COMPLEX CARBOHYDRATES: STRUCTURE, FUNCTION, SYNTHESIS

我们的长期目标是了解生物合成，调节和 膜糖蛋白的碳水化合物部分的功能。以来 N-连接和O-连接的寡糖被发现是许多 膜受体，如低密度脂蛋白、胰岛素和 乙酰胆碱，这些研究具有重要的意义，在各种 疾病状况，如动脉粥样硬化和糖尿病。 我们实现这些目标的战略将涉及多方面的办法。 我们将从猪主动脉中纯化几种我们认为的关键酶， 文献表明，可能是调节酶， N-连接寡糖的生物合成。纯化后， 同质性，抗体将针对蛋白质和寡核苷酸 将基于氨基末端或其它肽合成探针 序列的这些工具将使我们能够确定这些 酶及其mRNA在生长或发育的各个阶段， 正常动物或喂食高胆固醇饮食的动物。如果蛋白质在 主动脉平滑肌细胞与抗体交叉反应， 将在用各种抑制剂处理的这些细胞中研究酶。 这些研究将与抑制剂对 在培养的平滑肌细胞中形成脂质连接的寡糖。 另一种方法将是使用N-连接和O-连接的抑制剂， 寡糖的生物合成，以研究其形成和功能。我们有 几种新的N-连接寡糖加工抑制剂， 因为它们具有新的抑制特性， 提供有关结构-抑制关系的重要信息。在 目前还没有有用的O-连接寡糖抑制剂， 糖基转移酶因此，我们将开发一些快速的方法来寻找 这种抑制剂，并将测试一些抗体和其他糖苷 我们从各种渠道获得的信息。 最后，我们有令人信服的证据表明甘露糖-O-丝氨酸的存在 在培养物中生长的几种不同动物细胞系中的连接。我们将 确定将标记的甘露糖引入这些中的最佳条件， 蛋白质，然后做更详细的表征， 联动具有甘露糖基-丝氨酸的蛋白质将被纯化以 这样我们就可以制备抗体和寡核苷酸探针， 以确定蛋白质的位置和功能。的 将研究甘露糖基-丝氨酸的生物合成， 测定

Our long term goals are to understand the biosynthesis, regulation and function of the carbohydrate portion of membrane glycoproteins. Since N-linked and O-linked oligosaccharides are found as components of many membrane receptors, such as those for low density lipoprotein, insulin and acetylcholine, these studies have important implications in a variety of disease conditions such as atherosclerosis and diabetes. Our strategy to achieve these goals will involve a multifaceted approach. We will purify from pig aorta several key enzymes that we think, and the literature suggests, are likely to be regulatory enzymes in the biosynthesis of N-linked oligosaccharides. After purification to homogeneity, antibody will be made against the proteins and oligonucleotide probes will be synthesized based on the amino-terminal or other peptide sequences. These tools will enable us to determine the levels of these enzymes and their mRNAs at various stages of growth or development, and in normal animals or animals fed high cholesterol diets. If the proteins in aortic smooth muscle cells cross react with the antibodies, levels of enzymes will be studied in these cells treated with various inhibitors. These studies will be correlated with effects of inhibitors on the formation of lipid-linked oligosaccharides in cultured smooth muscle cells. Another approach will be to use inhibitors of N-linked and O-linked oligosaccharide biosynthesis to study formation and function. We have several new inhibitors of processing of N-linked oligosaccharides that we want to characterize since they have new inhibitory properties and will provide important information about structure-inhibition relations. At present there are no useful inhibitors of O-linked oligosaccharides or glycosyl transferases. Thus, we will develop some rapid methods to look for such inhibitors and will test a number of antibodies and other glycosides that we have obtained from various sources. Finally, we have compelling evidence for the presence of mannosyl-O-serine linkages in several different animal cell lines grown in culture. We will determine the optimum conditions for introducing labeled mannose into these proteins and then do more detailed characterization to establish this linkage. The protein(s) having the mannosyl-serine will be purified to homogeneity so that we can prepare antibody and oligonucleotide probes in order to establish the location and function of the protein. The biosynthesis of the mannosyl-serine will be studied and the mannosyl donor determined.