Regulation of Protein N-Glycosylation in the CNS

CNS 中蛋白质 N-糖基化的调节

• 批准号: 7492043
• 负责人: Charles J Waechter
• 金额: $ 31.25万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-06-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7492043
• 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; Numbers; Pathway interactions; Patients; Phosphoric Monoester Hydrolases; Phosphotransferases; Principal Investigator; Procedures; Protein Dephosphorylation; Protein Overexpression; Proteins; Reaction; Recycling; Regulation; Role; Route; Sepharose; Series; Site-Directed Mutagenesis; Staging; Structure; System; Tissues; Water; analog; base; benzophenone; design; dolichol monophosphate; glycosylation; in vivo; mannose-phosphate-citronellol; monolayer; novel; programs; reconstitution; research study

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(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翻译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、细胞内路线和功能是必不可少的。蛋白质N-糖基化在人类中的重要性被归类为先天性糖基化障碍(CDG)的一系列遗传性遗传缺陷的临床后果所强调。虽然低聚糖供体Glc3Man9GlcNAc2-P-P-Dol在内质网(ER)中生物合成的酶学和拓扑结构已经得到了广泛的研究，但对于这一途径中的酶是如何组织和调节的，仍存在许多空白。这项建议描述了进一步的研究，以了解更多的内质网相关蛋白在调节DOL-P的生物合成和循环中的作用，以及Man-P-DOL的跨双层运动。 利用生化、遗传和免疫化学方法设计了三个特定的目标：1)利用最近克隆的c/S-异丙二烯转移酶(c/S-IPTase)，通过研究其调控以及它与内质网潜在结合伙伴的关系，进一步了解催化DOL-P生物合成延伸阶段的长链c/S-IPTase；2)利用克隆的DK和Dol-P-P磷酸酶(Dol-P-P phatase)的编码基因，在体内外实验中阐明它们在Dol-P的合成和糖基载体脂的循环中的精确作用；3)纯化、鉴定和鉴定介导man-P-Dol在哺乳动物细胞中横向扩散的ER蛋白(S)。获得的关于Man-P-Dol Flippase结构的信息将与相关的膜蛋白相关，这些膜蛋白介导了蛋白O-、C-和N-糖基化、甘油磷脂、葡萄糖神经酰胺和糖基磷脂酰肌醇(GPI)锚定前体和潜在缺陷的跨双层运动。