MOLECULAR BIOLOGY OF ASPARAGINE-LINKED GLYCOSYLATION

天冬酰胺连接糖基化的分子生物学

• 批准号: 3295056
• 负责人: Mark Lehrman
• 金额: $ 20.42万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295056
• 关键词: CHO cells; antibody; asparagine; carbohydrate biosynthesis; dolichol; endoplasmic reticulum; enzyme inhibitors; enzyme mechanism; gel electrophoresis; gene expression; gene mutation; glycosylation; hamsters; high performance liquid chromatography; laboratory rabbit; laboratory rat; mannosidase; membrane proteins; molecular cloning; mutant; nucleic acid sequence; oligosaccharides; phosphotransferases; plasmids; sugar phosphates; tissue /cell culture; tunicamycin

This project will employ three distinct approaches to characterize the reactions in the endoplasmic reticulum of mammals which assemble 14-sugar dolichol pyrophosphate-linked oligosaccharides, the precursors of asparagine-linked oligosaccharides in glycoproteins. These approaches follow directly from the work of the previous funding period. First, a full-length cDNA clone for hamster tunicamycin-sensitive N-acetylglucosamine-l-phosphate transferase (GPT), which adds the first sugar (N-acetylglucosamine-l-phosphate) to the dolichol phosphate carrier, will be used to express and map functional domains on this enzyme. Second, a Chinese hamster ovary (CHO) cell mutant with a possible defect in mannose-P-dolichol translocation will be studied to gain information about mechanisms of dolichol "flipping" involved in assembly of the dolichol-linked oligosaccharide precursor. Third, a general strategy is being developed to allow the isolation of series of mutant CHO cells, each with a deficiency at a different step in the assembly of the dolichol-linked oligosaccharide. These mutants will facilitate both biochemical and genetic analyses of the proteins involved, many of which have been extremely difficult to study. It is anticipated that the information about the assembly of the asparagine-linked oligosaccharide precursors which will result from this work will provide important clues as to the functions of these carbohydrates, which are widespread among eukaryotes. In addition, general information will be obtained about interactions between membrane-bound enzymes and lipid substrates, and eventually for sorting or "retention signals" for resident endoplasmic reticulum membrane proteins.

该项目将采用三种不同的方法来表征 哺乳动物的内质网中的反应，这些哺乳动物组装14硫酸 多利醇焦磷酸二糖，是寡糖的前体 糖蛋白中天冬酰胺连接的寡糖。这些方法 直接从上一个资金期的工作中遵循。首先，a 全长cDNA克隆，用于仓鼠女衣霉素敏感 N-乙酰葡萄糖胺L-磷酸转移酶（GPT），该酶增加了第一个 糖（N-乙酰葡萄糖胺-L磷酸盐），磷酸二乙醇载体， 将用于在此酶上表达和映射功能域。第二， 中国仓鼠卵巢（CHO）细胞突变体，可能缺陷 将研究Mannose-P-Dolichol易位以获取有关 Dolichol的机制“翻转”参与组装 Dolichol连接的寡糖前体。第三，一般策略是 开发以允许隔离一系列突变的CHO细胞 在组装中的不同步骤中缺乏不足 多醇连接的寡糖。这些突变体将有助于两者 涉及的蛋白质的生化和遗传分析，其中许多 研究非常困难。 预计有关集会的信息 天冬酰胺连接的寡糖前体将因此 工作将为这些功能提供重要的线索 碳水化合物，在真核生物中广泛。另外，一般 将获得有关膜结合之间相互作用的信息 酶和脂质底物，最终用于分类或保留 信号“用于常驻内质网膜蛋白。