ASSEMBLY AND TRANSFER OF N-LINKED OLIGOSACCHARIDES

这个项目的长期目标是了解结构， 寡糖转移酶(OST)的功能和机制。OST 将高甘露低聚糖转移到天冬酰胺残基上 粗面内质网腔内的新生蛋白质。 在拟议的资助期内，将特别强调 关于该酶的生化、结构和分子表征 哺乳动物和真菌的OST复合体。犬类OST已解决 分成几种亚基组成不同的形式，并且 酶活性。一种高周转率的犬类OST 检测到缺少一个或多个亚单位，因此它似乎是 天然酶的催化核心。这个项目的一个目标是 比较核心酶和天然酶的酶活性 以生物化学均一的二羟基寡糖为原料的酶 施主底物。遗传和生化研究表明， 酵母寡糖基转移酶是一种异构体，由 以下三个亚络合物：Ost1p-Ost5p，Wbp1p-Swp1p-Ost2p和Stt3p- Ost3p-Ost4p。Stt3p-Ost3p-Ost4p亚复合体突变导致2-30 体外OST活性成倍降低。Stt3p的作用- Ost3p-Ost4p亚复合体将使用纯化的野生动物进行体外分析 类型和突变的OST复合体和纯化的供体底物。这个 这些实验的目的是确定Stt3p-Ost3p是否- Ost4p亚复合体选择完全组装的供体底物。小说 将开发鉴定供体和受体底物的检测方法 使用不依赖于的方法在OST中结合站点 糖肽的形成。第二个主要目标是调查 酵母内质网中的质量控制途径 低糖化蛋白质的折叠。酵母菌不能 表达非必需的DNAJ同源物Scj1p对 衣霉素、寡糖转移酶突变或突变 在ER葡萄糖苷酶中。低糖基化程度和精确度 天冬酰胺连接的寡糖的结构有助于 蛋白质折叠应激引起的低糖基化。一种组合 酵母菌分子生物学、生化和细胞生物学方法将 用于研究瞬时显示或永久保留是如何 N-连接低聚糖上的葡萄糖残基对折叠的影响 和酵母内质中低糖基化蛋白的成熟 网状结构。

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.