RPE cell surface proteins in normal and dystrophic rats.
Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.
D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000032:analysis*; D010857:Pigment Epithelium of Eye / Q000032:analysis* / Q000166:cytology; D051381:Rats; D012162:Retinal Degeneration / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D005643:Fucose; D005944:Glucosamine