
     
    In the course of our studies on the synthesis of amphiphilic chitoheptaose derivatives carrying binary long hydrocarbon chains at the reducing sugar, tetradecyl 4-O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-2-tetradecanamido-beta-D-glucopyranoside was prepared as a model. For general applicability, the 1,6-anhydro-2-deoxy-2-tetradecanamido-beta-D-glucopyranosyl moiety was employed as a precursor of the reducing end. Acetolysis of the 1,6-anhydro ring using triethylsilyl triflate gave an oxazoline intermediate as a major product, accompanied by alpha-glycosyl acetate as a by-product. Immediate treatment of the mixture with 1-tetradecanol and protic acid followed by a separation work-up efficiently led to tettadecyl beta-glycoside derivative.

Books etc

Conference Activities & Talks

MISC

Industrial Property Rights

Awards & Honors

  • 2023/03 John Wiley & Sons, Inc. Top Downloaded Article
     Top Downloaded Article 
    受賞者: Shogo Urakami;Hiroshi HINOU
  • 2022/03 Analysis & Sensing. Most accessed 03/2022
  • 2011 Best Poster Award
  • 2011 Very Important Paper (VIP)
  • 2007 高分子研究奨励賞
  • 2007 Poster Presentation Award

Research Grants & Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2022/04 -2026/03 
    Author : 比能 洋, 小林 純子
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2021/04 -2024/03 
    Author : 小林 純子, 工藤 正尊, 比能 洋
     
    黄体は妊娠の成立と維持に必須なプロゲステロンを産生する内分泌組織で、排卵後の卵胞壁細胞より形成される。妊娠が成立しない場合、ヒトでは、黄体は1週間ほど機能したのち、自発的に退行する。一方、妊娠が成立すると、胎盤より産生されるヒト絨毛性ゴナドトロピン（hCG）の作用により、黄体は退行を免れ妊娠黄体となって妊娠初期の数か月間機能を維持する。マウスやウシなどの動物では、子宮由来の因子が黄体の退行を誘導するが、ヒトでは子宮由来の因子は卵巣周期に影響を与えない。ヒトでは、黄体内で産生される因子が自発的に黄体の退行を制御すると考えられるがそのメカニズムは不明な点が多く残されている。 我々は、ヒト黄体では、beta-galactosideを認識するガレクチンのうち、galectin-1が機能黄体に、galectin-3が退行黄体に発現しており、ガレクチンと糖鎖との相互作用が機能制御に重要な役割を果たすことを報告してきた。ガレクチンと糖鎖との結合を阻害するalpha2,6シアル酸修飾は退行黄体で増加することから、ヒト黄体の機能制御に重要な役割を果たすと考えられるが、alpha2,6シアル酸修飾された糖鎖をもつタンパク質やその機能の詳細は明らかでない。 本研究では、ヒト黄体細胞において、退行時にalpha2,6シアル酸修飾をうける糖タンパク質を同定し、その機能を明らかにするとともに、質量分析イメージング装置を用いた切片上で糖鎖を検出できる技術の確立を目指し、黄体の機能制御に重要な糖鎖を探索することを目的とする。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2019/04 -2023/03 
    Author : 西村 紳一郎, 田中 良和, 比能 洋, 尾瀬 農之
     
    申請者らの独創的なアイデアおよび開発戦略によって作製されたED抗体（epitope-defined antibody）を用いて、がん領域におけるアンメットメディカルニーズに応え得る新たながん治療薬の開発を目的として本研究課題を設定した。2019年度（初年度）は多くのがん細胞表面に高発現してがんの増殖や転移を促進することが広く知られるMUC1の細胞外ドメインに生成する多様な動的エピトープを標的とするED抗体である抗MUC1モノクローナル抗体（SN-101, SN-121, およびSN-131）に関して以下の各項目に進展があった。（１）SN-101の作製、特性、構造および機能に関する基礎的研究の成果について論文を発表した（H. Wakui, et al., A straightforward approach to antibodies recognizing cancer specific glycopeptidic neoepitopes, Chemical Science, 2020, in press）。本論文は水溶液中でユニークなコンフォメーションを示す糖ペプチドエピトープの糖鎖とペプチド領域を同時に認識して結合する抗MUC1抗体として構造が示された世界初の事例であり、ED-抗体の概念と基本的な製法を記載した最初の論文としての意義が大きい。また、（２）SN-121およびSN-131についても同様の構造機能解析が進んでおり、SN-131の結晶構造解析を完了した。これらの抗体の高付加価値化を目指してエピトープ認識領域での変異導入による機能改変の試みが開始されている。さらに、（３）SN-131に新たな機能（がん細胞障害活性）を付与するため遺伝子工学的手法により新たな二重特異性抗体の作製を行い、MUC1発現がん細胞を標的として特異的な細胞障害活性を示す新規SN-131キメラ抗体の開発に成功した。以上のように本年度はED-抗体開発戦略の有効性とdynamic epitope理論に基づく新しい抗体デザインに関する基本原理である「抗原構造形成における新たな分子機構」の普遍性を証明する様々な研究が極めて順調に進行している。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : Toda Masako
     
