Ongoing comparative analysis of the protein compositions of the pigment epithelial (PE) cell plasma membrane from normal and dystrophic (RCS) rats will be extended in greater detail. The goal of this research has been to determine if the RCS mutation results in retinal degeneration by affecting the production of a PE plasma membrane protein that is involved in the recognition and phagocytosis of shed rod outer segment (ROS) membranes. Two dimensional (2D) electrophoretic analysis of the protein composition of plasma membrane enriched fractions from normal and dystrophic rat PE suggests that a 183,000 MW glycoprotein is underglycosylated in the RCS PE. In order to elucidate the chemical nature of this apparent decreased glycosylation, the carbohydrate compositions of the normal and RCS 183,000 MW proteins will be analyzed by sequential degradation with exo- and endoglycosidases, gel filtration chromatography and high performance liquid chromatography (HPLC). Cultured rat PE cells will be assayed for enzymes commonly found in the plasma membrane of other absorptive epithelia. Electroblots of 2D maps of PE cell membranes will be stained with antibodies specific for enzymes that test positive in the preliminary assays. Alternatively, subunits for some of the enzymes will be identified by use of specific radioactive affinity labels or histochemical stains. In this manner, specific functions will be identified for proteins previously characterized by molecular weight only in 2D electrophoretic analysis of PE plasma membrane proteins. This information will be valuable for narrowing down the number of spots in the complex 2D map that may be involved in recognition/phagocytosis of ROS membranes by the PE. Another important component of the PE cell plasma membrane, the receptor for serum retinol binding protein (RBP), will be detergent solubilized from swine PE. An assay for detergent solubilized receptor will be devised, and subsequently employed in purification of the receptor by lectin affinity chromatography, gel filtration chromatography, and HPLC. Antibodies will be prepared to the purified receptor. The anti-RBP receptor antibody will be used to determine if distribution of the receptor in the plasma membrane is altered as a secondary effect of retinal degeneration in the RCS rat. The long term goal of this research project is to elucidate, on a molecular level, the cause of inherited retinal degeneration by determining how alterations in the proteins of the PE plasma membrane are involved in both primary and secondary effects of inherited retinal degeneration.
| Clark, V M (1991) Normal and dystrophic rat retinal pigment epithelia display different sensitivities to plant lectins. Invest Ophthalmol Vis Sci 32:327-35 |
| Clark, V M; Zhou, X Y; Pfeffer, B A (1990) Partial characterization of fucosylated cell surface glycoproteins of cultured RPE. Curr Eye Res 9:977-86 |
| Clark, V M (1989) 3H-fucose incorporation into RPE cell surface proteins of normal and dystrophic rats. Invest Ophthalmol Vis Sci 30:1542-7 |
| Colley, N J; Clark, V M; Hall, M O (1987) Surface modification of retinal pigment epithelial cells: effects on phagocytosis and glycoprotein composition. Exp Eye Res 44:377-92 |
| Pfeffer, B A; Clark, V M; Flannery, J G et al. (1986) Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium. Invest Ophthalmol Vis Sci 27:1031-40 |
| Clark, V M; Hall, M O (1986) RPE cell surface proteins in normal and dystrophic rats. Invest Ophthalmol Vis Sci 27:136-44 |
