MOLECULAR BIOLOGY OF ASPARAGINE LINKED GLYCOSYLATION

天冬酰胺连接糖基化的分子生物学

• 批准号: 2179401
• 负责人: Mark Lehrman
• 金额: $ 21.98万
• 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/2179401
• 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描述 哺乳动物内质网中组装14-糖的反应 焦磷酸多萜醇连接的低聚糖， 糖蛋白中天冬酰胺连接的寡糖。这些方法 直接从上一个供资期的工作中得出。一是 仓鼠衣霉素敏感基因全长cDNA克隆 N-乙酰葡糖胺-L-磷酸转移酶（GPT），它添加了第一个 糖（N-乙酰葡糖胺-1-磷酸）与多萜醇磷酸载体， 将用于表达和映射这种酶的功能域。第二、 中国仓鼠卵巢（CHO）细胞突变体，可能存在以下缺陷： 将研究甘露糖-P-多萜醇易位，以获得关于 参与组装的多萜醇"翻转"机制， 长轴醇连接的寡糖前体。第三，总体战略是 正在开发允许分离一系列突变CHO细胞， 在装配的不同步骤中存在缺陷， 长轴醇连接的寡糖。这些突变体将促进 生物化学和基因分析的蛋白质，其中许多 非常难以研究。 预计，关于该组件的信息 天冬酰胺连接的寡糖前体，其将由此产生 工作将提供重要线索，这些功能， 碳水化合物，这是广泛存在于真核生物。此外，一般 将获得关于膜结合的 酶和脂质底物，并最终用于分选或"保留 信号"的常驻内质网膜蛋白。