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.
| Fliesler, S J; Alvarez, J; Hernandez, E et al. (1987) Effects of tunicamycin during early postnatal mammalian ocular development. Prog Clin Biol Res 247:551-69 |
| Pflugfelder, S C; Hernandez, E; Fliesler, S J et al. (1987) Intravitreal vancomycin. Retinal toxicity, clearance, and interaction with gentamicin. Arch Ophthalmol 105:831-7 |
