Studies on three-dimensional structures of the glycolytic enzymes responsible for the lysosomal diseases

负责溶酶体疾病的糖酵解酶的三维结构研究

基本信息

批准号：
12470486
负责人：
SATOW Yoshinori
金额：
$ 9.98万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

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 … More 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

法布里病是心脏和其他器官的溶酶体的分子病变。 α-N-乙酰氨基半乳糖苷酶引起另一种形式的血管角化瘤。为了阐明这些疾病的三维结构基础，这两种酶都被表达并进行了晶体学研究。人类α-半乳糖苷酶的重组体首先在酵母毕赤酵母中表达。巴斯德酵母成功地将糖基化和催化活性酶分泌到培养液中，以类似的方式获得和表征了突变酶的三种重组体。使用实验室和同步加速器源收集了游离形式和带有 1-脱氧半乳糖野尻霉素抑制剂的复合酶晶体。由此获得的三维结构表明α-半乳糖苷酶是同二聚体，由β-片层结构域和活性位点所在的α/β桶结构域组成。导致变异型疾病的两种重组体的结构表明它们与正常酶结构的差异非常小并且仅位于突变氨基酸附近，并且它们的特征表明它们达到了接近正常水平的活性，但引起经典型疾病的重组体的结构表明，突变氨基酸的侧链阻断了活性位点并完全阻碍了人类A重组体的活性。 α-N-乙酰氨基半乳糖苷酶的表达、纯化和结晶正在进行中，以阐明该酶的结构。