MOLECULAR BIOLOGY OF ASPARAGINE-LINKED GLYCOSYLATION

这个项目将使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 苯二酚焦磷酸连接的寡糖，其前体 糖蛋白中天冬酰胺连接的寡糖。这些方法 直接从上一个资助期的工作中跟进。首先，一个 金黄地鼠衣霉素敏感基因全长cDNA克隆 N-乙酰氨基葡萄糖-L-磷酸转移酶，它增加了第一 糖(N-乙酰氨基葡萄糖-L-磷酸盐)到磷酸多尔酚载体上， 将用于表达和定位该酶的功能结构域。第二, 中国仓鼠卵巢(CHO)细胞突变型 将研究甘露糖-P-二十二醇的易位以获得关于 Dolicol“翻转”机制参与的组装 二醇连接的低聚糖前体。第三，总体战略是 正在开发以允许分离一系列突变的CHO细胞，每个 在装配的不同步骤中存在缺陷 二十二醇连接的低聚糖。这些突变体将促进 对涉及的蛋白质进行生化和遗传分析，其中许多 已经非常难研究了。 预计有关组件的组装信息 天冬酰胺连接的寡糖前体将由此产生 这项工作将为了解这些基因的功能提供重要线索 碳水化合物，在真核生物中广泛存在。此外，一般 将获得关于膜结合之间相互作用的信息 酶和脂类底物，最终用于分选或“保留” 常驻内质网膜蛋白的“信号”。

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.