Research Center for Medical Glycoscience

Ibaraki, Japan

Research Center for Medical Glycoscience

Ibaraki, Japan
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Sato T.,Research Center for Medical Glycoscience | Sato T.,Glycoscience and Glycotechnology Research Group GGRG | Tateno H.,Cell Glycome Targeted Technology Res Group Of Biotechnology Research Institute For Drug Discovery | Kaji H.,Research Center for Medical Glycoscience | And 7 more authors.
Glycobiology | Year: 2017

Wisteria floribunda agglutinin (WFA) is a useful probe for distinguishing glycan structural alterations in diseases such as intrahepatic bile duct carcinoma and hepatic fibrosis; however, the gene encoding WFA has not been identified. Here, we identified the gene encoding WFA, and recombinant WFA (rWFA) was expressed in Escherichia coli and purified. The natural complementary DNA sequence obtained from wisteria seeds contained an open reading frame of 861 nucleotides encoding a WFA precursor, which included a hydrophobic signal peptide at the N-terminus, a propeptide at the C-terminus and a single cysteine (Cys) residue for dimer formation. We characterized the natural and rWFA by the glycoconjugate microarray and frontal affinity chromatography. rWFA exhibited glycan binding specificity similar to that of natural WFA: both bound to Gal- and N-acetylgalactosamine (GalNAc)-terminated glycans. Moreover, the engineered WFA with an amino acid substitution in Cys-272 yielded a recombinant monomeric lectin with limited binding specificity but wild-type affinity for GalNAc-terminated glycans, specifically GalNAcβ1,4GlcNAc. Thus, this engineered lectin may be applied to highly sensitive biomarker detection. © The Author 2017.

Ogawa H.,Aichi Medical University | Ogawa H.,Gifu University | Shionyu M.,Nagahama Institute of Bio-Science and Technology | Sugiura N.,Aichi Medical University | And 10 more authors.
Journal of Biological Chemistry | Year: 2010

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Ogawa H.,Aichi Medical University | Ogawa H.,Gifu University | Hatano S.,Aichi Medical University | Sugiura N.,Aichi Medical University | And 6 more authors.
PLoS ONE | Year: 2012

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. Based on its structural diversity in chain length and sulfation patterns, CS provides specific biological functions in cell adhesion, morphogenesis, neural network formation, and cell division. To date, six glycosyltransferases are known to be involved in the biosynthesis of chondroitin saccharide chains, and a hetero-oligomer complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is known to have the strongest polymerizing activity. Here, we generated and analyzed CSS2-/- mice. Although they were viable and fertile, exhibiting no overt morphological abnormalities or osteoarthritis, their cartilage contained CS chains with a shorter length and at a similar number to wild type. Further analysis using CSS2-/- chondrocyte culture systems, together with siRNA of CSS1, revealed the presence of two CS chain species in length, suggesting two steps of CS chain polymerization; i.e., elongation from the linkage region up to Mr ~10,000, and further extension. There, CSS2 mainly participated in the extension, whereas CSS1 participated in both the extension and the initiation. Our study demonstrates the distinct function of CSS1 and CSS2, providing a clue in the elucidation of the mechanism of CS biosynthesis. © 2012 Ogawa et al.

Takatsuki H.,Soka University | Takatsuki H.,Nagasaki University | Taguchi F.,Japan National Institute of Infectious Diseases | Taguchi F.,Nippon Veterinary and Life Science University | And 5 more authors.
Neuropathology | Year: 2010

