| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
1. Structure of the complexes of FGF-2 and regioselectively desulfated heparins
In order to elucidate the roll of the sulfate groups in the mechanism for FGF-2-heparin interaction, the complex formation between human FGF-2 and a series of regioselectively desulfated heparin was observed in terms of small angle X-ray diffraction. In the case of the FGF-2-native heparin complex, the globular FGF-2 molecules appeared to be arranged on the same side along the heparin chain, while the FGF-2 molecules appeared to be arranged randomly along both N-desulfated heparin and 6-O-desulfated heparin chains. In contrast, 2-O-desulfated heparin did not aggregate with FGF-2. The sulfate groups at O-2 of the iduronate residues of heparin molecule are most likely to be essential for the complex-formation with FGF-2.
The molecular modeling study indicated that removal of 2-O-sulfate groups from heparin restricts the conformation of the iduronate residues affecting the flexibility of the polysaccharide chain
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to inhibits the complex formation with FGF-2. The sulfate groups at O-6 of and 2-N of the glucosamine residues did not cause conformational change in the glucosamine residues and the flexibility of the heparin molecule, the 6-O-desulfation and N-desulfation thus allowing the complex formation with FGF-2.
2. Significance of an agarose-derived novel tetrasaccharide formation for the three-dimensional structure of agarose
In this study, we claim that the structure of a tetrasaccharide obtained by partial methanolysis of agarose reflects the three-dimensional structure of agarose and further suggests the gelation mechanism of agarose. The tetrasaccharide possesses a unique structure where the internal 3,6-anhydro-L-galactose and D-galactose residues are linked through a cyclic acetal bond.. The identical tetrasaccaride and the homologous tetrasaccharide carrying methyl groups at 0-2 of the anhydrogalactose residue and O-6 of galactose residue were also found in the partial methanolysis products from 6-sulfated agarose and 2,6-dimethylated agarose, respectively.
A strong hydrogen bond between the hydroxyl group at C-4 of 1,3-linked D-galactose residue and acetal oxygen atom of 1,4-linked 3,6-anhydro-L-galactose residue of agaorse has been observed by a previous NMR spectral study, and considered to be required for the gelation of agarose in a previous rheological work. Regarding the three-dimensional structure of agarose, the hydrogen bond appeared to cause a conformation favorable for the formation of the internal acetal bond in the tetrasaccharide. In contrast, β-carrageenan, possessing a covalent structure similar to agarose except that the 3,6-anhydro-L-galactose residues are replaced with D-enantiomers, did not afford a corresponding tetrasaccharide on partial methanolysis, while the the analogous hydrogen bond formation may be possible at least locally. Considering β-carrageenan does not make gel and that the hydrogen bonding is essential for both the gelation and tetrasaccharide-formation, we conclude that the tetrasaccharide is not an artifact firmed during the reaction but a product directly reflecting the three-dimensional structure of agarose. Less
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