GLYCOLIPID AND GLYCOPROTEIN METABOLISM IN EYE TISSUE

眼组织中的糖脂和糖蛋白代谢

• 批准号: 2157904
• 负责人: EDWARD L KEAN
• 金额: $ 34.92万
• 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 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2157904
• 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.

牛视紫红质的寡糖链是独特的， 与其他糖蛋白相比，它们的大小被缩短， 高甘露糖和复杂类型。 我们正在调查 引起这种情况的视网膜，即，什么是控制 存在半乳糖和另外的GlcNAc残基， 是寡糖链延伸反应的焦点，未发现 （除微量外）在成熟的视紫红质。 我们将扩大我们的 半乳糖基转移酶动力学的先前研究 这证明了视网膜酶对完整的 视紫红质和视蛋白。 我们现在用寡糖和糖肽 从视紫红质制备，以探讨蛋白质基质的影响 这些反应。 这些研究的参考化合物将是 光感受器间维甲酸结合蛋白（IRBP），一种天然的 视网膜的半乳糖化糖蛋白。 半乳糖基转移酶类 从牛视网膜、大鼠肝脏高尔基体和牛奶中纯化， 考察 我们将通过免疫细胞化学研究 半乳糖基转移酶在感光细胞使用光和 电镜 利用分子生物学技术， 调查是否有差异的相对数量的 基因编码的长和短形式的半乳糖基转移酶， 视网膜和感光细胞。 这些不同的亚型 差异定位在感光细胞;是同种型发现 在视网膜中主要涉及表面识别， 生物合成反应？ 将进行关于视网膜控制的研究， GlcNAc-转移酶II，一种可以将GlcNAc添加到α（1 - > 6）视紫红质低聚糖的甘露糖臂，因此可以是 扩链 这个反应的动力学，使用视紫红质，视蛋白， 视紫红质寡糖和糖肽作为受体， 使用通过重组DNA技术获得的酶进行检测。 我们 还应检查视网膜中半乳糖苷酶的存在， 氨基己糖苷酶对视紫红质相关类似物的活性。 我们将 尝试用免疫亲和法分离牛感光细胞 层析 人视紫红质寡糖的结构 将进行调查。 我们将继续调查有关控制启动 在视网膜中的多萜醇途径的反应。 我们将研究 甘露糖对GlcNAc-β-甘露醇变构激活的影响 脂质合成（我们已经发现的一种现象）通过其他关键 途径的中间体。 视紫红质基因突变的发现（一些位点 控制糖基化）在视网膜色素变性患者中具有 强调了理解化学的重要性， 视紫红质糖基化的生物化学。 调查 关于控制多萜醇途径的研究可能会进一步促进我们的基础研究， 了解糖蛋白生物合成的调控。