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; 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）： 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.这项工作很重要：I-G3M9GN2-P-P-DOL合成涉及新颖且令人兴奋的生化过程，从而产生了对其他途径的见解，II-一旦转移到蛋白质中，G3M9GN2单元就通过一系列酶（糖酶）处理。各种寡糖产品在ER中的蛋白质折叠和降解（“质量控制”）中起着作用。 G3M9GN2-P-P-DOL合成中的II-遗传缺陷形成了至少七个人类遗传疾病的家族，称为糖基化先天性疾病（CDG）LA-LG。该应用程序将利用当前资金期间的两项最近技术创新：氟辅助碳水化合物电泳（FACE）研究LLOS，以及基于链霉菌素-O（SLO）的体外系统，这些系统忠实地保留体内调节过程。这两种技术极大地改变了我们研究和思考这一途径的方式。该应用还将将新的见解汇总到展开的蛋白质反应（UPR），LEC35蛋白和两个功能上不同的Dolichoi-P（DOI-P）库中的作用中。这些对于通过GICNAC-1-P转移酶（GPT）催化的LLO合成步骤的调节尤其重要。目标1：使用SLO体外系统确定DOI-P的主要池和膜拓扑的途径的途径，DOI-P的次级池。 AIM 2：检验以下假设：鲁棒性ER应力触发展开的蛋白质反应会导致抑制涉及PERK和LEC35P的LLO合成的双重机制。 AIM 3：测试GPT直接与LEC35P相互作用的假设，而GPT的过表达通过以显性阴性的方式干扰LEC35P功能会导致LLO合成有缺陷。 AIM 4：检验以下假设：新型的糖 - -P-核酸酚和新型糖 - -P-二甲醇依赖性糖缀合物在动物细胞中仍有待鉴定。使用LEC35细胞的唯一特性和SLO系统的优势来识别这些糖缀合物。