Assembly and transfer of N-linked oligosaccharides

项目描述（申请人提供）：本项目的长期目标是了解真核生物寡糖基转移酶（OST）的生物学功能、机制和结构。OST将预组装的高甘露糖寡糖转移到粗面内质网腔内新生蛋白质的天冬酰胺残基上。在拟议的资助期间，将特别强调阐明由活性位点亚基（STT 3A或STT 3B）和一组共享的非催化亚基组成的人寡糖基转移酶的两种亚型的体内作用。这两种OST同工型在比活性和Dolichol连接的寡糖供体选择的严格性方面都有很大不同。STT 3A和STT 3B在人体组织中广泛表达，并且在迄今为止分析的所有细胞系中共表达。我们将测试的假设，哺乳动物OST亚型有重叠，但不相同的作用，在N-连接的糖基化蛋白质。将在已被操作以表达单个OST同种型的细胞中分析糖蛋白报告基因的低糖基化。同时siRNA介导的OST活性位点亚基（STT 3A或STT 3B）和ALG 6葡糖基转移酶的敲低将提供模型系统以分析OST同种型表达对在多萜醇连接的寡糖组装中受损的细胞中蛋白质低糖基化的影响。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源四聚体OST复合物，而不是由真菌和后生动物组装的七聚体或八聚体OST复合物。新的测定和生化探针将被用来分析结合的多萜连接的寡糖供体和肽受体的OST从不同的真核生物，包括病原性原生生物克氏锥虫，溶组织内阿米巴和阴道毛滴虫和致病性酵母隐球菌新生。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。这项研究将提供深入了解遗传性人类常染色体疾病家族，称为先天性糖基化障碍（CDG-I）。分析蛋白质N-糖基化的病原原生生物可能会揭示酶反应机制的差异，可能会被利用来生产有用的药物。

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.