| 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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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥7,900,000 (Direct Cost: ¥7,900,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1996: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | artificial proteoglycan / hyaluronidase / transglycosylation / sugar chain reconstruction / endo-beta-xylosidase / 糖鎖の組み換え / コンドロイチン硫酸 / ヒアルロン酸 / 酵素的糖鎖合成 / 細胞外マトリックス / 糖鎖工学 / エンド型グリコシダーゼ / 酵素的合成 / 糖鎖再構築 / 糖鎖導入のキャリヤ- / 4メチルウンベリフェロン誘導オリゴ糖 |
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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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