Molecular Biology of Asparagine-Linked Glycosylation

天冬酰胺连接糖基化的分子生物学

• 批准号: 7089852
• 负责人: Mark Lehrman
• 金额: $ 36.86万
• 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 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7089852
• 关键词: CHO cells; SDS polyacrylamide gel electrophoresis; asparagine; autoradiography; biosynthesis; dolichol; electrophoresis; endoplasmic reticulum; fluorescent dye /probe; gel electrophoresis; glycolipids; glycoproteins; glycosylation; intermolecular interaction; ionophores; oligosaccharides; phosphorylation; protein folding; thin layer chromatography; transferase

DESCRIPTION (provided by applicant): The long-term goal of this grant is to fully understand the biosynthesis, function, and clinical importance of a key molecule in the endoplasmic reticulum (ER) of mammalian cells, the lipid-linked oligosaccharide (LLO) glucose3mannose9N-Acetylglucosamine2-P-P-dolichol, or G3M9Gn2-P-P-Dol. This work is important: i- G3M9Gn2-P-P-Dol synthesis involves novel and exciting biochemical processes yielding insights into other pathways, ii- Once transferred to protein, the G3M9Gn2 unit is processed by a series of enzymes (glycosidases). The various oligosaccharide products play roles in protein folding and degradation ("quality control") in the ER. iii- Genetic defects in the synthesis of G3M9Gn2-P-P-Dol form a family of at least seven human genetic disorders called Congenital Disorders of Glycosylation (CDG) Type la-lg. This application will take advantage of two recent technical innovations from the current funding period: fluomphore-assisted carbohydrate electrophoresis (FACE) to study LLOs, and streptolysin-O (SLO)-based in vitro systems that faithfully preserve in vivo regulatory processes. These two techniques have dramatically changed the way we study and think about this pathway. The application will also bring together new insights into the roles of the Unfolded Protein Response (UPR), the Lec35 protein, and two functionally distinct pools of dolichoI-P (DoI-P) in control of LLO synthesis. These are especially important for the regulation of the initiating step of LLO synthesis, catalyzed by GIcNAc-1-P transferase (GPT). AIM 1: Use the SLO in vitro system to determine the route for recycling of the primary pool of DoI-P and the membrane topology the secondary pool of DoI-P. AIM 2: Test the hypothesis that triggering of the Unfolded Protein Response by robust ER stress leads to a dual mechanism for inhibition of LLO synthesis involving both PERK and Lec35p. AIM 3: Test the hypotheses that GPT interacts directly with Lec35p, and that overexpression of GPT causes defective LLO synthesis by interfering with Lec35p function in a dominant-negative manner. AIM 4: Test the hypothesis that novel sugar-P-dolichols and novel sugar-P-dolichol dependent glycoconjugates remain to be identified in animal cells. Use the unique properties of Lec35 cells and the advantages of the SLO system to identify these glycoconjugates.

描述(由申请人提供)： 这项资助的长期目标是全面了解哺乳动物细胞内质网(ER)中的一个关键分子--脂联寡糖(LLO)glucose3mannose9N-Acetylglucosamine2-P-P-dolichol，或G3M9Gn2-P-P-Dol的生物合成、功能和临床重要性。这项工作很重要：I-G3M9Gn2-P-P-Dol的合成涉及新颖和令人兴奋的生化过程，产生对其他途径的见解；II-一旦转移到蛋白质，G3M9Gn2单位由一系列酶(糖苷酶)处理。不同的寡糖产物在内质网中的蛋白质折叠和降解(“质量控制”)中起作用。III-G3M9Gn2-P-P-Dol合成中的遗传缺陷形成了至少七个人类遗传病家族，称为先天性糖基化紊乱(CDG)1a-lg型。这项应用将利用当前资助期的两项最新技术创新：用于研究LLO的荧光素辅助碳水化合物电泳法(FACE)，以及基于链溶酶-O(SLO)的体外系统，它忠实地保留了体内调节过程。这两种技术极大地改变了我们研究和思考这条途径的方式。该应用还将带来对未折叠蛋白反应(UPR)、Lec35蛋白和两个功能不同的DolichoI-P(DOI-P)池在控制LLO合成中的作用的新见解。这些对于GIcNAc-1-P转移酶(GPT)催化的LLO合成的起始步骤的调控尤为重要。目的1：利用体外SLO系统确定DOI-P初级池的再循环途径和次级池的膜拓扑结构。目的2：验证一种假设，即由强健的内质网应激触发的未折叠蛋白反应导致了包括PERK和Lec35p在内的抑制LLO合成的双重机制。目的：验证GPT与Lec35p直接相互作用以及GPT过表达通过显性-负性干扰Lec35p功能导致LLO合成缺陷的假说。目的4：验证在动物细胞中仍未发现新的糖-P-多利酚和新的糖-P-多利酚依赖的糖偶联物的假设。利用Lec35细胞的独特性质和SLO系统的优势来鉴定这些糖偶联物。