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Regulation of Protein N-Glycosylation in the CNS

CNS 中蛋白质 N-糖基化的调节

基本信息

批准号：
7923610
负责人：
Charles J Waechter
金额：
$ 11.26万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7923610
关键词：
Active Sites Affect Affinity Anabolism Antibody Formation Back Benzophenones Binding Binding Sites Biochemical Biochemical Genetics Cell Surface Receptors Cells Chinese Hamster Ovary Cell Clinical Complementary DNA Congenital Disorders Defect Diffuse Diffusion Dolichol Dolichol kinase Endoplasmic Reticulum Enzymatic Biochemistry Enzymes GPI Membrane Anchors Genes Glucosylceramides Glycerophospholipids Human In Vitro Inherited Ion Channel Learning Length Lipids Mammalian Cell Mediating Membrane Proteins Modeling Movement Mutation Nature Pathway interactions Patients Phosphoric Monoester Hydrolases Phosphotransferases Principal Investigator Procedures Protein Dephosphorylation Proteins Reaction Recycling Regulation Role Route Sepharose Series Site-Directed Mutagenesis Staging Structure System Tissues Water analog base design dolichol monophosphate glycosylation in vivo mannose-phosphate-citronellol monolayer novel overexpression programs reconstitution research study

项目摘要

DESCRIPTION (provided by applicant): The co-translational N-glycosylation of many important cell surface receptors, ion channels and lysosomal enzymes is essential for their correct folding, intracellular routing and function in the CNS and other mammalian cells. The vital importance of protein N-glycosylation in humans is emphasized by the clinical consequences of a series of inherited genetic defects in this pathway classified as Congenital Disorders of Glycosylation (CDG). While the enzymology and topology of the biosynthesis of Glc3Man9GlcNAc2-P-P-Dol, the oligosaccharyl donor, in the endoplasmic reticulum (ER) have been studied extensively, many gaps remain in the understanding of how the enzymes in this pathway are organized and regulated. This proposal describes further studies to learn more about the role of ER-associated proteins in the regulation of dolichyl phosphate (Dol-P) biosynthesis and recycling, and the transbilayer movement of Man-P-Dol. Three Specific Aims are planned using biochemical, genetic and immunochemical approaches designed: 1) To utilize a recently cloned cDNA to learn more about the long-chain c/s-isoprenyltransferase (c/s-IPTase) catalyzing the elongation stage in Dol-P biosynthesis by investigating its regulation and the nature of its association with potential binding partners in the ER; 2) To utilize cloned cDNAs encoding dolichol kinase (DK) and Dol-P-P phosphatase, an ER enzyme with a lumenally-oriented active site, in in vivo and in vitro experiments aimed at elucidating their precise roles in the ote novo synthesis of Dol-P and the recycling of the glycosyl carrier lipid and 3) To purify, identify and characterize the ER protein(s) mediating the transverse diffusion of Man-P-Dol in mammalian cells ("flippase"). The information gained on the structure of the Man-P-Dol flippase will be relevant to related membrane proteins mediating the transbilayer movement of other dolichyl-P-(P)-saccharide intermediates in protein O-, C- and N-glycosylation, glycerophospholipids, glucosylceramide and glycosylphosphatidylinositol (GPI) anchor precursors and potential defects in patients with Congenital Disorders of Glycosylation (CDG).

描述（由申请方提供）：许多重要细胞表面受体、离子通道和溶酶体酶的共翻译N-糖基化对于其在CNS和其他哺乳动物细胞中的正确折叠、细胞内路径和功能至关重要。蛋白质N-糖基化在人类中的至关重要性被归类为先天性糖基化障碍（CDG）的该途径中的一系列遗传性遗传缺陷的临床后果所强调。虽然已经广泛研究了内质网（ER）中寡糖基供体Glc 3 Man 9 GlcNAc 2-P-P-Dol生物合成的酶学和拓扑学，但是在理解该途径中的酶如何组织和调节方面仍存在许多空白。该建议描述了进一步的研究，以了解更多关于ER相关蛋白在调节磷酸多脂（Dol-P）生物合成和回收中的作用，以及Man-P-Dol的跨双层运动。本论文的主要目的是利用生物化学、遗传学和免疫化学的方法，研究长链顺式异戊二烯基转移酶的基因表达，包括：1）利用最近克隆的长链顺式异戊二烯基转移酶cDNA，进一步研究长链顺式异戊二烯基转移酶的基因表达（c/s-IPT酶）通过研究Dol-P生物合成中的延伸阶段的调节及其与ER中潜在结合配偶体的关联性质来催化Dol-P生物合成中的延伸阶段; 2）利用克隆的编码多萜醇激酶（DK）和Dol-P-P磷酸酶（一种具有内腔定向活性位点的ER酶）的cDNA，在体内和体外实验中，旨在阐明它们在Dol-P的从头合成和糖基载体脂质的再循环中的确切作用，以及3）纯化、鉴定和表征介导Man-P-Dol在哺乳动物细胞中横向扩散的ER蛋白（“翻转酶”）。获得的Man-P-Dol翻转酶结构信息将与介导蛋白质O-、C-和N-糖基化、甘油磷脂、葡糖神经酰胺和糖基磷脂酰肌醇（GPI）锚前体中其他长链-P-（P）-糖中间体跨双层运动的相关膜蛋白以及先天性糖基化障碍（CDG）患者的潜在缺陷相关。