GLYCOPROTEIN SYNTHESIS AND METABOLISM IN RETINA

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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.