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（由申请人提供）：许多重要的细胞表面受体，离子通道和溶酶体酶的共转换N-糖基化对于它们在中枢神经系统和其他哺乳动物细胞中的正确折叠，细胞内路由和功能至关重要。在该途径中，一系列遗传遗传缺陷的临床后果强调了蛋白质N-糖基化在人类中的至关重要性。虽然内质网（ER）中Glc3Man9GlCNAC2-P-P-DOL的生物合成的酶学和拓扑结构已进行了广泛的研究，但在这种途径中如何组织和调节了该途径中的酶的理解。该建议描述了进一步的研究，以了解更多有关ER相关蛋白在调节磷酸二甲基磷酸（DOL-P）生物合成和回收的作用以及人类P-DOL的跨贝贝运动中的作用。设计三个特定目标是使用设计的生化，遗传和免疫化学方法来计划的：1）利用最近克隆的cDNA，以更多地了解有关长链C/S-异源性转移酶（C/S-stiptase）的长链C/s-异源基因基转移酶（C/S-stipass），从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表征介导哺乳动物细胞中MAN-POL的横向扩散的ER蛋白（“ Flippase”）。获得的有关MAN-POL FLIPPase结构的信息将与介导其他多甲基 - P-（P） - 糖类中间体在蛋白O-，C-和N-糖基中的中间体的交通信号蛋白有关糖基化先天性疾病（CDG）患者的潜在缺陷。