Molecular Biology of Asparagine-Linked Glycosylation

描述（由申请人提供）： 这项资助的长期目标是充分了解哺乳动物细胞内质网（ER）中关键分子的生物合成，功能和临床重要性，脂质连接寡糖（LLO）葡萄糖3甘露糖9 N-乙酰葡萄糖胺2-P-P-dolichol或G3 M9 Gn 2-P-P-Dol。这项工作很重要：i-G3 M9 Gn 2-P-P-Dol合成涉及新颖且令人兴奋的生化过程，从而深入了解其他途径，ii-一旦转移到蛋白质中，G3 M9 Gn 2单元就会被一系列酶（糖苷酶）加工。各种寡糖产物在ER中的蛋白质折叠和降解（“质量控制”）中发挥作用。iii-G3 M9 Gn 2-P-P-Dol合成中的遗传缺陷形成至少七种人类遗传疾病的家族，称为先天性糖基化疾病（CDG）la-lg型。荧光团辅助碳水化合物电泳（FACE）研究LLO，和基于链球菌溶血素-O（SLO）的体外系统，其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。该应用程序还将汇集对未折叠蛋白质反应（UPR），Lec 35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。这些对于由GlcNAc-1-P转移酶（GPT）催化的LLO合成的起始步骤的调节尤其重要。目标1：使用SLO体外系统来确定DoI-P的初级池的再循环途径和DoI-P的次级池的膜拓扑结构AIM 2：检验以下假设：通过强ER应激触发未折叠蛋白质应答导致涉及PERK和Lec 35 p两者的LLO合成抑制的双重机制。目标3：测试GPT直接与Lec 35 p相互作用的假设，以及GPT的过表达通过以显性负性方式干扰Lec 35 p功能而导致LLO合成缺陷的假设。目的4：验证新的糖-β-多萜醇和新的糖-β-多萜醇依赖性糖缀合物仍有待于在动物细胞中鉴定的假设。利用Lec 35细胞的独特性质和SLO系统的优势来鉴定这些糖缀合物。

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.