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

α-半乳糖苷酶 A、B——分子/细胞机制

• 批准号: 2398048
• 负责人: Robert J Desnick
• 金额: $ 32.76万
• 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/2398048
• 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.

拟议研究的总体目标是调查 两种遗传相关的分子和细胞机制，但是 功能不同，溶酶体糖苷酶，α-半乳糖苷酶 A (α-Gal A) 和 α-N-乙酰半乳糖胺酶 (α-Gal B) 各自结构域、酶复合物的表征， 及缺乏症。 为了促进这些研究，我们 1) 分离并 表征了全长人类和小鼠 cDNA 以及完整的人类 编码 α-Gal A 和 α-Gal B 的基因组序列，2) 已开发 其高水平真核表达和纯化方法，3) 鉴定了导致其各自溶酶体的多种突变 疾病、法布里病和辛德勒病。 我们建议确定 这两种非常同源的糖苷酶的结构，其基因 推测是通过复制进化并分化为具有不同的底物 特异性、稳定性和 N 连接寡糖链。 大的 将产生一定量的重组 α-Gal A 和 α-Gal B 纯化用于其 3D 结构的结晶和溶解，以及 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 每个的结构，活性中的半乳糖识别和催化残基 位点域、二硫键中的半胱氨酸和亚基关联 域。 研究这些先天性的代谢和细胞病理学 糖脂和糖蛋白代谢错误、α-Gal A 或 α-Gal B缺陷小鼠将使用改变的小鼠基因组构建体产生 用于 ES 细胞中的同源重组，然后是囊胚 植入。 生化、形态学和 动物模型的病理生理学改变可能有助于了解 他们各自的人类溶酶体疾病的发病机制。 最后， α-Gal B 在神经轴突运输中的作用将通过 确定神经组织中的 α-Gal B 分布，表征 α-Gal B 抑制对坐骨神经轴突运输的影响 替代 α-Gal B 底物的制备和研究 特异性（例如，α-N-乙酰半乳糖氨基磷酸酯连接的残基 参与神经特异性细胞间粘附。 这些研究应该 提供对分子和细胞机制的基本理解 这两种相关的糖苷酶在健康和疾病中的作用。