Assembly and transfer of N-linked oligosaccharides

描述(申请人提供)：本项目的长期目标是了解真核寡糖基转移酶(OST)的生物学功能、机制和结构。OST将预先组装的高甘露糖低聚糖转移到粗面内质网腔内新生蛋白的天冬酰胺残基上。在拟议的供资期间，将特别强调阐明人类寡糖转移酶的两种异构体在体内的作用，这两种异构体由一个活性位点亚基(STT3A或STT3B)和一组共有的非催化亚基组成。这两种OST亚型在Dolicol连接的寡糖供体选择的比活性和严格性方面都有很大的不同。STT3A和STT3B在人体组织中广泛表达，到目前为止在所有分析的细胞系中都共同表达。我们将检验这一假设，即哺乳动物的OST亚型在蛋白质的N连接糖基化中具有重叠但不相同的作用。糖蛋白报告的低糖基化将在已经被操纵以表达单一OST亚型的细胞中进行分析。同时siRNA介导的OST活性部位亚单位(STT3A或STT3B)和ALG6葡萄糖基转移酶的敲除将为分析OST异构体表达对二羟基乙醇连接寡糖组装受损细胞中蛋白质低糖基化的影响提供一个模型系统。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复合体，而不是由真菌和后生动物组装的七聚体或八聚体OST复合体。新的检测方法和生物化学探针将被用来分析来自不同真核生物包括致病原生生物克氏锥虫、溶组织内阿米巴和阴道毛滴虫以及致病酵母新生隐球菌的Dolicol连接的寡糖供体和多肽受体与OST的结合。该项目的最终目标是通过电子结晶学获得哺乳动物OST的中到高分辨率结构。这里提出的研究将提供对遗传性人类常染色体疾病家族的洞察，这些疾病被称为先天性糖基化紊乱(CDG-I)。对致病原生生物蛋白质N-糖基化的分析可能揭示酶反应机制的不同，这可能被利用来生产有用的药理药物。

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.