MOLECULAR ANALYSIS OF ACID BETA-GALACTODIDASE

人类酸性β-半乳糖苷酶的遗传缺陷导致两种 非常独特的常染色体隐性遗传病：GM 1-神经节苷脂沉积症，a 神经系统疾病和莫基奥综合征B型，一种骨骼和 结缔组织疾病 显著的表型异质性， 这两种疾病之间的差异与 酸性β-半乳糖苷酶的两种天然底物的积累， 神经节苷脂-GM 1和硫酸角质素，在各自的疾病。 的 拟议研究的总体目标是阐明 和用于这种选择性底物积累的酶基础。 我们 最初的努力将集中在定义酸β- 半乳糖苷酶及其与其他必要的细胞蛋白质的相互作用 对于活动（即，保护蛋白和鞘脂激活素B）。 自然和 重组来源将用于纯化这些蛋白质。 过表达系统（即，杆状病毒和中国仓鼠 卵巢系统）将开发使用我们的可用cDNA克隆酸 β-半乳糖苷酶、保护蛋白和鞘脂激活蛋白原。 详细动力学 酸性β-半乳糖苷酶的纯二聚体形式的表征， 将进行酸性β-半乳糖苷酶/保护蛋白复合物 使用天然底物，在存在和不存在纯化的 天然激活剂（saposin B），以确定对 修饰剂、结合亲和力和周转率。 进一步界定 活性中心结构将通过详细的抑制剂 问题研究 将这些深入的动力学研究与定点 诱变和异源表达实验将导致 鉴定对酸功能完整性至关重要的残基 β-半乳糖苷酶。 表达的突变最初将包括那些 已在GM 1-神经节苷脂沉积症和Morquio综合征B型中定义 患者 这些研究将扩大到包括选定的氨基酸 为了开发一个结构/功能图的目标， 酸性β-半乳糖苷酶作为开发 诊断和治疗策略。 这些实验应该提供 更深入地了解分子病理学的广泛变化 表型由酸性β-半乳糖苷酶突变引起，作为一种 其他先天性代谢缺陷的原型，其酶具有 几种底物特异性。

The genetic deficiency of acid beta-galactosidase in humans causes two very distinct autosomal recessive diseases: GM1-gangliosidosis, a neurological disorder, and Morquio Syndrome Type B, a skeletal and connective tissue disorder. The marked phenotypic heterogeneity that exists between these two diseases is related to the differential accumulation of two natural substrates of acid beta-galactosidase, ganglioside-GM1 and keratan sulfate, in the respective diseases. The overall objective of the proposed research is to elucidate the molecular and enzymatic bases for this selective substrate accumulation. Our initial efforts will focus on defining the basic enzymology of acid beta- galactosidase and its interaction with other cellular proteins necessary for activity (i.e., the protective protein and saposin B). Natural and recombinant sources will be used for the purification of these proteins. Overexpression systems (i.e., the baculovirus based and Chinese hamster ovary systems) will be developed using our available cDNA clones for acid beta-galactosidase, protector protein and prosaposin. Detailed kinetic characterization of the pure dimeric form of acid beta-galactosidase and the acid beta-galactosidase/protector protein complex will be carried out using natural substrates in the presence and absence of the purified natural activator (saposin B) in order to determine response to the modifier, binding affinities, and turnover rates. Further definition of active site structure will be approached through detailed inhibitor studies. Coupling these in-depth kinetic studies with site-directed mutagenesis and heterologous expression experiments will lead to the identification of residues critical for the functional integrity of acid beta-galactosidase. Expressed mutations will initially include those already defined in GM1-gangliosidosis and Morquio Syndrome Type B patients. These studies will be expanded to include selected amino acid substitutions toward the goal of developing a structure/function map of acid beta-galactosidase as a rational foundation for development of diagnostic and therapeutic strategies. These experiments should provide greater insight into the molecular pathology for the widely varying phenotypes resulting from mutations in acid beta-galactosidase, as a prototype for other inborn errors of metabolism whose enzymes have several substrate specificities.