1998 Fiscal Year Final Research Report Summary
Enzymatic Synthesis of Artificial Proteoglycans Having Reconstructed Sugar Chains
| Project/Area Number |
08457032
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | HIROSAKI UNIVERSITY |
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Principal Investigator |
ENDO Masahiko Hirosaki University School of Medicine, Department of Biochemistry, Professor, 医学部, 教授 (20004616)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Toshiya Hirosaki University School of Medicine, Department of Biochemistry, Lecturer, 医学部, 講師 (00155847)
MUNAKATA Hidekazu Hirosaki University School of Medicine, Department of Biochemistry, Research Ass, 医学部, 助手 (80271807)
TAKAGAKI Keiichi Hirosaki University School of Medicine, Department of Biochemistry, Assistant Pr, 医学部, 助教授 (70163160)
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| Project Period (FY) |
1996 – 1998
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| Keywords | artificial proteoglycan / hyaluronidase / transglycosylation / sugar chain reconstruction / endo-beta-xylosidase |
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| Research Abstract |
To cover the deficiency of recombinant proteins produced by the present gene engineering having no carbohydrate chain, methods of the reconstruction of glycosaminoglycan (GAG) chains and the introduction of the chains into the recombinant protein using the transglycosylation reaction of endo-type glycosidases were investigated, and the following results were obtained.
1. A method of reconstructing GAG chains according to design was established. GAG (hyaluronic acid, chondroitin and chondroitin 4- and 6-sulfate) as donors, and pyridylaminated hexasaccharides prepared from the GAG as acceptors, and testicular hyaluronidase were incubated under optimal conditions (0.15 M Tris-HCl buffer pH 7.0 in the absence of NaCl at 37ﾟC). Systematic combinations of each of the donors and acceptors were used to synthesize natural and unnatural reconstructed GAG chains.
2. Some oligosaccharides effective as carriers to introduce the reconstructed GAG chains into a recombinant protein using endo-beta-xylosidase were discovered. The oligosaccharides were synthesized from xylosyl-(4-methylumbelliferone) as an initiator in culture medium of human skin fibroblasts. The oligosaccharides contained structures sensitive to endo-beta-xylosidase, Gal-Gal-Xyl- ( 4-methylumbelliferone).
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[Publications] Yasukochi, T., Fukase, K., Suda, Y., Takagaki, K., Endo, M., and Kusumoto, S.: "Enzymatic synthesis of 4-methylumbelliferyl glycosides of trisaccharide, and core tetrasaccharide, Gal(beta1-3)Gal(beta1-4)Xyl and GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl, corresponding to the linkage region of proteoglycans." Bull.Chem/Soc.Jpn.70. 2719-2725 (1997)
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[Publications] Takagaki, K., Munakata, H., Nakamura, W., Matsuya, H., majima, M., and Endo, M.: "Ion-spray mass spectrometry for identification of the nonreducing terminal sugar of glycosaminoglycan." Glycobiology. 8. 719-724 (1998)
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[Publications] Tazawa, T., Takagaki, K., Matsuya, H., Nakamura, T., Sasaki, H., and Endo, M.: "A novel 4-methylumbelliferyl-beta-D-xyloside derivative, sulfate-0-3-xylosyl-beta1-(4-metylumbelliferone), isolated from culture medium of human skin fihroblasts, and its role in 4-methylumbelliferone-initiated glycosaminoglycan biosynthesis.chondroitinase ABC treatment." Glycobiology. 8. 879-884 (1998)
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