C-TERMINAL PROCESSING OF NASCENT PROPROTEINS

通过糖基磷脂酰肌醇（GPI）结构的膜锚定是一种普遍存在的机制，许多功能不同的蛋白质与细胞表面连接，通过这种机制，任何感兴趣的蛋白质都可以在实验中附着在细胞上。从包括我们自己的实验室在内的几个实验室之前的工作中，提供GPI组装的生物合成途径已经被表征，并且编码介导这些反应的几种酶的基因已经被克隆。在最近的研究中，我们的实验室已经产生了一种新的GPI锚定缺陷突变系（指定为K），它积累了完整的GPI前体，但不会将它们转移到受体原蛋白上。我们以前的研究已经证实，这种突变体在介导这种转移的转酰胺反应中存在缺陷。我们克隆了受影响的基因，并表明它对应于hGPI8，酵母基因yGPI8的同源物，这似乎是转氨酶。在其他的合作工作中，我们克隆了第二个人类基因，称为hGAA1，它编码了转化所需的另一个因子。在进一步的工作中，我们已经表明，从转移的GPI中去除脂肪酸的去酰化反应与转酰胺反应密切相关。当前提案的目的是在这项工作的基础上进一步阐明GPI锚定的装配后步骤。具体来说，我们的目标是1)绘制hGpi8p/hGaa1p的活性位点并研究它们的底物特异性，2)利用互补的酵母和哺乳动物系统来寻找相关成分，3)开发一个可溶解/重构的系统来研究转酰胺反应，4)表征在转移后从GPI片段中去除肌醇连接的酰基链的脱酰基酶活性。获得的数据应该有助于对gpi锚定蛋白代谢的基本理解。它也可能与真菌和寄生虫传染病的治疗相关，以及与gpi锚定蛋白的工程细胞表面表达相关。

Membrane anchoring via glycosylphosphatidyl inositol (GPI) structures is a ubiquitous mechanism whereby many functionally diverse proteins are linked to cell surfaces, and by means of which any protein of interest can be experimentally attached to cells. From previous work by several labs including our own, the biosynthetic pathway that provides for GPI assembly has been characterized and the genes that encode several of the enzymes that mediate these reactions have been cloned. In recent studies, our laboratory has generated a novel GPI-anchoring-defective mutant line (designated K) which accumulates complete GPI precursors but does not transfer them to acceptor proproteins. Our previous studies have established that this mutant is defective in a transamidation reaction which mediates this transfer. We have cloned the affected gene and shown that it corresponds to hGPI8, a homologue of a yeast gene, yGPI8, which appears to be the transamidase. In other collaborative work we have cloned a second human gene termed hGAA1 which encodes another factor required for the transamidation. In further work we have shown that a deacylation reaction which removes a fatty acid from the transfered GPI is closely coupled to the transamidation reaction. The purpose of the current proposal is to build upon this work so as to further clarify the post-assembly steps in GPI anchoring. Specifically, our aims are 1) To map the active sites of hGpi8p/hGaa1p and investigate their substrate specificity, 2) To utilize complementary yeast and mammalian systems to search for associated components, 3) To develop a solubilized/reconstituted system to study the transamidation reaction, and 4) To characterize the deacylase activity that removes inositol-linked acyl chains from the GPI moiety after its transfer. The data obtained should contribute to basic understanding of GPI-anchored protein metabolism. It could also have therapeutic relevance for fungal and parasitic infectious diseases as well as relevance for engineering cell surface expression of GPI-anchored proteins.