GLYCOPROTEIN SYNTHESIS AND METABOLISM IN RETINAS

该项目的长期目标是进一步确定 糖蛋白合成和代谢的生物学意义 在正常和病理条件下的视网膜中。 这 提案描述了进一步调查的研究计划， 视杆细胞外节膜N-糖基化的作用 蛋白质在椎间盘膜形态发生过程中的作用。 这些 研究主要基于P.I.的实验室 涉及N-连接寡糖抑制剂的作用 生物合成和翻译后加工 两栖动物视网膜的体外形态发生。 工作 假设视紫红质的寡糖是必需的， 在同型或异型机制中的配体， 蛋白质-碳水化合物键合相互作用，其中蛋白质可以 是另一种视紫红质分子，一种利用 寡糖作为底物（例如，糖基转移酶或 糖苷酶）或凝集素。 假设将被检验为 具体如下：1）两栖类视紫红质寡糖的测定 组成和结构（推定的 必需配体）; 2）确定数量、位置和 两栖类碳水化合物附着位点的氨基酸序列 视紫红质; 3）评价潜在的寡糖结构 质膜中的视紫红质与成熟视紫红质之间的差异 ROS盘; 4）评价外源性 已知组成和结构的寡糖 形态发生; 5）评价凝集素和N- 末端定向抗视紫红质抗体对椎间盘形态发生的影响; 6)评估是否存在和分布 7）评估ROS中半乳糖基转移酶的存在和 内源性凝集素在ROS中的分布， 感光细胞间基质 这些研究将涉及现代 碳水化合物和蛋白质生化方法， 光学和电子显微镜和放射自显影， 免疫荧光和免疫细胞化学。 的潜在 这项研究与某些人类遗传性失明的相关性 研究结果（由P.I.和 合作者），这两个刻板的视网膜发育不良和 光感受器退化可以在动物实验中诱导 通过抑制生物合成中的酶， N-连接寡糖的合成途径。 等 处理导致ROS盘膜的异常组装， 体外以及体内ROS更新的停止。 这些发现 这表明一个或多个基因缺陷的可能性， 参与N-连接寡糖生物合成的酶 可能在某些遗传性视网膜病变的病因学中具有重要意义。 发育不良或视网膜变性。

The long-range objective of this project is to further define the biological significance of glycoprotein synthesis and metabolism in the retina, both in normal and pathological conditions. This proposal describes research plans for further investigations of the role of N-glycosylation of rod outer segment (ROS) membrane proteins in the process of disc membrane morphogenesis. These studies are largely based on previous studies in the P.I.'s lab involving the effect of inhibitors of N-linked oligosaccharide biosynthesis and post-translational processing on disc morphogenesis in amphibian retinas in vitro. The working hypothesis is that rhodopsin's oligosaccharides are essential ligands in either homotypic or heterotypic mechanisms involving protein-carbohydrate bonding interactions, where the protein may be either another rhodopsin molecule, an enzyme which utilizes oligosaccharides as substrates (e.g., a glycosyltransferase or a glycosidase), or a lectin. The hypothesis will be tested as follows: 1) determination of amphibian rhodopsin oligosaccharide composition and structure (characterization of the presumed essential ligands); 2) determination of the number, location, and amino acid sequence of carbohydrate attachment sites of amphibian rhodopsins; 3) evaluation of potential oligosaccharide structural differences between rhodopsins in the plasma membrane vs. mature ROS discs; 4) evaluation of the effect of exogenous oligosaccharides of known composition and structure on disc morphogenesis; 5) evaluation of the effect of lectins and N- terminal directed anti-rhodopsin antibodies on disc morphogenesis; 6) evaluation of the presence and distribution of galactosyltransferase in the ROS; 7) evaluation of the presence and distribution of endogenous lectins in the ROS and interphotoreceptor matrix. These studies will involve modern carbohydrate and protein biochemical methods, with correlative light and electron microscopy and autoradiography, immunofluorescence and immunocytochemistry. The potential relevance of this research to certain human hereditary blinding disorders is suggested by the finding (obtained by the P.I. and collaborators) that both a stereotypical retinal dysplasia and a photoreceptor degeneration can be induced experimentally in animals by pharmacologically inhibiting an enzyme in the biosynthetic pathway by which N-linked oligosaccharides are made. Such treatment results in aberrant assembly of ROS disc membranes in vitro as well as cessation of ROS renewal in vivo. These findings suggest the possibility that genetic defects in one or more of the enzymes involved in the biosynthesis of N-linked oligosaccharides may be significant in the etiology of some hereditary retinal dysplasias or retinal degenerations.