DOLICHOL PHOSPHATE METABOLISM AND GLYCOPROTEIN SYNTHESIS

磷酸多醇代谢和糖蛋白合成

• 批准号: 2178205
• 负责人: Charles J Waechter
• 金额: $ 21.49万
• 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/2178205
• 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.

该实验室的先前研究已经确定 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， 并描述了参与DOL-P代谢的几种酶。 这 应用提出了关于机械的研究的延续 DOL-P生物合成的细节和调节，以及控制的因素 多醇连接的寡糖中间生物合成速率 脑。 主要目标是：1）继续有关 培养的胚胎大鼠脑细胞中的脂质中间途径 将诱导DOL-P-的发育模式相关联 糖中间合成和聚异源二磷酸 合成酶（长链顺式 - 异源转移酶）一种活性 DOL-P的生物合成； 2）阐明酶机制 降低了多利果醇的α-异戊二烯单位，并识别多异丙肾 反应涉及的底物和还原剂； 3）选择 培养的神经母细胞瘤中多利果醇激酶的温度敏感突变体 通过原位菌落放射自显影的细胞。 激酶缺陷突变体将 研究以确定酶是否催化DE中的末端步骤 DOL-P或功能仅在磷酸化中的NOVO生物合成 n- 链接的糖蛋白被积极合成； 4）确定是否 聚异源二磷酸合酶，聚异源二磷酸α- 还原酶或多甲醇激酶会受到多利匹醇的反馈控制或 DOL-P在培养的神经母细胞瘤细胞中； 5）利用（C10）Citronellyl CIT-P-MAN和CIT-P的衍生物，作为DOL-P-MAN的水溶性类似物 和DOL-P研究膜蛋白在 细胞质之间中间体的横向扩散 传单和ER的Lumenal单层，6）完成 使用使用DOL-P磷酸酶和Dolichol激酶纯化 聚异源肾上腺素化的亲属10作为新的潜在亲和力支持。 这 提出的实验将扩展对DOL-P的当前理解 生物合成及调节dolichol结合寡糖的因素 大脑中的生物合成。 与CIT-P-Man的前瞻性研究可以 提供一种新颖的方法来表征介导的膜蛋白 DOL-P-糖和DOL-P在大脑和其他中的DOL-P-糖和DOL-P的转移运动 哺乳动物细胞。