Assembly and transfer of N-linked oligosaccharides

项目描述（由申请人提供）：本项目的长期目标是了解真核寡糖转移酶（OST）的生物学功能、机制和结构。OST将预先组装好的高甘露寡糖转移到粗内质网管腔中新生蛋白质的天冬酰胺残基上。在拟议的资助期内，将特别强调阐明由活性位点亚基（STT3A或STT3B）和一组共享的非催化亚基组成的人类低聚糖转移酶的两种同工异构体的体内作用。这两种OST异构体在特异性活性和寡糖供体选择的严格程度上都有很大的不同。STT3A和STT3B在人体组织中广泛表达，并在迄今为止分析的所有细胞系中共表达。我们将验证哺乳动物OST亚型在蛋白质的n链糖基化中具有重叠但不相同的作用的假设。糖蛋白报告基因的低糖基化将在表达单一OST异构体的细胞中进行分析。同时siRNA介导的OST活性位点亚基（STT3A或STT3B）和ALG6糖基转移酶的敲低将提供一个模型系统来分析OST异构体表达对蛋白低糖基化的影响，这些细胞在醇联寡糖组装中受损。蛋白质序列数据库搜索预测，大多数原生生物组装单体或异四聚体OST复合物，而不是真菌和后生动物组装的七聚体或八聚体OST复合物。新的检测方法和生化探针将用于分析各种真核生物（包括致病性原生生物克氏锥虫、溶组织内阿米巴和阴道毛滴虫以及致病性酵母新隐球菌）中dolicol -linked寡糖供体和肽受体与OST的结合。该项目的最终目标是通过电子晶体学获得哺乳动物OST的中高分辨率结构。这里提出的研究将提供深入了解被称为先天性糖基化疾病（CDG-I）的遗传性人类常染色体疾病家族。分析致病性原生生物中蛋白质n -糖基化可能揭示酶促反应机制的差异，从而有可能开发出有用的药物。

DESCRIPTION (provided by applicant): The long-term objective of this of this project is to understand the biological function, mechanism and structure of the eukaryotic oligosaccharyltransferase (OST). The OST transfers a preassembled high mannose oligosaccharide onto asparagine residues of nascent proteins in the lumen of the rough endoplasmic reticulum. During the proposed funding period particular emphasis will be placed on elucidating the in vivo roles of two isoforms of the human oligosaccharyltransferase that are composed of an active site subunit (STT3A or STT3B) and a shared set of non-catalytic subunits. The two OST isoforms differ greatly both with respect to specific activity and stringency of dolichol-linked oligosaccharide donor selection. STT3A and STT3B are widely expressed in human tissues, and are coexpressed in all cell lines analyzed to date. We will test the hypothesis that mammalian OST isoforms have overlapping but non-identical roles in N-linked glycosylation of proteins. Hypoglycosylation of glycoprotein reporters will be analyzed in cells that have been manipulated to express a single OST isoform. Simultaneous siRNA mediated knockdowns of an OST active site subunit (STT3A or STT3B) and the ALG6 glucosyltransferase will provide a model system to analyze the influence of OST isoform expression on protein hypoglycosylation in cells that are impaired in dolichol-linked oligosaccharide assembly. Protein sequence database searches predict that most protist organisms assemble monomeric or hetero-tetrameric OST complexes instead of the heptameric or octameric OST complexes assembled by fungi and metazoan organisms. Novel assays and biochemical probes will be used to analyze the binding of dolichol-linked oligosaccharide donor and peptide acceptors to the OST from diverse eukaryotic organisms including the pathogenic protists Trypanosoma cruzi, Entamoeba histolytica and Trichomonas vaginalis and the pathogenic yeast Cryptococcus neoformans. A final goal of this project is to obtain a mid to high-resolution structure of the mammalian OST by electron crystallography. The research proposed here will provide insight into the family of inherited human autosomal diseases known as congenital disorders of glycosylation (CDG-I). Analysis of protein N-glycosylation in pathogenic protists may reveal differences in enzymatic reaction mechanism that could potentially be exploited to produce useful pharmacological agents.