REGULATION OF PROTEIN N-GLYCOSYLATION IN THE CNS

N-连接寡糖链的共翻译加成对于许多膜糖蛋白的正确折叠、胞内移位和功能至关重要，所述膜糖蛋白介导中枢神经系统（CNS）中的基本神经生物学功能。由于这种修饰的重要性，本项目的长期目标是了解ER蛋白在脑中长羟磷酸（Dol-P）的生物合成、Glc 3 Man 9 GlcNAc 2-P-P-长羟的组装和长羟磷酸糖中间体的跨双层运动中的调节作用。 本申请提出了使用生物化学和遗传学方法的五个具体目的，旨在：1）使用新颖且有前景的水溶性类似物转运测定法，分离并更多地了解介导Man-P-Dol、Glc-P-Dol和ManSGlcNAc 2 P-P-Dol在脑中横向扩散的ER蛋白的结构; 2）利用生物化学和遗传学方法对M. luteus作为参与脑中ER中甘露脂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二烯基转移酶（cis-IPT酶）和多萜醇激酶的cDNA，这两种酶参与CNS中Dol-P的从头生物合成。 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激活的鼠B细胞中顺式-IPT酶和多萜醇激酶表达的发育变化; 4）分离多萜醇激酶的温度敏感性突变体，以测试该酶催化Dol-P生物合成的从头途径中的末端步骤的假设，以及5）通过研究脑中法尼醇（F-OH）和香叶基香叶醇（GG-OH）分别转化为“活化的”异戊二烯基供体--焦磷酸法尼酯（F-P-P）和焦磷酸香叶基香叶酯（GG-P-P）的酶促机制，拓展了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.