MOLECULAR BIOLOGY OF ASPARAGINE LINKED GLYCOSYLATION

天冬酰胺连接糖基化的分子生物学

• 批准号: 2179402
• 负责人: Mark Lehrman
• 金额: $ 23.45万
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179402
• 关键词: CHO cells; N acetylneuraminate; active sites; asparagine; confocal scanning microscopy; dolichol; endoplasmic reticulum; enzyme complex; enzyme mechanism; enzyme structure; fluorescence microscopy; gene expression; gene mutation; glycoprotein biosynthesis; glycosylation; glycosyltransferase; high performance liquid chromatography; laboratory rabbit; membrane proteins; molecular cloning; nucleic acid sequence; oligosaccharides; phosphotransferases; protein structure function; transfection

The goal of this proposal is to understand the structure and function of hamster GlcNAc-1-P transferase (GPT). GPT catalyzes the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichol-P to form GlcNAc-P-P-Dolichol. GlcNAc-PP-dolichol is then converted into oligosaccharide-PP-dolichol (with 14 sugars), the immediate precursor for N-linked glycans in eukaryotes. N-linked glycans are important in a broad range of functions, such as cell adhesion, organellar trafficking, and protein folding, and have been implicated in cancer and human diseases such as HEMPAS, CGDS, and I-cell disease. Recent amino acid sequence analyses have linked hamster GPT with a family of similar eukaryotic and prokaryotic sugar-1-P transferases which use UDP-GlcNAc or UDP-MurNAc-pentapeptide as a donor and a polyisoprenyl phosphate as an acceptor. It is essential that GPT activity be regulated within a narrowly defined window. Too little GPT activity prevents sufficient quantities of N-linked glycans from being produced. Too much GPT activity causes excessive competition between GlcNAc-P-P-dolichol synthesis and reactions synthesizing mannose-P-dolichol (MPD) and glucose-P-dolichol (GPD). Insufficient MPD and GPD results in abnormal N-linked glycosylation and defective glycosylphosphatidylinositol (GPI) anchor synthesis. Furthermore, recent data indicate that binding of unfolded proteins to the ER chaperone calnexin, which specifically recognizes Glc1Man9GlcNAc2, would be inhibited. Five specific aims are proposed to address the following questions about hamster GPT: (1) What are the functions of the conserved sequences found in members of the UDP-GlcNAc/MurNAc transferase family? (2) What determines specificity for different sugar donors and lipid acceptors in the UDP-GlcNAc/MurNAc family of glycosyltransferases? (3) Rather than mediating bind of dolichol, does the "potential dolichol recognition sequence" (PDRS) promote a complex of enzymes which act early in the dolichol pathway? (4) How is GPT stabilized by its sequence-specific, bipartite C-terminus? (5) What is the topological orientation of GPT in the ER membrane? The answers to these questions will not only provide new fundamental information about GPT and its role in N-linked glycosylation, but will also have direct implications for a number of structurally related proteins.

本提案的目标是了解 仓鼠GlcNAc-1-P转移酶（GPT）。 谷丙转氨酶催化 GlcNAc-1-P从UDP-GlcNAc转化为Dolichol-P以形成GlcNAc-P-P-Dolichol。 然后将GlcNAc-PP-多萜醇转化为寡糖-PP-多萜醇 (with 14糖），N-连接聚糖的直接前体， 真核生物N-连接聚糖在广泛的功能中是重要的， 如细胞粘附、细胞器运输和蛋白质折叠， 与癌症和人类疾病如HEMPAS，CGDS， 和I细胞疾病 最近的氨基酸序列分析表明， 仓鼠谷丙转氨酶与一个类似的真核和原核糖-1-P家族 使用UDP-GlcNAc或UDP-MurNAc-五肽作为供体的转移酶 和作为受体的聚异戊二烯基磷酸酯。 至关重要的是，GPT活性应在严格定义的 窗口太少的GPT活性会阻止足够数量的N-连接 聚糖的产生。 过多的GPT活性会导致过度的 GlcNAc-P-P-dolichol合成与反应之间的竞争 合成甘露糖-β-多萜醇（MPD）和葡萄糖-β-多萜醇（GPD）。 不足的MPD和GPD导致异常的N-连接糖基化， 糖基磷脂酰肌醇（GPI）锚合成缺陷。 此外，最近的数据表明，未折叠的蛋白质结合到 ER伴侣钙连接蛋白，其特异性识别Glc 1 Man 9 GlcNAc 2， 会受到抑制。 提出了五个具体目标，以解决以下问题： 仓鼠谷丙转氨酶：（1）发现的保守序列的功能是什么 在UDP-GlcNAc/MurNAc转移酶家族的成员中？(2)什么 确定不同糖供体和脂质受体的特异性， UDP-GlcNAc/MurNAc糖基转移酶家族？(3)而不是 介导长醇的结合，是否“潜在的长醇识别 序列”（PDRS）促进一种酶的复合物，这种酶在细胞生长的早期起作用。 多萜醇途径？(4)GPT是如何通过其序列特异性， 二分C末端？(5)中GPT的拓扑方向是什么 ER膜？ 这些问题的答案将不仅提供新的 关于GPT及其在N-连接糖基化中的作用的基本信息， 但也将对一些结构性的 相关蛋白质