DOLICHOL PHOSPHATE METABOLISM AND GLYCOPROTEIN SYNTHESIS

这个实验室之前的研究已经证实，多利基 磷酸盐(Dol-P)作为糖基载体脂类在体内起着至关重要的作用。 N连接的糖蛋白在脑中的组装，如在其他哺乳动物组织中一样， 并描述了参与Dol-P代谢的几种酶。这 应用提出了对机构学研究的继续 DOL-P生物合成的细节、调控及其控制因素 二醇连接低聚糖中间体生物合成的速率 大脑。主要目标是：1)继续开展发展研究 体外培养大鼠胚胎脑细胞脂质中间途径的研究 Dol-P诱导的发育模式 糖类中间体的合成及聚异戊二烯基二磷酸 合成酶(长链顺式异戊二烯转移酶) Dol-P的生物合成；2)阐明了Dol-P的酶合成机制 二元醇的α-异戊二烯基团被还原并鉴定为聚异戊二烯 底物和参与反应的还原剂；3)选择 培养的神经母细胞瘤中温度敏感型多巴胆碱激酶突变体 细胞原位集落放射自显影。有激酶缺陷的突变体会 以确定该酶是否催化脱氧核糖核酸的末端步骤 Dol-P的新生物合成或仅在磷酸化中起作用 在发育期间预制的多元醇的储备池 连接的糖蛋白被活跃地合成；4)确定 聚异戊二磷酸合成酶，聚异丙二磷酸 还原酶或Dolicol Kinase受Dolicol或Dolicholk反馈控制 DOL-P在培养神经母细胞瘤细胞中的作用；5)利用(C10)香茅基 Cit-P-Man和Cit-P作为Dol-P-Man的水溶性类似物 和Dol-P研究膜蛋白在细胞周期中的潜在作用。 甘露糖脂中间体在胞质间的横向扩散 叶和内质网的管腔单层和6)完成 Dol-P磷酸酶和Dol-ol-Kinase的纯化 聚异戊二烯基化的Affi-Gel 10作为一种新的潜在的亲和力载体。这个 拟议的实验将扩展目前对DOL-P的理解 二醇结合低聚糖的生物合成及其调控因素 大脑中的生物合成。Cit-P-Man的前瞻性研究可能 提供了一种新的方法来表征介导膜蛋白。 Dol-P-糖和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.