    The aim of this study was to identify immunological function of mannose-based molecules and its action mechanisms. Among α-mannooligosaccharides, long chain molecules such as α-Man-(1→6)-Man4 inhibited lipopolysaccharide (LPS)-stimulated activation of bone marrow derived murine dendritic cells (BMDC). In contrast, among β-mannooligosaccharides, β-Man-(1→4)-Man activated BMDC via engagement of toll-like receptor 4. We also found that α-mannan from yeast promotes anti-inflammatory responses (e.g., IL-10 production) in BMDC when it was stimulated with LPS. Metabolome analysis showed that α-mannan altered levels of many metabolites in BMDC, e.g., an increased level of lactic acid, the final product of glycolysis, and a reduced level of succinic acid, an inflammatory metabolite of TCA cycle. The results suggest that α-mannan induces reprogram of cellular metabolism and thereby promotes anti-inflammatory response in BMDC.
  • JST：A-STEP
    Date (from‐to) : 2021/04 -2022/03 
    Author : Hiroshi Hinou
  • JST：A-STEP
    Date (from‐to) : 2020/11 -2022/03 
    Author : Hiroshi Hinou
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2017/04 -2020/03 
    Author : Hinou Hiroshi
     
    Regarding the interaction between glycopeptides and lectins, mucin-type glycopeptides and O-mannose-type glycopeptides were used as models for interaction analysis by microarray and conformational analysis by NMR. In mucin-type glycopeptides, hydrogen bonds and hydrophobic interactions between glycans and peptides are strongly involved in conformations of both sugar chains and peptides. Both of the glycan and peptide moiety is markedly restricted near the binding sites of the glycan on the peptides. It was found that the binding property of lectin changes with the conformational change. On the other hand, it was confirmed that the O-mannose type glycopeptide does not cause conformational restriction of glycan, but that the conformational restriction of peptides occurs with the addition of a specific sugar residue. Corresponding to this peptide conformational change, the NMR signal and lectin binding pattern also altered.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)
    Date (from‐to) : 2013/05 -2018/03 
    Author : Nishimura Shin-Ichiro
     
    To establish the first disease-related glycome database, we have challenged the large-scale glycomics using more than 3500 patient’s sera provided from clinical teams. An innovative “automated glycan analytical system” facilitated rapid and large-scale serum glycomics and quantitative glycan profiling toward new biomarker discovery. For example, we succeeded in the discovery of novel N-glycan biomarkers as a significant recurrence factor based on whole serum glycan profiling of 369 patients of hepatocellular carcinoma(HCC)(Hepatology 2013 57, 2314-2325; Hepatology 2014 59, 355-356), N-glycans as promising prognostic biomarkers in renal cell carcinoma(The Prostate 2014 74, 1521-1529), and N-glycans associated significantly with metastatic castration-resistant status in prostate cancer(J. Urology 2014 191, 805-813). These results allowed the construction of the first database of the disease-related glycome feasible for the development of new diagnostic and therapeutic technologies.
  • 科学技術振興機構(JST)：A-STEP機能検証フェーズ（旧・地域産学バリュープログラム）(A-STEP機能検証フェーズ)
    Date (from‐to) : 2015/06 -2016/03 
    Author : 比能洋
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2012/04 -2015/03 
    Author : KIMURA Katsuki, HINOU Hiroshi, AIZAWA Tomoyasu
     
    Enrichment of polysaccharides by glyco-blotting, in which polysaccharides are specifically purified via interactions between the aldehydes in the polysaccharides and aminooxy groups on glycoblotting beads, enabled MALDI-TOF/MS analysis at a high resolution. Structures of polysaccharides extracted from fouled membranes used in a pilot-scale MBR and those in the supernatant of the mixed liquor suspension in the MBR were investigated. It was demonstrated that the overlap between polysaccharides found in the supernatants and those extracted from the fouled membrane was limited, implying that polysaccharides that dominate in supernatants may not be important in membrane fouling in MBRs. Analysis using a bacterial carbohydrate database suggested that capsular polysaccharides (CPS) and/or lipo-polysaccharides (LPS) play an important role in the evolution of membrane fouling in MBRs.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2011/04 -2014/03 
    Author : HINOU HIROSHI
     
    Cyclic anti-freeze glydopeptide (AFGP) found by our group has different conformation from linear AFGP found from polar fish. To elucidate the structural feature of the cyclic AFGP, we developed novel cyclization method for peptide and glycopeptide by use of hydrogen-bonding property of fluorinated alcohols. Pure cyclic AFGP was obtained by the new method in high yield from solution of corresponding corresponding linear precursor. Interestingly, common cyclization method using DPPA induce C-terminal epimerization. NMR and X-ray crystallographic experiments for cyclic AFGP and corresponding cyclic peptide skeleton revealed anti-parallel beta-sheet structure of the both compound, and glycan modification induce a replacement of amino acid position in the structure.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)
    Date (from‐to) : 2007 -2009 
    Author : HINOU Hiroshi
     
