ASSEMBLY AND TRANSFER OF N-LINKED OLIGOSACCHARIDES

N-连接低聚糖的组装和转移

• 批准号: 6180417
• 负责人: JAMES REID GILMORE
• 金额: $ 29.51万
• 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 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180417
• 关键词: Saccharomyces cerevisiae; binding proteins; biosensor device; carbohydrate transport; dogs; endoplasmic reticulum; enzyme activity; enzyme biosynthesis; glycosylation; high performance liquid chromatography; immunoprecipitation; laboratory rabbit; lipid bilayer membrane; membrane permeability; membrane proteins; nucleic acid sequence; oligosaccharides; protein biosynthesis; protein folding; protein reconstitution; protein structure function; protein transport; synthetic peptide; western blottings

The long term objective of this project is to understand the structure, function and mechanism of the oligosaccharyltransferase (OST). The OST transfers high mannose oligosaccharides onto the asparagine residues of nascent proteins in the lumen of the rough endoplasmic reticulum. During the proposed funding period, particular emphasis will be placed on a biochemical, structural and molecular characterization of the mammalian and fungal OST complexes. The canine OST has been resolved into several forms that differ with respect to subunit composition and enzymatic activity. A high turnover form of the canine OST has been detected that lacks one or more subunits, hence it appears to be the catalytic core of the native enzyme. One objective of this project is to compare the enzymatic activity of the core enzyme and the native enzyme using biochemically homogeneous dolichol-oligosaccharides as donor substrates. Genetic and biochemical studies indicate that the yeast oligosaccharyltransferase is a hetero-ocatamer composed of the following three subcomplexes: Ost1p-Ost5p, Wbp1p-Swp1p-Ost2p and Stt3p- Ost3p-Ost4p. Mutations in the Stt3p-Ost3p-Ost4p subcomplex cause 2-30 fold reductions in the in vitro OST activity. The role of the Stt3p- Ost3p-Ost4p subcomplex will be analyzed in vitro using purified wild type and mutant OST complexes and purified donor substrates. The objective of these experiments is to determine whether the Stt3p-Ost3p- Ost4p subcomplex selects the fully assembled donor substrate. Novel assays will be developed to identify donor and acceptor substrate binding sites in the OST using methods that are not dependent upon glycopeptide formation. A second major objective is to investigate a quality control pathway in the yeast endoplasmic reticulum that mediates the folding of hypoglycosylated proteins. Yeast strains that do not express the non-essential DnaJ homologue Scj1p are hypersensitive to tunicamycin, mutations in the oligosaccharyltransferase, or mutations in the ER glucosidases. The extent of hypoglycosylation and precise structure of the asparagine-linked oligosaccharides contribute to the protein folding stress caused by hypoglycosylation. A combination of yeast molecular biological, biochemical and cell biological methods will be used to investigate how the transient display or permanent retention of glucose residues on N-linked oligosaccharides influences the folding and maturation of hypoglycosylated proteins in the yeast endoplasmic reticulum.

该项目的长期目标是了解结构， 寡糖胆碱转移酶（OST）的功能和机制。 OST 将高甘露糖寡糖转移到芦笋残基 粗糙内质网的腔内中的新生蛋白。 在拟议的资金期间，将特别强调 在生化，结构和分子表征上 哺乳动物和真菌OST复合物。 犬OST已解决 分为几种在亚基组成方面不同的形式和 酶活性。 犬Ost的高离职形式是 检测到缺少一个或多个亚基，因此 天然酶的催化核心。这个项目的一个目的是 比较核心酶和天然的酶活性 使用生物化学上均质的多醇 - 寡糖作为酶为酶 供体底物。 遗传和生化研究表明 酵母寡糖胆碱转移酶是一种异质 - 蛋白酶 以下三个子复合物：OST1P-OST5P，WBP1P-SWP1P-OST2P和STT3P- OST3P-OST4P。 stt3p-ost3p-ost4p子复合物中的突变导致2-30 体外OST活性的折叠减少。 stt3p-的作用 OST3P-OST4P子复合物将在体外使用纯化的野生进行分析 类型和突变体OST复合物以及纯化的供体底物。 这 这些实验的目的是确定stt3p-ost3p-是否是否 OST4P子复合选择完全组装的供体底物。 小说 将开发测定以识别捐赠者和受体基板 使用不依赖于OST的OST绑定位点 糖肽形成。 第二个主要目标是调查 酵母内质网中的质量控制途径介导 降糖基化蛋白的折叠。酵母菌菌株不 表达非必需的DNAJ同源性SCJ1P对 皮甲霉素，寡素胆脂式杂化酶中的突变或突变 在ER糖苷酶中。 降糖基化和精确的程度 天冬酰胺连接的寡糖的结构有助于 由低糖基化引起的蛋白质折叠应激。 组合 酵母分子生物学，生化和细胞生物学方法将 用于研究瞬态显示或永久保留方式 N连接的寡糖上的葡萄糖残基会影响折叠 酵母内质中低糖基化蛋白的成熟 网状。