MAPPING FUNCTIONAL DOMAINS OF LYSOSOMAL ENZYMES

溶酶体酶对其底物的可及性受到以下因素的影响 它们的亚细胞区室化。 酶的路线通过 细胞已被证明受到发生在 溶酶体酶以及其他蛋白质。 由于缺乏 足够数量的均质正常酶和突变酶 生化和结构研究，我们认为有必要 通过体外诱变分离编码特定酶的 cDNA 重现突变对酶活性和结构的影响。 为了实现这一目标，我们最初使用戈谢病作为模型， 分离并测序了编码所有人类葡萄糖脑苷脂酶的cDNA。 我们已经描述了该酶的前导序列，因此有 确定了葡萄糖脑苷脂酶中影响易位的部分 酶到达内质网池。 为了映射 负责寡糖添加和加工的其他域， 亚状态水解、溶酶体路径和膜关联，我们有 合成的寡核苷酸用于诱变 cDNA 葡萄糖脑苷脂酶。 使用逆转录病毒构建体，将 cDNA 转移 哺乳动物宿主细胞系以重建戈谢变体。 这个 提供了一种体外细胞培养模型，其中区室化 转移蛋白的功能可以直接与 cDNA 以及蛋白质结构域的特定变化。 这项研究将 为治疗策略的制定提供更合理的依据 使用基因或基因产物替代。

The accessibility of lysosomal enzymes to their substrates is affected by their subcellular compartmentalization. Routing of enzymes through the cell has been shown to be influenced by mutations occurring in the lysosomal enzyme as well as in other proteins. Due to the lack of sufficient quantities of homogeneous normal and mutant enzymes for biochemical and structural studies, we felt that it was necessary to isolate the cDNA encoding specific enzymes and by in vitro mutagenesis recreate the consequences of mutations on enzyme activity and structure. To accomplish this end, initially using Gaucher's disease as a model, we isolated and sequenced the cDNA encoding all of human glucocerebrosidase. We have described the leader sequence for this enzyme and thus have identified that portion of glucocerebrosidase that effects translocation of the enzyme to the cisternae of the endoplasmic reticulum. In order to map the other domains responsible for oligosaccharide addition and processing, substate hydrolysis, lysosomal routing and membrane association, we have synthesized oligonucleotides that are being used to mutagenize the cDNA for glucocerebrosidase. Using retroviral constructs, the cDNA was transferred to mammalian host cell lines to reconstruct Gaucher variants. This provides an in vitro cell culture model in which the compartmentalization and function of the transferred protein can be directly correlated with specific changes in the cDNA and hence protein domains. This research will provide a more rational basis for the development of therapeutic strategies using gene or gene product replacement.