ASSEMBLY AND TRANSFER OF N-LINKED OLIGOSACCHARIDE

这里描述的研究集中在天冬酰胺连接的糖基化 粗面内质网中新合成的蛋白质。 特别强调的是：（一）隔离 寡糖基转移酶，（ii）生物化学和分子表征 的寡糖基转移酶和（iii）检查的机制， 脂质连接的寡糖的跨双层转运。 长期 该项目的目标是提供深入了解结构和 天冬酰胺连接的糖基化装置的功能组织 粗面内质网 寡糖基转移酶将是 分离自胰腺和酵母微粒体膜。 纯化的 蛋白质将使用生物化学、分子生物学和生物化学的组合来表征。 和细胞生物学方法。 糖基化位点的作用 将使用以下方法检查寡糖附着中的结合蛋白（GSBP） 寡糖基转移酶和寡糖基转移酶的纯化制剂， 糖基化位点结合蛋白。 细胞膜内容物和细胞内 酶的位置将通过用探针进行蛋白质印迹来确定， 寡糖基转移酶特异性抗体。 的氨基酸序列 寡糖基转移酶将通过分离和 对来自酵母和犬胰腺cDNA文库的cDNA克隆进行测序。 这两种酶的序列分析应该揭示了 功能意义、分选序列和跨膜跨越 片段 将蛋白质重构为磷脂囊泡 含有脂质连接的寡糖将用于研究 底物识别 组装中的生物合成中间体 脂连接的寡糖已经显示出不对称性 分布在粗糙的细胞质和内腔面之间 内质网膜 最大的中间检测上 哺乳动物微粒体膜的细胞质面（Man 5GlcNAc 2-PP- 长萜醇）显然是跨膜运输， 延伸至Glc 3 Man 9 GlcNAc 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.酿酒酵母作为模型 系统 Man 5GlcNAc 2-PP-多萜醇的易位将在 体外使用从头合成的脂质连接的寡糖和合成的 三肽作为寡糖受体。 特定中间体 组装和运输将被正常的扰动所限制 组装过程。 Man 5GlcNAc 2-PP-多萜醇将被掺入到 微粒体膜的细胞质面，以便可以监测转运 在重新合成脂连接寡糖的条件下， 不需要维护。 这些研究的目的是确定 脂联寡糖的转运是否是蛋白介导的， 自发过程

The research described here is focused upon asparagine-linked glycosylation of newly synthesized proteins in the rough endoplasmic reticulum. Particular emphasis will be placed on (i) the isolation of the oligosaccharyltransferase, (ii) biochemical and molecular characterization of the oligosaccharyltransferase and (iii) examination of the mechanism of transbilayer transport of lipid-linked oligosaccharides. 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. Oligosaccharyltransferase will be isolated from both pancreatic and yeast microsomal membranes. The purified protein will be characterized using a combination of biochemical, molecular and cellular biological approaches. The role of the glycosylation site binding protein (GSBP) in oligosaccharide attachment will be examined using purified preparations of the oligosaccharyltransferase and the glycosylation site binding protein. The membrane content and intracellular location of the enzyme will be determined by probing Western blots with oligosaccharyltransferase-specific antibodies. The amino acid sequence of the oligosaccharyltransferase will be determined by the isolation and sequencing of cDNA clones from yeast and canine pancreas cDNA libraries. Sequence analysis of the two enzymes should reveal conserved regions of functional significance, sorting sequences and transmembrane spanning segments. Reconstitution of the protein into phospholipid vesicles containing lipid-linked oligosaccharides will be used to investigate substrate recognition. 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 apparently transported across the membrane for subsequent elongation to Glc3Man9GlcNAc2-PP-Dol. The membrane topology and transbilayer transport of lipid-linked oligosaccharides will be investigated using microsomal membranes from S. cerevisiae as a model system. Translocation of Man5GlcNAc2-PP-dolichol will be investigated in vitro using de novo synthesized lipid-linked oligosaccharide and synthetic tripeptides as oligosaccharide acceptors. Specific intermediates in assembly and transport will be trapped by perturbation of the normal assembly process. Man5GlcNAc2-PP-dolichol will be incorporated into the cytoplasmic face of microsomal membranes so that transport can be monitored under conditions where de novo synthesis of lipid-linked oligosaccharide need not be maintained. The objective of these studies is to determine whether transport of lipid-linked oligosaccharides is a protein mediated or spontaneous process.