| Project/Area Number |
13672409
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用薬理学・医療系薬学
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| Research Institution | Tokyo Metropolitan Organization for Medical Research |
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Principal Investigator |
KASE Ryoichi The Tokyo Metropolitan Institute of Medical Science, 東京都臨床医学総合研究所, 研究員 (20150203)
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| Co-Investigator(Kenkyū-buntansha) |
SIMMOTO Michie The Tokyo Metropolitan Institute of Medical Science, 東京都臨床医学総合研究所, 研究員 (20216237)
SAKURABA Hitoshi The Tokyo Metropolitan Institute of Medical Science, 東京都臨床医学総合研究所, 研究員 (60114493)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Fadry Disease / α-Galactosidase / Lysosome / Enzyme Replacement / α-ガラクトシターゼ |
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| Research Abstract |
The genetic defect of α-galactosidase encoded by a gene localized to the X- chromosomal region, Xq22, results in Fabry disease. This disease is characterized by the systemic intralysosomal accumulation of neutral sphingolipids, predominantly globotriaosylceramide. In our basic studies of α-galactosidase enzyme replacement therapy, first, we constructed the clones of the methylotropic yeast Pichia pastois. Then, the glyco-chains of the recombinant α-galactosidase was trimmed by α-mannosidase, and the effective up-take by Fabry fibroblasts was observed. The administrated α-galactosidase could be recaptured via the mannose 6-phosphate receptors by cells, a phenomenon commonly referred as receptor-mediated endocytosis. Second, we introduced site-directed mutations into α-galactosidase. X-ray crysrarographic analysis revealed that two Asp (170 and 231) residues were involved in catalitic-site of the enzyme. We made Asp (D) to Glu (E) changed mutants D170E, D231E, and D170E/D231E (double mutation), respectively. These mutants were Vmax mutants. The kinetic studies revealed that D231 played the important role in the rate limiting step of the α-galactosidase reaction
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