CELL BIOLOGY OF INHERITED RETINAL DEGENERATION

基于我们之前的工作(与Con A和Lch)表明甘露糖 残基在营养不良的RPE上更容易获得，并且岩藻糖连接 含甘露糖的糖偶联物可能是一个重要的决定因素，我们将 检验这样的假设，即类似的糖结合物在 营养不良的RPE。我们将通过电子显微镜(EM)和 计算机辅助定量，凝集素-铁蛋白结合位点的数量 营养不良与正常RPE膜的比较 依赖岩藻糖的甘露糖结合凝集素(PEA凝集素)。如果 在营养不良的RPE中，PEA凝集素位点的数量显著增加 将通过包衣测试它们是否更适合吞噬细胞相互作用 含PEA凝集素的乳胶珠和计数珠子摄取量。其次，我们将 不同方法对正常RPE膜的前处理效果分析 糖苷酶对PEA、ConA和Lch凝集素-铁蛋白结合数的影响 网站。我们将测试正常的RPE膜有这些的假设 甘露醇特异性凝集素结合糖共轭，由其他 低聚糖链中的糖残留物。第二组实验 将测试酶的预处理是否会影响吞噬细胞对 豌豆、ConA和LCH包裹的珠子。第三，我们将按细胞进行分析 RPE的分级和聚丙烯酰胺凝胶电泳法 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 用正常的凝集素染色的PAGE-BLOT来检测是否有一个或多个 营养不良时，糖蛋白发生改变。这些特征将 目的是在营养不良的RPE中识别一类特定的糖蛋白 这些膜将用于未来的抗体定位研究。 最后，我们将分析营养不良的RPE是否还有其他缺陷 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和小麦胚芽凝集素WGA-过氧化物酶 染色，这已经被用来证明未成熟的存在 近端与成熟低聚糖组成的对比 远端高尔基体室，这可能表明染色的差异 营养不良的和正常的RPE高尔基体系统之间。我们预料到这一数据 将有助于理解分子基础。 遗传性视网膜变性的吞噬细胞缺陷。

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.