LIPID LINKED SACCHARIDES IN GLYCOPROTEIN SYNTHESIS

糖蛋白合成中的脂联糖

• 批准号: 2137620
• 负责人: Alan D Elbein
• 金额: $ 17.88万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2137620
• 关键词: SDS polyacrylamide gel electrophoresis; active sites; enzyme activity; enzyme inhibitors; glycoprotein biosynthesis; glycosyltransferase; high performance liquid chromatography; mannose; mutant; nucleic acid probes; oligonucleotides; paroxysmal nocturnal hemoglobinuria; protein purification; swine

A large number of eucaryotic proteins are anchored in the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor including the variable surface antigen of Trypanosomes, acetylcholinesterase, trehalase, 5'- nucleotidase, decay accelerating factor, etc., of animal cells, and various proteins in yeast and plants. Although the specific role of this anchoring mechanism has not been established, GPI anchors may be involved in 1). Targeting proteins to apical or basolateral surfaces, 2). Signal transduction, 3). Antigenic variation, 4). Facilitating the lateral movement of proteins in the membrane, 5). Providing a means to modulate the expression of proteins at the cell surface. At least one human disease, paroxysmal nocturnal hemoglobinuria (PNH) is due to an inability to synthesize the glycan portion of the GPI anchor which is essential for decay accelerating factor and CD59 to be anchored at the erythrocyte surface to protect these cells from the lytic action of complement. The biosynthetic pathway for the formation of the glycan portion of GPI anchors has been well established, but none of the glycosyltransferases has yet been purified or characterized. We are in an excellent position to purify the GlcNAc transferase that forms GlcNAc-PI since we have a rapid and reliable assay for this enzyme, and we have prepared several photoaffinity analogs of UDP-GlcNAc. Thus, we should be able to locate the specific catalytic protein on SDS gels. This is the enzyme proposed to be missing in PNH. Once we have this enzyme purified, we will prepare antibodies and oligonucleotide probes. In collaboration with Dr. Ed Yeh, we will use these probes to determine if the lesion in one of the lymphoma cell mutants that are unable to make GlcNAc-PI is in the GlcNAc transferase. We will also collaborate with Dr. Rosse to identify the missing enzyme in PNH cells. We also have a good assay for the first mannosyltransferase and expect to be able to purify this enzyme. Previously, we identified the site of inhibition of mannosamine in GPI glycan assembly at one of the mannosyltransferases. We have several new inhibitors that prevent mannose incorporation into the GPI intermediates and we will determine their site of action. We have also set up in vivo and in vitro screens to identify other inhibitors and to determine where and how they inhibit. Once we have purified transferases available, we will test any demonstrated inhibitors on these enzymes. The GPI anchored trehalase has been purified to homogeneity from pig kidney and we have antibody against this enzyme. Thus, we can also screen for inhibitors that block anchor assembly and therefore prevent attachment of trehalase to the cell surface. One of these inhibitors may mimic the effect of the PNH defect and allow us to set up a model system of this disease.

大量真核蛋白质锚定在质膜上 通过糖基磷脂酰肌醇 (GPI) 锚定，包括变量 锥虫表面抗原、乙酰胆碱酯酶、海藻糖酶、5'- 动物细胞的核苷酸酶、腐烂加速因子等， 酵母和植物中的各种蛋白质。 虽然这个的具体作用 锚定机制尚未建立，可能涉及GPI锚定 在1）中。将蛋白质靶向顶端或基底外侧表面，2)。信号 转导，3)。抗原变异，4)。促进横向 膜中蛋白质的运动，5)。提供调节手段 细胞表面蛋白质的表达。 至少一名人类 阵发性睡眠性血红蛋白尿症 (PNH) 是由于无法 合成 GPI 锚的聚糖部分，这对于 腐烂加速因子和 CD59 锚定在红细胞上 表面以保护这些细胞免受补体的裂解作用。 GPI 聚糖部分形成的生物合成途径 锚点已经很好地建立，但是没有一个糖基转移酶 尚未被纯化或表征。 我们处于有利地位 纯化形成 GlcNAc-PI 的 GlcNAc 转移酶，因为我们有 对该酶进行快速可靠的测定，我们准备了几种 UDP-GlcNAc 的光亲和类似物。 因此，我们应该能够找到 SDS 凝胶上的特定催化蛋白。 这是提议的酶 在 PNH 中失踪。 一旦我们纯化了这种酶，我们就可以准备 抗体和寡核苷酸探针。 与Ed Yeh博士合作， 我们将使用这些探针来确定病变是否属于淋巴瘤之一 不能产生 GlcNAc-PI 的细胞突变体位于 GlcNAc 中 转移酶。 我们还将与 Rosse 博士合作，确定 PNH 细胞中缺少酶。 我们也有一个很好的第一个分析 甘露糖基转移酶并期望能够纯化该酶。 之前，我们确定了 GPI 中甘露糖胺的抑制位点 在一种甘露糖基转移酶处进行聚糖组装。 我们有几个新的 防止甘露糖掺入 GPI 中间体的抑制剂 我们将确定他们的行动地点。 我们在vivo也设置了 和体外筛选，以确定其他抑制剂并确定在哪里 以及它们如何抑制。 一旦我们有了纯化的转移酶，我们 将测试这些酶的任何已证实的抑制剂。 GPI锚定 海藻糖酶已从猪肾中纯化至同质，我们有 针对这种酶的抗体。 因此，我们还可以筛选抑制剂 阻止锚固组件，从而防止海藻糖酶的附着 到细胞表面。 这些抑制剂之一可能会模仿 PNH 缺陷使我们能够建立这种疾病的模型系统。