ASSEMBLY AND TRANSFER OF N-LINKED OLIGOSACCHARIDES

这个项目的长期目标是了解结构， 寡糖基转移酶（OST）的功能和机制。 的OST 将高甘露糖寡糖转移到 粗面内质网腔内的新生蛋白质。 在拟议的供资期间， 生物化学，结构和分子特征的研究， 哺乳动物和真菌OST复合物。 犬OST已解决 分成几种不同的亚基组成形式， 酶活性 一个高营业额形式的狗OST一直是 检测到缺乏一个或多个亚基，因此它似乎是 天然酶的催化核心。该项目的一个目标是 为了比较核心酶和天然酶的酶活性， 使用生物化学上均质的长萜低聚糖作为 施主衬底 遗传和生物化学研究表明， 酵母寡糖基转移酶是一种杂八聚体， Ost 1 p-Ost 5 p、Wbp 1 p-Swp 1 p-Ost 2 p和Stt 3 p三个亚复合体。 Ost3p-Ost4p。 Stt 3 p-Ost 3 p-Ost 4p亚复合物中的突变导致2-30 体外OST活性的倍数降低。 Stt 3 p的作用- Ost 3 p-Ost 4p亚复合物将使用纯化的野生型在体外分析。 型和突变体OST复合物和纯化的供体底物。 的 这些实验的目的是确定Stt 3 p-Ost 3 p- Ost 4p亚复合物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分析方法 OST中的结合位点使用不依赖于 糖肽形成。 第二个主要目标是调查一个 酵母内质网中的质量控制途径， 低糖基化蛋白质的折叠。酵母菌株不 表达非必需的DnaJ同源物Scj 1 p对 衣霉素、寡糖基转移酶中的突变或突变 在内质网葡萄糖苷酶中。 低糖基化程度和精确 天冬酰胺连接的寡糖的结构有助于 低糖基化引起的蛋白质折叠应激。 的组合 酵母分子生物学、生物化学和细胞生物学方法将 用于研究短暂显示或永久保留 N-连接寡糖上的葡萄糖残基影响折叠 和酵母内质中低糖基化蛋白质的成熟 网状细胞。

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.