MOLECULAR ANALYSIS OF ACID BETA-GALACTOSIDASE

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：GM1神经节苷脂沉积症， 神经功能障碍和B型Morquio综合征，一种骨骼和 结缔组织紊乱。显著的表型异质性 这两种疾病之间的存在与辨证分型有关 酸性β-半乳糖苷酶两种天然底物的积累， 神经节苷脂-GM1和角蛋白硫酸盐，在各自的疾病中。这个 这项拟议研究的总体目标是阐明分子 以及这种选择性底物积累的酶基础。我们的 最初的努力将集中在定义酸性贝塔的基本酶学- 半乳糖苷酶及其与其他细胞蛋白的相互作用 对于活性(即，保护蛋白和皂苷B)。自然而自然 重组来源将用于这些蛋白质的纯化。 过度表达系统(即基于杆状病毒的中国仓鼠 卵巢系统)将使用我们现有的酸的cDNA克隆进行开发 β-半乳糖苷酶、保护蛋白和丙皂苷。详细动力学 酸性β-半乳糖苷酶纯二聚体的性质和性质 将进行酸性β-半乳糖苷酶/保护蛋白复合体 在存在和不存在纯化的情况下使用天然底物 天然激活剂(皂苷B)，以确定对 修饰剂、结合亲和力和周转率。进一步定义 将通过详细的抑制剂来探讨活性中心结构 学习。将这些深入的动力学研究与现场定向相结合 诱变和异源表达实验将导致 对酸的功能完整性至关重要的残基的鉴定 β-半乳糖苷酶。表达的突变最初将包括 已经在神经节苷脂沉积症和B型Morquio综合征中定义 病人。这些研究将扩大到包括选定的氨基酸 对制定一份结构/功能图的目标的替代 酸性β-半乳糖苷酶作为开发乳酸菌的合理基础 诊断和治疗策略。这些实验应该提供 对差异很大的疾病的分子病理学有更深入的了解 酸性β-半乳糖苷酶突变导致的表型，如 其他先天代谢错误的原型，其酶具有 几种底物的特性。

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.