Viral spread during the early stages after infection was compared between a highly neurovirulent mouse hepatitis virus (MHV), JHMV cl-2 strain (cl-2), and its low-virulent mutant, soluble-receptor-resistant (srr)7. The infection of cells with srr7 (soluble-receptor-resistant mutant 7) is dependent on a known MHV receptor (MHVR), carcinoembryonic cell adhesion molecule 1a, whereas cl-2 shows MHVR-independent infection. Initial viral antigens were detected between 12 and 24 h post-inoculation (p.i) in the infiltrating cells that appeared in the subarachnoidal space of mouse brains infected with viruses. There were no significant differences in the intensity or spread of viral antigens in the inflammatory cells between the two viruses. However, 48 h after infection with cl-2, viral antigen-positive cells in the grey matter with the shape of neurons, which do not express MHVR, were detected, while srr7 infection was observed primarily in the white matter. Some of the viral antigen-positive inflammatory cells found in the subarachnoidal space during the early phase of infection reacted with anti-F4/80 or anti-CD11b monoclonal antibodies. Syncytial giant cells (SGCs) expressing viral and CD11b antigens were also detected among these inflammatory cells. These antigen-positive cells appeared in the subarachnoidal space prior to viral antigen spread into the brain parenchyma, indicating that viral encephalitis starts with the infection of infiltrating monocytes which express MHVR. Furthermore, the observation indicates that viral infection has cytopathic effects on the monocyte lineage, which plays a critical role in innate immunity, leading to the rapid spread of viruses during the early stage of infection. © 2009 Japanese Society of Neuropathology.

Yoshimura Y.,Research Center for New Fuels and Vehicle Technology | Suzuki K.,Bioproduction Research Institute | Gotoh M.,Research Center for Medical Glycoscience
AIST Today (International Edition) | Year: 2014

AIST continues to make significant efforts to encourage Japanese research institutes to initiate better R&D activities by networking with overseas research institutes, penetrating global markets and conducting global business operations. AIST works jointly with Thailand's National Science and Technology Development Agency (NSTDA) and the Thailand Institute of Scientific and Technological Research (TISTR) to develop fundamental technology to produce transportation fuel from non-edible biomass as part of the Science and Technology Research Partnership for Sustainable Development (SATREPS) being implemented by the Japan Science and Technology Agency and the Japan International Cooperation Agency. AIST has also established a framework for international joint research by three parties, including AIST and Bridgestone Corporation to initiate such technology development activities.

Chen Y.,University of Tokyo | Hu D.,University of Tokyo | Yabe R.,Research Center for Medical Glycoscience | Tateno H.,Research Center for Medical Glycoscience | And 4 more authors.
Molecular Biology of the Cell | Year: 2011

Malectin was first discovered as a novel endoplasmic reticulum (ER) - resident lectin from Xenopus laevis that exhibits structural similarity to bacterial glycosylhydrolases. Like other intracellular lectins involved in glycoprotein quality control, malectin is highly conserved in animals. Here results from in vitro membrane-based binding assays and frontal affinity chromatography confirm that human malectin binds specifically to Glc 2Man 9GlcNAc 2 (G2M9) N-glycan, with a K a of 1.97 × 10 5 M -1, whereas binding to Glc 1Man 9GlcNAc 2 (G1M9), Glc 3Man 9GlcNAc 2 (G3M9), and other N-glycans is barely detectable. Metabolic labeling and immunoprecipitation experiments demonstrate that before entering the calnexin cycle, the folding-defective human α1-antitrypsin variant null Hong Kong (AT NHK) stably associates with malectin, whereas wild-type α1-antitrypsin (AT) or N-glycan-truncated variant of AT NHK (AT NHK-Q3) dose not. Moreover, malectin overexpression dramatically inhibits the secretion of AT NHK through a mechanism that involves enhanced ER-associated protein degradation; by comparison, the secretion of AT and AT NHK-Q3 is only slightly affected by malectin overexpression. ER-stress induced by tunicamycin results in significantly elevated mRNA transcription of malectin. These observations suggest a possible role of malectin in regulating newly synthesized glycoproteins via G2M9 recognition. © 2011 Chen et al.

Sugiura N.,Aichi Medical University | Shioiri T.,Aichi Medical University | Chiba M.,Aichi Medical University | Sato T.,Research Center for Medical Glycoscience | And 3 more authors.
Journal of Biological Chemistry | Year: 2012

Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-D-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing> 95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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