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，其中许多功能不同的蛋白质连接到细胞表面，并且通过该机制，任何感兴趣的蛋白质可以实验性地附着到细胞。 从包括我们自己的实验室在内的几个实验室以前的工作来看，为GPI组装提供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，我们的实验室产生了一种新型的GPI锚定缺陷突变系（命名为K），它积累了完整的GPI前体，但不会将它们转移到受体前蛋白上。 我们以前的研究已经确定，这种突变体是有缺陷的转酰胺反应介导这种转移。 我们已经克隆了受影响的基因，并表明它对应于hGPI 8，酵母基因yGPI 8的同源物，这似乎是转酰胺酶。 在其他合作工作中，我们克隆了第二个人类基因，称为hGAA 1，它编码转酰胺所需的另一个因子。 在进一步的工作中，我们已经表明，从转移的GPI中除去脂肪酸的脱酰反应与转酰胺反应密切相关。 本提案的目的是在这项工作的基础上进一步澄清GPI锚定的组装后步骤。 具体而言，我们的目标是1）定位hGpi 8 p/hGaa 1 p的活性位点并研究其底物特异性，2）利用互补酵母和哺乳动物系统来寻找相关组分，3）开发溶解/重构系统来研究转酰胺反应，以及4）表征脱酰酶活性，该脱酰酶活性在GPI部分转移后从其上去除肌醇连接的酰基链。 获得的数据应有助于对GPI锚定蛋白质代谢的基本了解。 它也可能具有治疗真菌和寄生虫感染性疾病的相关性，以及与GPI锚定蛋白的工程化细胞表面表达的相关性。

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.