ASSEMBLY AND TRANSFER OF N-LINKED OLIGOSACCHARIDE

N-连接低聚糖的组装和转移

• 批准号: 3302806
• 负责人: JAMES REID GILMORE
• 金额: $ 14.67万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302806
• 关键词: Saccharomyces cerevisiae; asparagine; binding proteins; carbohydrate transport; complementary DNA; dogs; enzyme mechanism; glycosylation; high performance liquid chromatography; laboratory mouse; laboratory rabbit; lipid bilayer membrane; membrane permeability; membrane proteins; microsomes; oligosaccharides; pancreas; protein biosynthesis; protein purification; protein reconstitution; protein sequence; protein structure function; protein transport; radionuclides; transferase

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.

此处描述的研究集中于天冬酰胺连接的糖基化 粗糙内质网中新合成的蛋白质的。 特别强调（i）隔离 寡素胆碱转移酶，（ii）生化和分子表征 寡素胆碱转移酶和（iii）检查机理 脂质连接寡糖的跨贝尔运输。 长期 该项目的目的是提供有关结构性和 天冬酰胺连接的糖基化设备的功能组织 粗糙的内质网。 寡糖胆碱转移酶将是 从胰腺和酵母微粒体膜中分离出来。 纯化 蛋白质将使用生化，分子的组合来表征 和细胞生物学方法。 糖基化位点的作用 将使用寡糖附着中的结合蛋白（GSBP）使用 寡素胆碱转移酶的纯化制剂和 糖基化位点结合蛋白。 膜含量和细胞内 酶的位置将通过探测与 寡糖胆碱转移酶特异性抗体。 氨基酸序列 寡糖胆碱转移酶将由分离和 从酵母和犬胰腺cDNA文库中对cDNA克隆的测序。 两种酶的序列分析应揭示 功能意义，分类序列和跨膜跨度 细分市场。 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 脂质连接的寡糖已被证明是不对称的 分布在粗糙的细胞质和流体面之间 内质网膜。 检测到的最大的中间体 哺乳动物微粒体膜的细胞质面（MAN5GLCNAC2-PP-- Dolichol）显然在整个膜上运输以进行后续 伸长到Glc3Man9GlCNAC2-PP-DOL。 膜拓扑和 脂质连接的寡糖的跨贝伊勒运输将是 使用酿酒酵母的微粒体膜作为模型进行了研究 系统。 MAN5GLCNAC2-PP-DOLICHOL的易位将在研究中进行研究 使用从头合成脂质连接的寡糖和合成的体外体外 将三肽作为寡糖受体。 特定的中间体 组装和运输将被正常的扰动所困扰 组装过程。 MAN5GLCNAC2-PP-DOLICHOL将合并到 微粒体膜的细胞质面，以便可以监测运输 在从头合成脂质连接寡糖的条件下 不需要维护。 这些研究的目的是确定 脂质连接寡糖的转运是介导的蛋白质还是 自发过程。