GLYCOPROTEIN SYNTHESIS AND METABOLISM IN RETINAS

这项计划的长远目标是进一步界定 糖蛋白合成与代谢的生物学意义 在视网膜中，在正常和病理条件下。这 提案描述了进一步调查的研究计划 杆状外节(ROS)膜N-糖基化的作用 在盘膜形态发生过程中的蛋白质。这些 这些研究在很大程度上是基于P.I.S实验室之前的研究 涉及N-连接低聚糖抑制剂的作用 光盘上的生物合成和翻译后加工 两栖动物视网膜的体外形态发生。在工作中 假设视紫红质的低聚糖是必不可少的 同型或异型机制中的配体包括 蛋白质-碳水化合物的相互作用，其中蛋白质可以 或者是另一种视紫红质分子，一种利用 作为底物的低聚糖(例如糖基转移酶或 糖苷酶)或凝集素。这一假设将被检验为 1)两栖类视紫红质低聚糖的测定 组成和结构(推定的 必需配体)；2)数量、位置和 两栖动物碳水化合物附着部位的氨基酸序列 视紫红质；3)潜在低聚糖结构的评价 质膜视紫红质与成熟视紫红质的差异 ROS盘；4)外源效应的评估 盘上已知组成和结构的低聚糖 形态发生；5)凝集素和N-半乳糖胺的作用评价。 末端导向的抗视紫质抗体对视盘形态发生的影响； 6)对存在和分布的评价 ROS中的半乳糖基转移酶；7)存在的评估和 内源性凝集素在ROS和ROS中的分布 光感受器间矩阵。这些研究将涉及现代 碳水化合物和蛋白质生化方法，以及相关 光学显微镜、电子显微镜和放射自显影， 免疫荧光和免疫细胞化学。潜力 这项研究与某些人类遗传性失明的相关性 这一发现(由P.I.和P.I.获得)表明存在精神障碍 合作者)既有典型的视网膜发育不良，也有 光感受器退化可以通过实验在动物身上诱导 通过药物抑制生物合成中的一种酶 制造N-连接的低聚糖的途径。是这样的 治疗导致ROS盘膜的异常组装 体外以及体内停止ROS更新。这些发现 表明一种或多种基因缺陷的可能性 N-连接低聚糖生物合成中的酶 可能在某些遗传性视网膜的病因学上有重要意义 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.