DOLICHOL PHOSPHATE METABOLISM AND GLYCOPROTEIN SYNTHESIS

磷酸多醇代谢和糖蛋白合成

• 批准号: 3289667
• 负责人: Charles J Waechter
• 金额: $ 20.64万
• 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 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289667
• 关键词: affinity chromatography; alcohol phosphotransferase; autoradiography; clone cells; developmental neurobiology; dolichol; embryo /fetus tissue /cell culture; enzyme mechanism; glycoprotein biosynthesis; glycoproteins; glycosylation; hexosyltransferase; laboratory rat; lipid biosynthesis; lipid transport; membrane proteins; phosphatidate phosphatase; protein purification; radiotracer; temperature sensitive mutant

Previous studies in this laboratory have established that dolichyl phosphate (Dol-P) plays a critical role as a glycosyl carrier lipid in the assembly of N-linked glycoproteins in brain, as in other mammalian tissues, and described several enzymes involved in Dol-P metabolism. This application proposes a continuation of the studies on the mechanistic details and regulation of Dol-P biosynthesis, and the factors controlling the rate of dolichol-linked oligosaccharide intermediate biosynthesis in brain. The major objectives are to: 1) continue developmental studies on the lipid intermediate pathway in cultured embryonic rat brain cells by correlating the developmental patterns for the induction of Dol-P- saccharide intermediate synthesis and for polyisoprenyl diphosphate synthase (long-chain cis-isoprenyltransferase) an activity required for the biosynthesis of Dol-P; 2) elucidate the enzymatic mechanism by which the alpha-isoprene unit of dolichol is reduced and identify the polyisoprenyl substrate and the reductant involved in the reaction; 3) select a temperature-sensitive mutant of dolichol kinase in cultured neuroblastoma cells by in situ colony autoradiography. The kinase-defective mutant will be studied to determine if the enzyme catalyzes the terminal step in the de novo biosynthesis of Dol-P or functions only in the phosphorylation of reserve pools of preformed dolichol during developmental periods when N- linked glycoproteins are actively synthesized; 4) determine if polyisoprenyl diphosphate synthase, polyisoprenyl diphosphate alpha- reductase or dolichol kinase are subject to feedback control by dolichol or Dol-P in cultured neuroblastoma cells; 5) utilize the (C10) citronellyl derivatives, Cit-P-Man and Cit-P, as water-soluble analogues of Dol-P-Man and Dol-P to investigate the potential role of membrane proteins in the transverse diffusion of the mannolipid intermediate between the cytoplasmic leaflet and the lumenal monolayer of the ER and 6) complete the purification of Dol-P phosphatase and dolichol kinase using polyisoprenylated Affi-Gel 10 as a new potential affinity support. The proposed experiments will extend the current understanding of Dol-P biosynthesis and the factors regulating dolichol-bound oligosaccharide biosynthesis in brain. The prospective studies with Cit-P-Man could provide a novel approach to characterize membrane proteins mediating the transbilayer movement of Dol-P-saccharides and Dol-P in brain and other mammalian cells.

该实验室之前的研究已经证实，多醇 磷酸盐 (Dol-P) 作为糖基载体脂质在 与其他哺乳动物组织一样，大脑中 N 连接糖蛋白的组装， 并描述了几种参与 Dol-P 代谢的酶。 这 申请建议继续进行机械研究 Dol-P生物合成的细节和调控以及控制因素 多萜醇连接的寡糖中间体生物合成的速率 脑。 主要目标是： 1) 继续进行发展研究 培养的胚胎大鼠脑细胞中的脂质中间途径 关联 Dol-P-诱导的发育模式 糖类中间体合成和聚异戊二烯基二磷酸酯 合成酶（长链顺式异戊二烯基转移酶）所需的活性 Dol-P 的生物合成； 2）阐明酶促机制 多醇的 α-异戊二烯单元被还原并鉴定出聚异戊二烯基 底物和参与反应的还原剂； 3) 选择一个 培养神经母细胞瘤中多萜醇激酶的温度敏感突变体 通过原位集落放射自显影法检测细胞。 激酶缺陷突变体将 进行研究以确定该酶是否催化 de 的最终步骤 Dol-P 的新生物合成或仅在磷酸化中发挥作用 当 N- 发育期间，预先形成的多醇储备池 连接的糖蛋白被主动合成； 4) 判断是否 聚异戊二烯二磷酸合酶，聚异戊二烯二磷酸α- 还原酶或多醇激酶受到多醇或多醇的反馈控制 培养的神经母细胞瘤细胞中的 Dol-P； 5)利用(C10)香茅基 衍生物，Cit-P-Man 和 Cit-P，作为 Dol-P-Man 的水溶性类似物 和 Dol-P 研究膜蛋白在 甘露糖脂中间体在细胞质之间的横向扩散 ER 的小叶和腔单层，6) 完成 使用Dol-P磷酸酶和dolichol激酶的纯化 聚异戊二烯化 Affi-Gel 10 作为新的潜在亲和支持物。 这 拟议的实验将扩展目前对 Dol-P 的理解 多萜醇结合寡糖的生物合成及调节因素 大脑中的生物合成。 Cit-P-Man 的前瞻性研究可以 提供了一种表征介导膜蛋白的新方法 Dol-P-糖和 Dol-P 在大脑和其他部位的跨双层运动 哺乳动物细胞。