Previous studies from other laboratories have indicated the presence of mucin-type glycoproteins (containing an O-glycosidic linkage) in retinal tissue, primarily in the interphotoreceptor matrix (IPM). These studies, however, provided only partial biochemical characterization and no information on the metabolism of these golycoproteins. In many other systems, a malfunction in the production of the mucins has been implicated in certain diseases. While the function of the mucins in the retina is presently unknown there is evidence that they might assist in adhesion between the retina and retinal pigment epithelium (RPE) and in the normal phagocytosis of the shed rod discs by the RPE. A lack of the mucins might be involved in some animal retinal dystrophies. The studie described in this proposal will attempt to characterize this potentially very important class of glycoproteins more fully. The number of mucins present and the cell(s) responsible for their biosynthesis will be evaluated following incubations of either the eyecup (initially) or isolated cells in culture with labeled precursors of the mucins. This evaluation will utilize gel electrophoretic techniques (e.g., electrofocusing and native and denaturing electrophoresis) and analytical density gradient centrifugation. The mucins found to be most abundant will then be isolated and purified using procedures suited to the unique properties of the mucins (e.g., gel filtration on porous agarose and density gradient centrifugation). They will then be characterized in terms of composition, number of O-linked sugar chains and linkage amino acid, isoelectric point and molecular weight. Their oligosccharide chains will then be fully characterized, following release by alkaline sodium borohydride treatment and separation using lectin affinity and HPLC technques. The structure of the isolated oligosaccharide-alditols will be determined by a combination of various sensitive techniques, such as sequential exoglycosidase digestion, fast atom bombardment mass spectroscopy, gas chromatography/mass spectroscopy of the permethylated mono- and oligosaccharides, and NMR spectroscopy. These studies should provide a basis for future investigations of the function of the mucins in the retina and their possible involvement in retinal disease.
