ASSEMBLY AND TRANSFER OF N LINKED OLIGOSACCHARIDE

该项目的长期目标是提供对 结构和功能组织的天冬酰胺连接 粗面内质网的糖基化装置。 特别 重点将放在（一）生物化学和分子 哺乳动物和真菌寡糖基转移酶的表征， (ii)酿酒酵母鉴定与特性研究 基因产物在功能上与 寡糖基转移酶和（iii）检查的机制， 脂质连接低聚糖的跨双层转运。 从哺乳动物和酵母中分离的寡糖基转移酶 微粒体膜将使用以下组合进行表征： 生物化学、分子和细胞生物学方法。 的氨基酸 酵母寡糖基转移酶的亚基的序列将是 通过基因组克隆的分离和测序来确定。 的 将评估酵母寡糖基转移酶亚基的功能 通过条件突变体的分析。 的空间和时间 蛋白质转运机制和蛋白质之间的关系 糖基化装置将在S.啤酒。 酵母基因 与寡糖基转移酶相互作用的产物将 通过基因筛选确定高拷贝抑制基因， 寡糖基转移酶突变体。 生物合成中间体 已经证明，脂质连接的寡糖的组装 不对称分布在细胞质和内腔面之间， 粗面内质网膜。 最大的中间体 在哺乳动物微粒体膜的细胞质表面上检测到 （Man 5GlcNAc 2-PP-多萜醇）跨膜转运， 随后延伸为Glc 3 Man 9 GlcNA 2-PP-Dol。 膜拓扑结构和 脂质连接的寡糖的跨双层转运将是 使用来自S.啤酒。 易位 Man 5GlcNAc 2-PP-多萜醇的体外研究将使用内源性 和从头合成的脂连接的寡糖作为寡糖 供体和作为寡糖受体的合成三肽。 干扰脂质相关的实验扰动 寡糖转运将被用作研究 运输过程。 这些研究的目的是确定是否 脂质连接寡糖的转运是蛋白质介导的， 自发过程， 糖缺乏糖蛋白综合征 是一种最近描述的多系统性人类疾病， 由新合成的蛋白质的多效性糖基化不足引起， 内质网 导致CDGS的病变尚未被发现。 定义，但可能涉及脂质连接的组装缺陷， 寡糖供体或寡糖基转移酶缺陷。 因此，在本发明中， 本提案中描述的研究与人类直接相关， 疾病

The long-term objective of this project is to provide insight into the structural and functional organization of the asparagine-linked glycosylation apparatus of the rough endoplasmic reticulum. Particular emphasis will be placed on (i) a biochemical and molecular characterization of the mammalian and fungal oligosaccharyltransferases, (ii) the identification and characterization of Saccharomyces cerevisiae gene products that functionally interact with the oligosaccharyltransferase and (iii) an examination of the mechanism of transbilayer transport of lipid-linked oligosaccharides. Oligosaccharyltransferase isolated from both mammalian and yeast microsomal membranes will be characterized using a combination of biochemical, molecular and cellular biological approaches. The amino acid sequence of the subunits of the yeast oligosaccharyltransferase will be determined by the isolation and sequencing of genomic clones. The function of the yeast oligosaccharyltransferase subunits will be evaluated in vivo by the analysis of conditional mutants. The spatial and temporal relationships between the protein translocation machinery and the protein glycosylation apparatus will be evaluated in S. cerevisiae. Yeast gene products that interact with the oligosaccharyltransferase will be identified by genetic screens for high-copy suppressors of an oligosaccharyltransferase mutant. Biosynthetic intermediates in the assembly of lipid-linked oligosaccharide have been shown to be asymmetrically distributed between the cytoplasmic and lumenal faces of the rough endoplasmic reticulum membrane. The largest intermediate detected upon the cytoplasmic face of mammalian microsomal membranes (Man5GlcNAc2-PP-dolichol) is transported across the membrane for subsequent elongation to Glc3Man9GlcNA2-PP-Dol. The membrane topology and transbilayer transport of lipid-linked oligosaccharides will be investigated using microsomal membranes from S. cerevisiae. Translocation of Man5GlcNAc2-PP-dolichol will be investigated in vitro using endogenous and de novo synthesized lipid-linked oligosaccharides as oligosaccharide donors and synthetic tripeptides as oligosaccharide acceptors. Experimental perturbations that interfere with lipid-linked oligosaccharide transport will be used as tools to investigate the transport process. The objective of these studies is to determine whether transport of lipid-linked oligosaccharides is a protein-mediated or spontaneous process., Carbohydrate deficient glycoprotein syndrome (CDGS) is a recently described multi-systemic human disease that appears to be caused by pleiotropic underglycosylation of newly synthesized proteins in the endoplasmic reticulum. The lesion responsible for CDGS has not been defined, but could involve a defect in the assembly of the lipid-linked oligosaccharide donor or a defect in the oligosaccharyltransferase. Thus, the research described in this proposal is of direct relevance to a human disease.