CELL BIOLOGY OF INHERITED RETINAL DEGENERATION

基于我们以前的工作（用Con A和LcH）表明甘露糖 残基在营养不良的RPE上更容易接近， 含甘露糖的糖缀合物可能是一个重要的决定因素，我们将 测试假设，类似的糖缀合物也更多地暴露在 营养不良性RPE。 我们将通过电子显微镜（EM）进行分析， 计算机辅助定量，凝集素-铁蛋白结合位点的数量 与正常RPE膜相比，使用另一种 甘露糖结合凝集素（PEA凝集素）是岩藻糖依赖性的。 如果 PEA凝集素位点的数量在营养不良的RPE上显著更高，我们 将测试它们是否更容易通过涂层进行吞噬作用 乳胶珠与PEA凝集素和计数珠摄取。 其次，我们将 分析不同浓度的预处理对正常RPE膜的影响， 糖基化酶对PEA、Con A和LcH凝集素-铁蛋白结合数的影响 网站. 我们将测试正常RPE膜具有这些功能的假设。 甘露糖基特异性凝集素结合糖缀合物，其被其他 寡糖链中的糖残基。 第二组实验 将测试酶预处理是否会影响吞噬细胞对 PEA、Con A和LcH包被微珠。 第三，我们将分析细胞 RPE分级分离和聚丙烯酰胺凝胶电泳（PAGE） 微绒毛膜，以测试是否有特定类别的 与正常膜相比，营养不良的膜上存在糖蛋白。 我们 将比较营养不良的RPE膜的蛋白质和糖蛋白部分 用凝集素染色的PAGE印迹法检测是否有一个或多个 糖蛋白在营养不良中改变。 这些特征将 目的在于鉴定营养不良性RPE中的特定类别的糖蛋白 膜，将用于未来的抗体定位研究。 最后，我们将分析营养不良性RPE是否存在其他缺陷 涉及通过高尔基体的糖基化阶段的改变 系统 我们将使用刀豆蛋白A和麦胚凝集素WGA-过氧化物酶 染色，已被用来证明存在未成熟的 与成熟寡糖组成，分别在近端与 远侧高尔基室，并可能揭示染色差异 营养不良和正常RPE高尔基体系统之间的差异。 我们预计这些数据 将有助于理解的分子基础， 遗传性视网膜变性中的吞噬细胞缺陷。

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.