MOLECULAR BIOLOGY OF ASPARAGINE LINKED GLYCOSYLATION

这项建议的目标是了解 仓鼠谷氨酸氨基转移酶(GPT)。谷丙转氨酶催化转运蛋白 GlcNAc-1-P由UDP-GlcNAc转变为Dolicol-P，形成GlcNAc-P-P-Dolicol。 然后将GlcNAc-PP-DOLICHOL转化为寡糖-PP-DOLICHOL (含14种糖)，N-连接的葡聚糖的直接前体 真核生物。N-连接的葡聚糖在广泛的功能中是重要的， 如细胞黏附、细胞器运输和蛋白质折叠，以及 与癌症和人类疾病有关，如HEMPAS，CGDS， 和i-细胞疾病。最近的氨基酸序列分析表明 仓鼠GPT具有一个类似的真核和原核糖-1-P家族 以UDP-GlcNAc或UDP-MurNAc-五肽为供体的转移酶 和作为受体的聚异戊二烯基磷酸酯。 至关重要的是，GPT活动应在狭义的 窗户。GPT活性太低会阻止N-连接的足够数量 多聚糖不能生产。GPT活动过多导致过度 GlcNAc-P-对苯二醇合成与反应的竞争 合成甘露糖-对-二醇(MPD)和葡萄糖-对-二醇(GPD)。 MPD和GPD不足会导致N-连接糖基化异常和 缺陷糖基磷脂酰肌醇(GPI)锚定合成。 此外，最近的数据表明，未折叠的蛋白质与 特异性识别Glc1Man9GlcNAc2的伴侣蛋白calnexin， 都会被抑制。 提出了五个具体目标，以解决以下问题 仓鼠GPT：(1)所发现的保守序列有哪些功能 在UDP-GlcNAc/MurNAc转移酶家族成员中？(2)什么 确定不同糖供体和脂类受体的特异性 UDP-GlcNAc/MurNAc糖基转移酶家族？(3)而不是 调节二羟基二苯醚的结合，做了“潜在的二羟基胆碱识别 序列“(PDR)促进一种酶的复合体，这些酶在早期起作用 Dolicol途径？(4)GPT是如何被其序列特异性稳定的， 二分C-末端？(5)GPT的拓扑方向是什么 急诊室膜？这些问题的答案不仅将提供新的 关于谷丙转氨酶及其在N-连接糖基化中的作用的基本信息， 但也将对许多结构上的 相关蛋白质。

The goal of this proposal is to understand the structure and function of hamster GlcNAc-1-P transferase (GPT). GPT catalyzes the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichol-P to form GlcNAc-P-P-Dolichol. GlcNAc-PP-dolichol is then converted into oligosaccharide-PP-dolichol (with 14 sugars), the immediate precursor for N-linked glycans in eukaryotes. N-linked glycans are important in a broad range of functions, such as cell adhesion, organellar trafficking, and protein folding, and have been implicated in cancer and human diseases such as HEMPAS, CGDS, and I-cell disease. Recent amino acid sequence analyses have linked hamster GPT with a family of similar eukaryotic and prokaryotic sugar-1-P transferases which use UDP-GlcNAc or UDP-MurNAc-pentapeptide as a donor and a polyisoprenyl phosphate as an acceptor. It is essential that GPT activity be regulated within a narrowly defined window. Too little GPT activity prevents sufficient quantities of N-linked glycans from being produced. Too much GPT activity causes excessive competition between GlcNAc-P-P-dolichol synthesis and reactions synthesizing mannose-P-dolichol (MPD) and glucose-P-dolichol (GPD). Insufficient MPD and GPD results in abnormal N-linked glycosylation and defective glycosylphosphatidylinositol (GPI) anchor synthesis. Furthermore, recent data indicate that binding of unfolded proteins to the ER chaperone calnexin, which specifically recognizes Glc1Man9GlcNAc2, would be inhibited. Five specific aims are proposed to address the following questions about hamster GPT: (1) What are the functions of the conserved sequences found in members of the UDP-GlcNAc/MurNAc transferase family? (2) What determines specificity for different sugar donors and lipid acceptors in the UDP-GlcNAc/MurNAc family of glycosyltransferases? (3) Rather than mediating bind of dolichol, does the "potential dolichol recognition sequence" (PDRS) promote a complex of enzymes which act early in the dolichol pathway? (4) How is GPT stabilized by its sequence-specific, bipartite C-terminus? (5) What is the topological orientation of GPT in the ER membrane? The answers to these questions will not only provide new fundamental information about GPT and its role in N-linked glycosylation, but will also have direct implications for a number of structurally related proteins.