GLYCOPROTEIN SYNTHESIS AND METABOLISM IN RETINAS

视网膜中的糖蛋白合成和代谢

基本信息

批准号：
3261974
负责人：
Steven J. Fliesler
金额：
$ 24.56万
依托单位：
THE EYE INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-03-01 至 1992-07-31
项目状态：
已结题

项目摘要

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.

该项目的长期目标是进一步明确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杆外节 (ROS) 膜 N-糖基化的作用椎间盘膜形态发生过程中的蛋白质。这些研究主要基于 P.I. 实验室之前的研究涉及 N-连接寡糖抑制剂的作用光盘上的生物合成和翻译后处理两栖动物视网膜的体外形态发生。工作中假设视紫质的寡糖是必需的同型或异型机制中的配体涉及蛋白质-碳水化合物键合相互作用，其中蛋白质可能是另一个视紫红质分子，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。该假设将被检验为如下：1)两栖类视紫红寡糖的测定成分和结构（假定的表征重要配体）； 2）数量、位置的确定两栖动物碳水化合物附着位点的氨基酸序列视紫质； 3) 潜在寡糖结构评估质膜中的视紫红质与成熟的视紫红质之间的差异 ROS 光盘； 4) 外源效应评价盘上已知组成和结构的寡糖形态发生； 5) 凝集素和N-的效果评价末端定向抗视紫红质抗体对椎间盘形态发生的影响； 6) 评估存在和分布 ROS 中的半乳糖基转移酶； 7）评估存在和内源性凝集素在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，以及相关的光学和电子显微镜以及放射自显影术，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致盲的相关性该发现表明存在疾病（由 P.I. 和合作者）认为，既是典型的视网膜发育不良又是可以在动物身上通过实验诱导光感受器变性通过药理学抑制生物合成中的酶 N-连接寡糖的制造途径。这样的治疗导致 ROS 盘膜异常组装体外以及体内ROS更新的停止。这些发现表明一种或多种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的病因学中具有重要意义发育不良或视网膜变性。