| Project/Area Number |
12470486
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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 |
Physical pharmacy
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SATOW Yoshinori The University of Tokyo, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学研究科, 教授 (30150014)
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| Co-Investigator(Kenkyū-buntansha) |
MIZUTANI Ryuta The University of Tokyo, Graduate School of Pharmaceutical Sciences, Research Associate, 大学院・薬学研究科, 助手 (70272482)
NOGUCHI Shuji The University of Tokyo, Graduate School of Pharmaceutical Sciences, Research Associate, 大学院・薬学研究科, 助手 (60237823)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥15,600,000 (Direct Cost: ¥15,600,000)
Fiscal Year 2001: ¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 2000: ¥9,000,000 (Direct Cost: ¥9,000,000)
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| Keywords | Lysosomal Disease / Fabry Disease / Human α-Galactosidase / X-ray Crystallography / Structural Biology / Three-Dimensional Structure / Genetic Mutation / Hereditary Disease / ファブリ-病 / アセチルガラクトサミニダーゼ |
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| Research Abstract |
Fabry disease is an X-linked hereditary disorder of lysosomal α-galactosidase. This enzyme hydrolyzes terminal α galactoside linkages in various glycolipids, and its genetic mutations decrease enzymatic activities which result in deficiencies of glycolipid metabolism and hence accumulates such substrate as glycosphingolipid of globotriaosylceramide in heart and other organs. The molecular lesion of lysosomal α-N-acetylgalactosaminidase causes another form of angiokeratoma. In order to elucidate three-dimensional structural bases of these diseases, both the enzymes have been expressed and subjected to crystallographic studies.
A recombinant of human α-galactosidase was first expressed in the yeast Pichia pastoris that successfully secretes a glycosilated and catalytically active enzyme into a culture broth. Three recombinants for the mutated enzymes were similarly obtained and characterized by biophysical and biochemical analyzes. Crystals of the enzymes both in free forms and in complex
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es with a inhibitor of 1-deoxygalactonojirimycin were obtained. X-ray diffraction data from these crystals were collected using laboratory and synchrotron sources, and analyzed with the molecular replacement method.
The three-dimensional structures thus obtained show that α-galactosidase is homodimeric, consisting of a β-sheet domain and an α/β barrel domain in which the active site is located. The structures of the two recombinants responsible for the variant-form disease indicate that their differences from the structure of the normal enzyme are very small and localized only near the mutated amino acids, and their characterizations indicate that they attain nearly normal levels of the activities but lose their activities rapidly. The structure of the recombinant causing the classical-form disease show that the side chain of the mutated amino-acid blocks the active site and completely hinders the activity. A recombinant of human α-N-acetylgalactosaminidase was similarly expressed, purified, and crystallized. Optimization of qualities of the obtained crystals is in progress so as to elucidate the structure of this enzyme. Less
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