GLYCOPROTEIN SYNTHESIS AND METABOLISM IN RETINA

该项目的长期目标是评估 糖缀合物的结构，生物合成和代谢在正常 以及脊椎动物视网膜的病理生理功能。 本 （修订）提案解决了蛋白质糖基化在 视杆外节（ROS）椎间盘膜形态发生的机制。 基于我们以前关于体外效应的研究， 糖蛋白合成和盘组装加工的抑制剂， 和寡糖结构测定的视紫红质，我们建议 （1）体外实验表明，外源性 凝集素和ROS蛋白特异性抗体对椎间盘形态发生的影响 分离的两栖动物视网膜;（2）存在，分布，和 脊椎动物内源凝集素的生化特性 视网膜，特别是那些可能与视紫红质和其他 ROS驻留糖蛋白;（3）两栖动物视网膜的能力， 用半乳糖瞬时修饰视紫红质寡糖链 和/或岩藻糖;以及（4）“新”和“旧”的混合比例， 糖蛋白在新生的ROS光盘，使用衣霉素扰动 椎间盘形态发生作为模型系统。 这些研究将采用 生物化学和形态学/细胞生物学方法，包括： 用放射性标记的糖体外放射性标记糖蛋白， 氨基酸;亚细胞分级;蛋白质分析和 制备程序（SDS-PAGE、荧光法、HPLC、FPLC、亲和层析法） 光学和电子显微镜和放射自显影 (LM光镜和电镜凝集素细胞化学 免疫细胞化学和定量图像分析。 这些 这些研究将为分子事件提供基本的新见解， 参与ROS椎间盘组装和椎间盘结构保存， 是维持ROS超微结构和功能的基础。 等 信息最终可能有助于理解 某些形式的视网膜变性， 光感受器外节的退化和丧失是 感光细胞死亡和失明。

The long-term goal of this project is to evaluate the role of glycoconjugate structure, biosynthesis, and metabolism in the normal and pathophysiological function of the vertebrate retina. The present (revised) proposal addresses the role of protein glycosylation in the mechanism of rod outer segment (ROS) disc membrane morphogenesis. Building upon our previous studies concerning the in vitro effects of inhibitors of glycoprotein synthesis and processing on disc assembly, and oligosaccharide structure determination of rhodopsins, we propose to evaluate the following: (1) the in vitro effects of exogenous lectins, and ROS protein-specific antibodies, on disc morphogenesis in isolated amphibian retinas; (2) the presence, distribution, and biochemical characteristics of endogenous lectins in vertebrate retinas, particularly those that may bind to rhodopsin and other ROS-resident glycoproteins; (3) the capacity of amphibian retinas to transiently modify rhodopsin's oligosaccharide chains with galactose and/or fucose; and (4) the mixing proportions of "new" and "old" glycoproteins in nascent ROS discs, using tunicamycin perturbation of disc morphogenesis as a model system. These studies will employ biochemical and morphological/cell biological methodologies, including: in vitro radiolabeling of glycoproteins with radiolabeled sugars and amino acids; subcellular fractionation; protein analytical and preparative procedures (SDS-PAGE, fluorography, HPLC, FPLC, affinity chromatography); light and electron microscopy and autoradiography (LM/ARG, EM/ARG); light and electron microscopic lectin cytochemistry and immunocytochemistry; and quantitative image analysis. These studies will provide fundamental new insights into the molecular events involved in ROS disc assembly and preservation of disc structure, which underlie the maintenance of ROS ultrastructure and function. Such information may ultimately contribute to a rationale for understanding certain forms of retinal degeneration that involve progressive deterioration and loss of photoreceptor outer segments as a prelude to photoreceptor cell death and blindness.