LIPID LINKED SACCHARIDES IN GLYCOPROTEIN SYNTHESIS

糖蛋白合成中的脂联糖

• 批准号: 2414771
• 负责人: Alan D Elbein
• 金额: $ 18.68万
• 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/2414771
• 关键词: SDS polyacrylamide gel electrophoresis; active sites; enzyme activity; enzyme inhibitors; glycoprotein biosynthesis; glycosylphosphatidylinositols; 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.

大量的真核蛋白质锚定在质膜上 通过糖基磷脂酰肌醇（GPI）锚，其包括可变的 锥虫表面抗原、乙酰胆碱酯酶、海藻糖酶、5 '- 核苷酸酶、衰变加速因子等，动物细胞，以及 酵母和植物中的各种蛋白质。 虽然这其中的具体作用 锚定机制尚未建立，可能涉及GPI锚 在1）中。将蛋白质靶向至顶端或基底外侧表面，2）。信号 转导，3）。抗原变异，4）。促进横向 蛋白质在膜中的运动，5）。提供一种调节 蛋白质在细胞表面的表达。 至少一种人 阵发性睡眠性血红蛋白尿症（PNH）是由于无法 以合成GPI锚的聚糖部分，其对于 衰变加速因子和CD 59锚定在红细胞上 保护这些细胞免受补体的溶解作用。 GPI聚糖部分形成的生物合成途径 锚已经很好地建立，但没有一个糖基转移酶 还没有被纯化或鉴定。 我们处在一个很好的位置 为了纯化形成GlcNAc-PI的GlcNAc转移酶， 这种酶的快速和可靠的测定，我们已经准备了几个 UDP-GlcNAc的光亲和类似物。 因此，我们应该能够找到 SDS凝胶上的特异性催化蛋白。 这是一种酶， 在PNH失踪。 一旦我们纯化了这种酶，我们将准备 抗体和寡核苷酸探针。 与艾德叶博士合作， 我们将使用这些探针来确定是否在一个淋巴瘤病变， 不能产生GlcNAc-PI的细胞突变体在GlcNAc中 转移酶 我们还将与Rosse博士合作， PNH细胞中缺少酶。 我们也有一个很好的分析， 甘露糖基转移酶，并期望能够纯化该酶。 先前，我们确定了GPI中甘露糖胺的抑制位点， 在甘露糖基转移酶之一处的聚糖组装。 我们有几个新的 阻止甘露糖掺入GPI中间体的抑制剂 我们会确定他们的行动地点 我们还在体内建立了 并在体外筛选，以确定其他抑制剂， 以及它们如何抑制。 一旦我们有了纯化的转移酶， 将测试这些酶的抑制剂 GPI锚定 海藻糖酶已从猪肾中纯化至均一， 对这种酶的抗体。 因此，我们也可以筛选抑制剂， 该阻挡锚组件并因此防止海藻糖酶附着 到细胞表面。 这些抑制剂中的一种可以模拟 PNH缺陷，并允许我们建立这种疾病的模型系统。