ASSEMBLY AND TRANSFER OF N-LINKED OLIGOSACCHARIDE

这里描述的研究主要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。 将特别强调：(I)隔离 寡糖转移酶，(II)生化和分子特征 低聚糖基转移酶的研究和(Iii)低聚糖转移酶作用机制的研究 脂联寡糖的跨双层转运。从长远来看 本项目的目标是提供对结构和 天冬酰胺连接的糖基化装置的功能组织 粗面内质网。寡糖转移酶将是 从胰腺和酵母微粒体膜中分离得到。纯净的 蛋白质将使用生化、分子和生物化学相结合的方法进行表征 以及细胞生物学方法。糖基化位点的作用 寡糖结合中的结合蛋白(GSBP)将使用 低聚糖基转移酶的纯化制剂及 糖基化结合蛋白。膜含量和细胞内 酶的位置将通过探测Western blotts和 寡糖转移酶特异性抗体。该蛋白的氨基酸序列 寡糖转移酶将通过分离和鉴定 酵母和犬胰腺cdna文库的克隆测序。 这两种酶的序列分析应该揭示出保守的区域 功能意义、排序序列和跨膜跨越 细分市场。蛋白质在磷脂囊泡中的重组 含有脂质连接的寡糖将被用来研究 底物识别。生物合成中间体在组装中的应用 脂联寡糖已被证明是不对称的 分布在粗面的胞质和管腔面之间 内质网膜。检测到的最大中间体 哺乳动物微粒体膜的细胞质表面(Man5GlcNAc2-PP- Dolicol)显然是跨膜运输，以便随后 伸长率为Glc3Man9GlcNAc2-PP-Dol。膜的拓扑结构和 脂联寡糖的跨双层转运将是 以酿酒酵母微粒体膜为模型进行研究 系统。将研究Man5GlcNAc2-PP-Dolicol的移位 体外使用从头合成的脂联寡糖和人工合成 作为寡糖受体的三肽。中的特定中间体 组装和运输将被正常的扰动所困 组装过程。Man5GlcNAc2-PP-Dolicol将被整合到 微体细胞膜的细胞质表面，以便可以监测运输 在从头合成脂联寡糖的条件下 不需要维护。这些研究的目的是确定 脂联低聚糖的转运是蛋白质介导的还是 自发的过程。

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.