REGULATION OF PROTEIN N-GLYCOSYLATION IN THE CNS

在中枢神经系统（CNS）中，n -连接寡糖链的共翻译添加对于许多膜糖蛋白的正确折叠、细胞内易位和功能至关重要，这些膜糖蛋白介导重要的神经生物学功能。由于这种修饰的重要性，本项目的长期目标是了解内质网蛋白在调节多羟基磷酸（dolp）的生物合成、glc3man9glcnac2 -p -p -多醇的组装以及多羟基磷酸糖中间体在脑内的跨双层运动中的作用。该应用程序提出了五个特定的目标，使用生化和遗传方法设计：1)使用一种新的和有前途的水溶性类似物的转运试验，分离和了解更多关于内质网蛋白介导Man-P-Dol， Glc-P-Dol和ManSGlcNAc2 P-P-Dol在大脑中的横向扩散的结构；2)利用生物化学和遗传方法对黄豆中膜锚定甘露聚糖的组装进行拓扑研究，作为参与脑内质网中甘露聚糖中间体合成和跨双层运动的膜蛋白的模型系统；3)克隆编码长链顺式异戊烯基转移酶（cis- IPTase）和醇激酶的cdna，这两种酶参与中枢神经系统中dolp的新生生物合成。这些克隆的cdna将提供编码序列信息和探针，用于评估胚胎大鼠脑培养物和lps激活的小鼠B细胞中顺式iptase和醇激酶表达的发育变化；4)分离一个醇醇激酶的温度敏感突变体，以验证该酶是否催化了dolp生物合成新途径的末端步骤；5)通过研究法尼醇（F-OH）和香叶香醇（GG-OH）在脑内分别转化为“活化”的异戊二烯基供体——法尼基焦磷酸（F-P-P）和香叶香叶基焦磷酸（GG-P-P）的酶促机制，拓展类异戊二烯生物合成的新进展。

The co-translational addition of N-linked oligosaccharide chains is critical for the proper folding, intracellular translocation and function of many membrane glycoproteins mediating essential neurobiological functions in the central nervous system (CNS). Because of the importance of this modification, the long-term goal of this project has been to understand the role of ER proteins in the regulation of biosynthesis of dolichyl phosphate (Dol-P), the assembly of Glc3Man9GlcNAc2-P-P-dolichol and the transbilayer movement of dolichyl-phospho-saccharide intermediates in brain. This application proposes five Specific Aims using biochemical and genetic approaches designed to: 1) isolate and learn more about the structures of ER proteins mediating the transverse diffusion of Man-P-Dol, Glc-P-Dol and ManSGlcNAc2 P-P-Dol in brain using a novel and promising transport assay with water-soluble analogues; 2) use biochemical and genetic approaches in a topological study of the assembly of a membrane-anchored mannan in M. luteus as a model system for membrane proteins involved in the synthesis and transbilayer movement of mannolipid intermediates in the ER in brain; 3) clone cDNAs that encode the long-chain cis-isoprenyltransferase (cis- IPTase) and dolichol kinase, two enzymes involved in the de novo biosynthesis of Dol-P in the CNS. These cloned cDNAs will provide coding sequence information and probes to be used to assess developmental changes in the expression of cis-IPTase and dolichol kinase in embryonic rat brain cultures and LPS-activated murine B cells; 4) isolate a temperature-sensitive mutant for dolichol kinase to test the hypothesis that this enzyme catalyzes the terminal step in the de novo pathway for Dol-P biosynthesis and 5) to extend the study of a new development in isoprenoid biosynthesis by investigating the enzymatic mechanism by which farnesol (F-OH) and geranylgeraniol (GG-OH) are converted to the "activated" isoprenyl donors, farnesyl pyrophosphate (F-P-P) and geranylgeranyl pyrophosphate (GG-P-P) , respectively in brain.