    I have tried to develop a novel method of inhibitor design of such enzymes exemplified to a neuraminidase Vibrio cholerae neuraminidase (VCNA) of which crystal structure was reported in 1994 and most potent inhibitor was reported in 1971. As a result, a potent inhibitor for Vibrio cholerae neuramnidase was developed using mechanism-based labeling information of key amino acid residues, design of a skeleton of focused library based on labeling information, screening of the focused library, and elimination of promiscuous inhibitor by detergent-based assay to afford potent (Ki=73nM) and selective inhibitor among viral, bacterial, and mammal neuraminidases.
  • 環状化合物の合成、制御、利用
    Date (from‐to) : 2008
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2005 -2007 
    Author : NISIMURA Shin-ichitro, NAGAHORI Noriko, HINOU Hiroshi
     
    this research was performed to understand the functions of glycoconjugates at the molecular level, that is to say, to develop a new methodology for hunting and identifying the partner molecules with target carbohydrates by using the gold colloidal nanoparticle and the mass spectrometer. in this study, the modification of nanoparticles was performed using specific affinity with a metallic surface and thiol group. Strong interaction of carbohydrate-displaying nanoparticle with a partner molecule is expected, because several hundred of carbohydrate molecules are displayed onto a surface of nanoparticle (carbohydrate cluster effect). In addition, the system that could display any carbohydrates was built by using chemoselective reaction with aminooxy1 group and a reducing end of carbohydrate. A library of carbohydrate presentation nano particle was made using this system. In addition, the surfaces of the nanoparticles were modified with suicide substrate type carbohydrate derivatives that could anchor the target proteins with covalent bonds. The presentation of carbohydrates on the nanoparticles was evaluated by MALDI-TOF MS using that the laser cleaved metal-thiol bond. In order to reduce the non-specific adhesion of proteins, a thiol compound with zwitterionic head group was designed and synthesized. All carbohydrate-displaying metal nanoparticles prepared in this study showed high water solubility and stability. The highly water-soluble nanoparticles could be directly subjected to a various biochemical assays and MALDI-TOF MS measurement after the capture of partner molecules. Incubate nanoparticle and sample solution containing partner molecules, and wash the particle with buffer, then the particle was just applied to SDS-PAGE. The protein identification was possible according to the ordinary method of proteomics. In this way, a high sensitivity by reduced sample loss and a promotion of efficiency by simplification of activity process were achieved, compared with traditional approaches such as the affinity chromatography which needed elution by a competing carbohydrate.
  • 糖鎖・糖ペプチド自動合成システムを活用した創薬シーズの探索研究
    Date (from‐to) : 2006
  • 酵素阻害剤の開発
    Date (from‐to) : 2000
  • 天然有機資源の有効活用
    Date (from‐to) : 1994

Educational Activities

Teaching Experience

  • The World of Science and Technology Frontier in Life ScienceThe World of Science and Technology Frontier in Life Science Hokkaido University
  • Transdisciplinary Life Science/Soft Matter ScienceTransdisciplinary Life Science/Soft Matter Science Hokkaido University
  • Laboratory Work on Bio-macromolecular Science III
    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 : 物理学実験，化学分析実験，有機合成実験，生化学実験, 分子生物学実験, 細胞生物学実験，生物物理学実験
  • Research Work in Bio-macromolecular Science
    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 : 実験研究
  • Seminar in Bio-macromolecular Science
    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 : 物理学実験，化学分析実験，有機合成実験，生化学実験, 分子生物学実験, 細胞生物学実験，生物物理学実験
  • Organic Chemistry for Life Science
    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 : 生命科学と有機化学、分子軌道、物理的性質、酸と塩基、命名法、立体化学、配座、配位、熱力学と速度論、アルケンの反応
  • The World of Science and Technology
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 生命科学　細胞　分子　生物　化学　物理　体　病気　薬　医療
  • Laboratory Work on Bio-macromolecular Science II
    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 : 物理学実験，化学分析実験，有機合成実験，生化学実験, 分子生物学実験，細胞生物学実験, 生物物理学実験
  • Experimental Biological Science
    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 : 高分子機能学，実験技術，生命科学，物質科学，情報科学，融合科学
  • Exercise on Scientic English
    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 : 科学英語, 英文論文の作製, 英文読解
  • Chemistry and English for Life Science
    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 : ケミカルバイオロジ― 生命科学 英語と化学
  • Laboratory Work on Bio-macromolecular Science I
    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 : 物理学実験，化学分析実験，有機合成実験，生化学実験, 分子生物学実験，細胞生物学実験, 生物物理学実験
  • Fundamental Laboratory Work on Bio-macromolecular Science
    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 : 基礎実験手法，物理学実験，化学分析実験，有機合成実験，生化学実験，安全教育，法令

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