Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants|
|Research Institution||Kobe Pharmaceutical University|
SUGAHARA Kazuyuki KOBE PHARMACEUTICAL UNIVERSITY, Dept. of Biochemistry, Professor, 薬学部, 教授 (60154449)
YOSHIDA Keiichi Seikagaku Corporation, Glycoforum Project, Team Leader, 糖鎖プロジェクト, チームリーダー
YAMADA Shuhei KOBE PHARMACEUTICAL UNIVERSITY, Dept. of Biochemistry, Instructor, 薬学部, 助手 (70240017)
KITAGAWA Hiroshi KOBE PHARMACEUTICAL UNIVERSITY, Dept. of Biochemistry, Lecturer, 薬学部, 講師 (40221915)
木下 亜希子 神戸薬科大学, 薬学部, 助手 (30289037)
|Project Period (FY)
1997 – 1999
Completed(Fiscal Year 1999)
|Budget Amount *help
¥10,500,000 (Direct Cost : ¥10,500,000)
Fiscal Year 1999 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1998 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1997 : ¥4,200,000 (Direct Cost : ¥4,200,000)
|Keywords||chondroitin sulfate / heparin / heparin sulfate / dermatan sulfate / sulfated oligosaccharides / glycosaminoglycan / heparinase / chondroitinase / 硫酸化 / 線虫 / コンドロイチナーゼC / マンノサミン / C2-エピメリゼーション / NMR / デルマタン流酸 / 2-アミノベンズアミド / 螢光標識|
Various growth/differentiation factors exhibit their biological activities through binding to the respective high affinity receptors on cell surface. For some of these factors to do so, they need to first bind to glycosaminoglycan (GAG) chains on cell surface. However, the structural analysis especially the sequencing of these GAG chains has been hampered by the complicated structure. Therefore, we have carried out the following studies to develop simple and wide-applicable sequencing methods for sulfated GAG chains.
(1) Five novel sulfated tetrasaccharide sequences have been isolated from squid cartilage chondroitin sulfate and structurally defined.
(2) A sulfated tetrasaccharide, a pentasaccharide, eight hexasaccharides, three octasaccharides and four octasaccharide serines have been isolated from porcine intestinal heparin and structurally defined as novel structures. In addition, several sulfated tetra- and hexasaccharides have been isolated from dermatan sulfate chains which contami
nated the porcine intestinal heparin preparation described above.
(3) Eight novel sulfated tetrasaccharides and five hexasaccharides have been isolated from hagfish notochord and structurally defined.
(4) It has been demonstrated during the course of the above structural analysis of heparin oligosaccharides that N-sulfated glucosamine is converted into N-sulfated mannosamine non-enzymatically under mild alkaline conditions.
(5) Substrate specificities of a novel heparinase isolated from Prevotella h. and chondroitinase C isolated from Flavobacterium h., both of which are tools for structural studies of GAG chains, have been clarified.
(6) A microanalytical method for GAG chains has been developed by fluorescent labeling of GAG chains at their reducing termini with a fluorophore 2-aminobenzamide. Furthermore, a sequencing method for GAG chains with high sensitivity has been developed by combining this labeling method and enzymatic digestion.
(7) Using the newly developed method described above, nonsulfated chondroitin and highly sulfated heparan sulfate have been demonstrated in C. elegans. Less