GLYCOLIPID AND GLYCOPROTEIN METABOLISM IN EYE TISSUE

眼组织中的糖脂和糖蛋白代谢

• 批准号: 2157903
• 负责人: EDWARD L KEAN
• 金额: $ 33.41万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2157903
• 关键词: Golgi apparatus; RNase protection assay; carbohydrate structure; chick embryo; cow; dolichol; electron microscopy; enzyme activity; galactosyltransferases; glycolipids; glycoprotein biosynthesis; immunoaffinity chromatography; immunocytochemistry; isozymes; laboratory mouse; laboratory rat; light microscopy; lipid metabolism; messenger RNA; oligosaccharides; recombinant DNA; retina; retinoid binding proteins; rhodopsin; rod cell

The oligosaccharide chains of bovine rhodopsin are unique when compared to other glycoproteins in that they are abridged in size and are hybrids of high mannose and complex types. We are investigating mechanisms used by the retina that give rise to this situation, i.e., what controls are present such that galactose and additional GlcNAc residues, that would be foci for oligosaccharide chain extension reactions, are not found (except in trace amounts) in mature rhodopsin. We shall extend our previous investigations on the kinetics of the galactosyltransferases that demonstrated the low efficiency of the retina enzyme toward intact rhodopsin and opsin. We now will use oligosaccharides and glycopeptides prepared from rhodopsin to explore the influence of the protein matrix on these reactions. A reference compound for these studies will be the interphotoreceptor retinoid binding protein (IRBP), a naturally galactosylated glycoprotein of the retina. Galactosyltransferases purified from bovine retina, from rat liver Golgi, and from milk will be examined. We shall investigate by immunocytochemistry the localization of galactosyltransferase in the photoreceptor cell using both light and electron microscopy. Using the techniques of molecular biology we shall investigate whether there are differences in the relative amounts of the mRNAs that code for the long and short forms of galactosyltransferase in the retina and the photoreceptor cell. Are these different isoforms differentially located in the photoreceptor cell; is the isoform found in the retina concerned mainly with surface recognition in contrast to biosynthetic reactions? Investigations will be carried out concerning control by the retina of GlcNAc-transferase II, an enzyme that could add a GlcNAc to the alpha(1 - > 6) mannose arm of the rhodopsin oligosaccharides and thus be a site for chain extension. The kinetics of this reaction, using rhodopsin, opsin, rhodopsin oligosaccharides and glycopeptides as acceptors, will be examined using the enzyme obtained by recombinant DNA techniques. We shall also examine for the presence in the retina of galactosidase and hexosaminidase activity toward pertinent analogs of rhodopsin. We shall attempt the isolation of bovine photoreceptor cells by immunoaffinity chromatography. The structure of the oligosaccharides of human rhodopsin will be investigated. We shall continue our investigations concerning control of the initiating reactions of the dolichol pathway in the retina. We shall examine the influence on the allosteric activation by dolichol-P-mannose of GlcNAc- lipid synthesis (a phenomenon which we have discovered) by other key intermediates of the pathway. The findings of mutations in the rhodopsin gene (some in sites controlling glycosylation) in patients with retinitis pigmentosa have highlighted the importance of understanding the chemistry and biochemistry of the glycosylation of rhodopsin. The investigations concerning control of the dolichol pathway may further our basic understanding of the regulation of glycoprotein biosynthesis.

比较时，牛Rhodopsin的寡糖链是独一无二的 到其他糖蛋白，因为它们的尺寸缩小并且是杂种 高甘露糖和复杂类型。 我们正在调查使用的机制 通过引起这种情况的视网膜，即哪些控制是什么 目前是半乳糖和其他GlcNAC残基，这将 找不到寡糖链扩展反应的焦点 （痕量除外）在成熟的视紫红质中。 我们将延长我们的 先前对半乳糖基转移酶动力学的研究 这表明视网膜酶朝着完整的效率低 Rhodopsin和Opsin。 现在，我们将使用寡糖和糖肽 从视紫红质制备以探索蛋白质基质的影响 关于这些反应。 这些研究的参考化合物将是 球型维生素类维生素类似结合蛋白（IRBP），一种天然 视网膜的半乳糖化糖蛋白。 半乳糖基转移酶 从牛视网膜，大鼠肝高尔基体和牛奶中纯化 检查。 我们将通过免疫细胞化学调查定位 使用光和光感受器细胞中的半乳糖基转移酶 电子显微镜。 使用分子生物学的技术，我们将 调查相对量是否存在差异 mRNA代码为长而短的半乳糖基转移酶中的密码 视网膜和感光细胞。 这些不同的同工型 差异位于感光细胞中；是找到的同工型 在视网膜中，主要与表面识别有关 生物合成反应？ 将对视网膜控制的调查 GlcNAC-转移酶II，一种可以在α上添加GlcNAC的酶（1 - > 6）视紫红质寡糖的甘露糖臂，因此是一个地点 链扩展。 使用Rhodopsin，Opsin，该反应的动力学， 视紫红质寡糖和糖肽作为受体将是 使用通过重组DNA技术获得的酶检查。 我们 还应检查半乳糖苷酶视网膜的存在和 己糖胺酶对视紫红质相关类似物的活性。 我们将 尝试通过免疫亲和力分离牛感光细胞 色谱法。 人视紫红质寡糖的结构 将被调查。 我们将继续有关控制启动的调查 视网膜中Dolichol途径的反应。 我们将检查 多利希尔 - p-甘露糖的变构激活的影响 其他钥匙的脂质合成（我们发现的现象） 路径的中间体。 视紫红质基因中突变的发现（某些位点 在色素性视网膜炎患者中控制糖基化） 强调了理解化学和 视紫红质糖基化的生物化学。 调查 关于对Dolichol途径的控制可能会进一步进一步 了解糖蛋白生物合成的调节。