COMPLEX CARBOHYDRATES--STRUCTURE, FUNCTION, SYNTHESIS

复杂碳水化合物——结构、功能、合成

• 批准号: 2215072
• 负责人: Alan D Elbein
• 金额: $ 23.94万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1975
• 资助国家: 美国
• 起止时间: 1975-02-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2215072
• 关键词: N glycosidase; aminosugar; antibody formation; aorta; carbohydrate biosynthesis; carbohydrate structure; castanospermine; cross immunity; dolichol; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; glycolipids; glycoprotein biosynthesis; glycoprotein structure; glycoproteins; glycosyltransferase; liposomes; low density lipoprotein; mannose; membrane activity; membrane proteins; oligosaccharides; protein purification; protein structure function; receptor; serine; swine; tissue /cell culture; vascular smooth muscle

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.

我们的长期目标是了解生物合成、调控和 膜糖蛋白碳水化合物部分的功能。自从 N-连接和O-连接寡糖被发现是许多物质的组成部分 膜受体，例如低密度脂蛋白、胰岛素和 乙酰胆碱，这些研究在多种方面具有重要意义 动脉粥样硬化和糖尿病等疾病。 我们实现这些目标的战略将涉及多方面的方法。 我们将从猪主动脉中纯化出我们认为的几种关键酶，以及 文献表明，可能是调节酶 N-连接寡糖的生物合成。纯化后至 同质性，将针对蛋白质和寡核苷酸制备抗体 探针将根据氨基末端或其他肽合成 序列。这些工具将使我们能够确定这些的水平 生长或发育各个阶段的酶及其 mRNA，以及 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发生交叉反应， 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。 另一种方法是使用 N-连接和 O-连接的抑制剂 寡糖生物合成以研究形成和功能。我们有 我们发现了几种新的 N-连接寡糖加工抑制剂 想要表征，因为它们具有新的抑制特性并且会 提供有关结构抑制关系的重要信息。在 目前没有有用的 O-连接寡糖抑制剂或 糖基转移酶。因此，我们将开发一些快速方法来寻找 此类抑制剂并将测试许多抗体和其他糖苷 我们从各种来源获得的信息。 最后，我们有令人信服的证据证明甘露糖基-O-丝氨酸的存在 培养物中生长的几种不同动物细胞系中的连接。我们将 确定将标记甘露糖引入其中的最佳条件 蛋白质，然后进行更详细的表征以确定这一点 连锁。具有甘露糖基-丝氨酸的蛋白质将被纯化为 均质性，以便我们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。这 将研究甘露糖基丝氨酸的生物合成和甘露糖基供体 决定。