LIPID LINKED SACCHARIDES IN GLYCOPROTEIN SYNTHESIS

大量的真核蛋白锚定在质膜上。 通过糖基化磷脂酰肌醇(GPI)锚，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、衰变加速因子等，以及 酵母和植物中的各种蛋白质。尽管这一点的具体作用 锚定机制尚未建立，可能涉及GPI锚定 在1)中。将蛋白质定位于顶端或基底外侧表面，2)。讯号 转导，3)。抗原变异，4)。促进侧向 蛋白质在膜中的运动，5)。提供了一种手段来调节 蛋白质在细胞表面的表达。至少有一个人类 疾病，阵发性睡眠性血红蛋白尿(PNH)是由于 为了合成GPI锚点的多糖部分，这是 衰变加速因子和CD59锚定在红细胞上 表面保护这些细胞免受补体的溶解作用。 糖蛋白多糖部分形成的生物合成途径 锚已经被很好地建立了，但没有糖基转移酶 尚未被提纯或表征。我们处于一个非常有利的位置 为了纯化形成GlcNAc-PI的GlcNAc转移酶，因为我们有一个 快速、可靠地检测这种酶，我们已经准备了几种 UDP-GlcNAc的光亲和类似物。因此，我们应该能够定位 十二烷基硫酸钠上的特异性催化蛋白形成凝胶。这就是提出的酶 在PNH失踪。一旦我们提纯了这种酶，我们就会准备 抗体和寡核苷酸探针。与叶爱德博士合作， 我们将使用这些探针来确定其中一种淋巴瘤的病变 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，确定 PNH细胞中缺少酶。我们也有一个很好的第一个测试 甘露糖基转移酶，并有望提纯该酶。 此前，我们确定了甘露糖胺在GPI中的抑制部位 在甘露糖基转移酶中的一种上组装多糖。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 并在体外筛选以识别其他抑制剂并确定在哪里 以及它们是如何抑制的。一旦我们有纯化的转移酶可用，我们 将测试这些酶上任何已证实的抑制剂。GPI锚定在 海藻糖酶已经从猪肾中纯化成均一组分，我们有 抗这种酶的抗体。因此，我们还可以筛选出抑制物 从而防止海藻糖酶附着 到细胞表面。这些抑制剂中的一种可能会模拟 PNH缺陷，为建立该病的模型系统奠定了基础。

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.