PLASMA MEMBRANE ASSOCIATED PROTEINS OF MAMMALIAN RPE CEL

This proposal is directed towards the biochemical characterization of the plasma membrane associated proteins of cultured human and RCS rat (normal and dystrophic) retinal pigment epithelial cells (RPE) and human corneal epithelial cells. These proteins and glycoproteins will be metabolically labeled with S35-methionine H3-sugars. The plasma membranes are isolated by subcellular fractionation of the post-nuclear supernatant on discontinuous sucrose density gradients. The purity of the membranes are assessed by marker assays including: DNA, RNA, 5'-nucleotidase, Ouabain binding, galactosyltransferase, succinate dehydrogenase, glucose-6-phosphatase and by electron microscopy. Alternatively, the proteins and glycoproteins are labeled (in vitro) by acetimidation, lactoperoxidase-catalyzed iodination, or reduction, after galactose oxidase treatment, with sodium borohydride. In addition, the integral membrane labeled proteins are specifically extracted with Triton X-114, or the Triton X-100 insoluble cytoskeleton are pelleted to elucidate these proteins by gel electrophoresis. Two-dimensional gel electrophoresis is used to separate both the acidic and basic labeled proteins: first by their isoelectric points and second by their molecular weights. Comparisons of the 2D fluorograms of the labeled proteins and glycoproteins are expected to reveal species specific, epithelial cell specific and, in the case of the animal model with inherited retinal dystrophy (RCS rat), genetically aberrant protein differences in concentration and/or position, probably associated with phagocytosis. Further identification of reference proteins will be achieved by specific immunostaining of the Western blots from whole cell protein gels with antibodies against actin, tubulin, vimentin and cytokeratin via I125 protein A. The glycoconjugates in 2D gels are further defined by their ability to bind specific lectins, concanavalin A and wheat germ agglutinin. It is hoped that these studies with human and RCS rat RPE cells will eventually lead to the production of a reference protein profile with some identifiable and characterized internal markers, some of which are associated with the plasma membrane, that will be compared to an analogous pattern obtained from donors exhibiting various forms of genetic retinal degeneration. In this way, abnormal proteins may become apparent and help to localize the molecular site of the disease. This is to be tested in the RCS rat animal model system where the primary genetic site of the disease is known to reside in the RPE.