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，通过这种机制，许多功能不同的蛋白质被连接到细胞表面，通过这种机制，任何感兴趣的蛋白质都可以实验地连接到细胞上。根据包括我们自己在内的几个实验室之前的工作，为GPI组装提供的生物合成途径已经被表征，并且编码几种介导这些反应的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个新的GPI锚定缺陷突变株(命名为K)，它积累了完整的GPI前体，但不将它们转移到受体前体蛋白。我们之前的研究已经证实，这个突变体在介导这种转移的转酰胺化反应中是有缺陷的。我们已经克隆了受影响的基因，并证明它对应于hGPI8，一个酵母基因的同源物，yGPI8，似乎是转氨酶。在其他合作工作中，我们克隆了第二个人类基因，称为hGAA1，它编码另一种转氨基作用所需的因子。在进一步的工作中，我们已经证明了从转移的GPI中去除脂肪酸的脱酰化反应与转酰胺化反应密切耦合。本提案的目的是在这项工作的基础上再接再厉，以便进一步澄清GPI锚定的装配后步骤。具体地说，我们的目标是1)绘制hGpi8p/hGaa1p的活性位点图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寻找相关成分，3)开发一个增溶/重组系统来研究转酰化反应，以及4)表征转移后从GPI部分去除肌醇连接的酰链的脱酰基酶的活性。所获得的数据应该有助于对GPI锚定蛋白代谢的基本了解。它还可能对真菌和寄生虫感染疾病具有治疗意义，以及与工程细胞表面表达GPI锚定蛋白有关。

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.