DOLICHOL PHOSPHATE METABOLISM AND GLYCOPROTEIN SYNTHESIS

本实验室先前的研究已经确定， 磷酸（Dol-P）作为糖基载体脂质在细胞内起关键作用。 在脑中组装N-连接的糖蛋白，如在其他哺乳动物组织中， 并描述了参与Dol-P代谢的几种酶。 这 应用程序建议继续研究的机制 Dol-P生物合成的细节和调节，以及控制因素 长轴醇连接寡糖中间体生物合成速率 个脑袋 主要目标是：1）继续发展研究， 培养的胚胎大鼠脑细胞中脂质中间体途径 将发育模式与Dol-P- 糖中间体合成和用于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）的活性所需的 Dol-P的生物合成; 2）阐明酶促机制， 长萜醇的α-异戊二烯单元被还原并鉴定聚异戊二烯基 底物和参与反应的还原剂; 3）选择一种 培养神经母细胞瘤中多萜醇激酶温度敏感突变体 原位集落放射自显影。 激酶缺陷突变体将 研究以确定酶是否催化降解过程中的末端步骤。 Dol-P的重新生物合成或仅在 在发育期间，当N- 连接的糖蛋白被活跃地合成; 4）确定是否 聚异戊二烯基二磷酸合酶，聚异戊二烯基二磷酸α- 还原酶或多萜醇激酶受到多萜醇的反馈控制， Dol-P在培养的神经母细胞瘤细胞中的作用; 5）利用（C10）香茅基 作为Dol-P-Man的水溶性类似物的衍生物Cit-P-Man和Cit-P 和Dol-P来研究膜蛋白在细胞凋亡中的潜在作用。 甘露糖脂中间体在细胞质之间的横向扩散 小叶和ER的内腔单层，以及6）完成 使用纯化Dol-P磷酸酶和多萜醇激酶 聚异戊二烯化的Affi-Gel 10作为新的潜在亲和载体。 的 提出的实验将扩展目前对Dol-P的理解 结合寡糖的生物合成及其调控因素 脑内的生物合成 Cit-P-Man的前瞻性研究可能 提供了一种新的方法来表征膜蛋白介导的 Dol-P-ATP和Dol-P在脑和其他组织中的跨双层运动 哺乳动物细胞

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.