LIPID-LINKED SACCHARIDES IN GLYCOPROTEIN SYNTHESIS

N-连接糖蛋白是许多真核细胞中的重要受体， 这些类型的分子与各种识别 包括细胞-细胞相互作用、宿主-寄生物和宿主-共生体的现象 相互作用，以及许多生物重要分子的结合。 因此，这些类型的糖蛋白可能在这种疾病中具有特别重要的意义。 疾病状况如癌症、动脉粥样硬化和糖尿病。 主要 本提案的目的是了解生物合成的机制 和加工这些N-连接的 糖蛋白 在这些研究中采取的方法是双重的。 在 在第一种情况下，我们将溶解尽可能多的糖基转移酶， 尽可能地加工糖苷酶，在一定程度上纯化它们，并研究 他们的财产。 我们将特别关注 关于所使用的底物和所形成的产物的性质 在每个反应中。 在初步研究中，我们已经发现， 合成脂质连接低聚糖的活性可以是 通过用NP-40处理大豆细胞膜来溶解。 我们也有 脂质连接的寡糖用作这些酶的受体， 我们可以用可溶性的形式来分析它们。 因此，我们准备继续进行 一些净化研究。 我们还检测到葡萄糖苷酶， 甘露糖苷酶活性的溶解级分，并可以测定这些 酶和寡糖底物。 所以我们 应该也可以在这里进行提纯。 这些酶将 特别感兴趣的是， 利用低聚糖。 在第二种方法中，我们将提取 各种野生植物生长在得克萨斯州，并被称为是有毒的， 家畜，寻找糖蛋白加工的抑制剂。 这种想法是 基于发现苦马豆素，一种溶酶体的抑制剂， α-甘露糖苷酶和糖蛋白加工，是发现在几个有毒的 植物，即，澳大利亚植物苦马豆和美国 疯草 由于苦马豆素具有3个不对称的羟基， 其他种类的疯草，或其他植物， 苦马豆素的立体异构体，其可抑制其它糖苷酶。

N-linked glycoproteins are important receptors in many eucaryotic cells, and these types of molecules have been implicated in various recognition phenomena including cell-cell interactions, host-parasite and host-symbiote interactions, and the binding of many biologically-important modlecules. Thus, these types of glycoproteins may be of especial significance in such disease conditions as cancer, atherosclerosis and diabetis. The major objective of this proposal is to understand the mechanism of biosynthesis and processing of the oligosaccharide portion of these N-linked glycoproteins. The approach to be taken in these studies is twofold. In the first case, we will solubilize as many of the glycosyl transferases and processing glycosidases as possible, purify them to some extent, and study their properties. We will be particularly concerned with the specificity with regard to substrate utilized and to the nature of the product formed in each reaction. In preliminary studies, we have already found that some of the activity for synthesizing the lipid-linked oligosaccharides can be solubilized by treating soybean cell membranes with NP-40. We also have lipid-linked oligosaccharides to use as acceptors with these enzymes so that we can assay them in soluble form. Thus we are ready to proceed with some purification studies. We have also detected glucosidase and mannosidase activities in the solubilized fraction and can assay these enzymes with oligosaccharide substrates that we have available. So we should also be able to proceed with purification here. These enzymes will be of special interest with regard to substrate specificity of the oligosaccharides utilized. In the second type of approach, we will extract various wild plants that grow in Texas, and are known to be toxic for livestock, to look for inhibitors of glycoprotein processing. This idea is based on the finding that swainsonine, an inhibitor of lysosomal Alpha-mannosidase and glycoprotein processing, is found in several toxic plants, i.e., the australian plant Swainsona canescens and american locoweed. Since swainsonine has 3 assymetric hydroxyl groups, it seems likely that other species of locoweed, or other plants, will have stereoisomers of swainsonine which may inhibit other glycosidases.