ALPHA-GALACTOSIDASES A, B--MOLECULAR/CELLULAR MECHANISM

α-半乳糖苷酶 A、B——分子/细胞机制

• 批准号: 2701066
• 负责人: Robert J Desnick
• 金额: $ 33.8万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701066
• 关键词: Fabry's disease; N acetylgalactosamine; active sites; alpha galactosidase; biotechnology; cell adhesion; computer assisted sequence analysis; crosslink; disease /disorder model; disulfide bond; embryonic stem cell; enzyme activity; enzyme complex; enzyme structure; enzyme substrate; gene mutation; gene therapy; genetic disorder diagnosis; genetic manipulation; glycolipids; glycoproteins; glycosylation; human tissue; inborn lysosomal enzyme disorder; intracellular transport; laboratory mouse; laboratory rat; lysosomes; molecular cloning; molecular genetics; molecular pathology; neuronal transport; nonhuman therapy evaluation; nucleic acid sequence; oligosaccharides; orphan disease /drug; polymerase chain reaction; protein metabolism; protein sequence; protein structure function; synthetic enzyme; transfection

The overall objective of the proposed research is to investigate the molecular and cellular mechanisms of two genetically related, but functionally different, lysosomal glycosidases, alpha-galactosidase A (alpha-Gal A) and alpha-N-acetylgalactosaminidase (alpha-Gal B) by characterization of their respective structural domains, enzyme complexes, and deficiency diseases. To facilitate these studies, we 1) isolated and characterized the full-length human and murine cDNAs and the complete human genomic sequences encoding alpha-Gal A and alpha-Gal B, 2) developed methods for their high-level eukaryotic expression and purification, 3) identified a variety of mutations that result in their respective lysosomal disorders, Fabry and Schindler diseases. We propose to determine the structures of these two remarkably homologous glycosidases whose genes presumably evolved by duplication and diverged to have different substrate specificities, stabilities and N-linked oligosaccharide chains. Large amounts of recombinant alpha-Gal A and alpha-Gal B will be produced and purified for crystallization and solution of their 3D structures, and for the biochemical and computer-assisted analyses of their respective structural domains, including N-glycosylation sites and the oligosaccharide structures of each, galactose recognition and catalytic residues in active site domains, cysteines in disulfide bridges, and subunit association domains. To investigate the metabolic and cellular pathologies of these inborn errors of glycolipid and glycoprotein metabolism, alpha-Gal A or alpha-Gal B deficient mice will be generated using murine genomic constructs altered for homologous recombination in ES cells followed by blastocyst implantation. Characterization of the biochemical, morphologic and pathophysiologic alterations in the animal models may provide insights into the pathogenesis of their respective human lysosomal disease. Finally, the role of alpha-Gal B in neuroaxonal transport will be explored by determining the alpha-Gal B distribution in neural tissues, characterizing the effect of alpha-Gal B inhibition on axonal transport in sciatic nerve preparations, and studies of alternative alpha-Gal B substrate specificities (e.g., alpha-N-acetylgalactosaminylphosphate-linked residues involved in neural-specific cell-cell adhesion. These studies should provide fundamental understanding of the molecular and cellular mechanisms of these two related glycosidases in health and disease.

拟议研究的总体目的是调查 两个遗传学相关的分子和细胞机制，但 功能上不同的溶酶体糖苷酶，α-半乳糖苷酶A （alpha-gal A）和α-N-乙酰乳糖苷酶（Alpha-gal b）通过 其各自的结构结构域的表征，酶复合物， 和缺陷疾病。 为了促进这些研究，我们1）孤立和 表征了全长的人和鼠cdnas以及完整的人 编码α-gal A和alpha-gal B的基因组序列，2） 其高级真核表达和纯化的方法，3） 确定了导致其各自溶酶体的各种突变 疾病，法布里和辛德勒疾病。 我们建议确定 这两个非常同源糖苷酶的结构，其基因 大概是通过复制而发展的，并具有不同的基板 特异性，稳定性和N连接的寡糖链。 大的 将产生重组α-GAL A和α-GAL B的数量，并且 纯化以结晶和解决其3D结构的溶液，并用于 对其各自的生化和计算机辅助分析 结构域，包括N-糖基化位点和寡糖 每个结构，半乳糖识别和活性的催化残基 位点域，二硫键中的半胱氨酸和亚基关联 域。 研究这些天生的代谢和细胞病理 糖脂和糖蛋白代谢的错误，α-gal A或α-gal b使用鼠基因组构建体改变了不足的小鼠 对于ES细胞中的同源重组，然后是胚泡 植入。 生化，形态学和 动物模型中的病理生理改变可能会为 它们各自的人类溶酶体疾病的发病机理。 最后， Alpha-gal B在神经交通中的作用将由 确定神经组织中的α-GAL B分布，表征 α-GAL B抑制对坐骨神经中轴突转运的影响 准备工作以及替代α-gal b底物的研究 特异性（例如，α-N-乙酰乳糖酰二氨基磷酸磷酸残基 参与神经特异性细胞粘附。 这些研究应该 提供对分子和细胞机制的基本理解 在健康和疾病中的这两种相关糖苷酶中。