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.

该提议的目的是了解 仓鼠GLCNAC-1-P转移酶（GPT）。 GPT催化 从UDP-GLCNAC到Dolichol-P形成GlcNAC-P-P-P-Dolichol。 然后将GlcNAC-PP-Dolichol转换为寡糖-PP-Dolichol （含14种糖），是N连接聚糖的直接前体 真核生物。 N连接的聚糖在广泛的功能中很重要， 例如细胞粘附，细胞器运输和蛋白质折叠，以及 与癌症和人类疾病有关，例如Hempas，CGD， 和I细胞疾病。 最近的氨基酸序列分析已连接 仓鼠GPT与类似的真核和原核糖的家族 使用UDP-GLCNAC或UDP-Murnac-Pentapeptide作为供体的转移酶 和聚异源磷酸作为受体。 必须在狭义的定义中调节GPT活动至关重要 窗户。 GPT活动太少会阻止足够数量的N链接 从生产中产生的聚糖。 过多的GPT活动会导致过度 GlcNAC-P-P-P-Dolichol合成与反应之间的竞争 合成的甘露糖-P-Dolichol（MPD）和葡萄糖-P-Dolichol（GPD）。 MPD和GPD不足会导致N-连接糖基化异常，并且 缺陷糖基磷脂酰肌醇（GPI）锚固合成。 此外，最近的数据表明，展开的蛋白质与 ER伴侣calnexin，专门识别Glc1Man9GlCNAC2， 会被抑制。 提出了五个具体目标来解决以下问题 仓鼠GPT：（1）找到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 糖基转移酶的UDP-GLCNAC/Murnac家族？ （3）而不是 Dolichol的介导结合，“潜在的Dolichol识别 序列”（PDR）促进了一种在早期起作用的酶 Dolichol Pathway？ （4）如何通过其序列特异性稳定GPT， 两分C末端？ （5）GPT的拓扑取向是什么 ER膜？ 这些问题的答案不仅会提供新的 有关GPT及其在N连接糖基化中的作用的基本信息， 但也将对许多结构上有直接影响 相关蛋白质。