Based on our previous work (with Con A and LcH) indicating that mannose residues are more accessible on dystrophic RPE and that fucose linked mannose-containing glycoconjugates may be an important determinant, we will test the hypothesis that similar glycoconjugates are also more exposed on dystrophic RPE. We will analyze by electron microscopy (EM) and computer-assisted quantitation, the number of lectin-ferritin binding sites on dystrophic as compared to normal RPE membranes using another mannose-binding lectin (PEA lectin) that is fucose-dependent. If the number of PEA lectin sites is significantly higher on dystrophic RPE, we will test if they are more available for phagocytic interaction by coating latex beads with PEA lectin and counting bead uptake. Secondly, we will analyze the effects of pretreating normal RPE membranes with various glycosidases on the number of PEA, Con A and LcH lectin-ferritin binding sites. We will test the hypothesis that normal RPE membranes have these mannosyl-specific lectin binding glycoconjugates that are covered by other sugar residues in the oligosaccharide chain. A second set of experiments will test whether the enzyme pretreatments will affect phagocytic uptake of PEA, Con A and LcH-coated beads. Thirdly, we will analyze by cell fractionation and polyacrylamide gel electrophoresis (PAGE) the RPE microvillous membranes in order to test whether a particular class of glycoproteins is present on dystrophic as compared to normal membranes. We will compare protein and glycoprotein moieties of dystrophic RPE membranes with the normal by lectin stained PAGE-blots to test whether one or more glycoproteins is altered in the dystrophic. These characterizations will be aimed at identifying a specific class of glycoproteins in dystrophic RPE membranes that will be used in future antibody localization studies. Lastly, we will analyze whether there is another defect in dystrophic RPE involving an alteration in the glycosylation stages through the Golgi system. We will use Con A and wheat germ agglutinin WGA-peroxidase staining, which have been used to demonstrate the presence of immature versus mature oligosaccharide composition, respectively, in proximal versus distal Golgi compartments and which may reveal differences in staining between dystrophic and normal RPE Golgi systems. We anticipate this data will contribute to an understanding of the molecular basis of the phagocytic defect in inherited retinal degeneration.
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