Fadool J M, Linser P J
Whitney Laboratory, University of Florida, St. Augustine 32086.
J Neurochem. 1993 Apr;60(4):1354-64. doi: 10.1111/j.1471-4159.1993.tb03296.x.
The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell-cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P-40-solubilized retina membranes had two components as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 45.5-kDa doublet and a 69-kDa polypeptide. Immunoreactive bands of 46-50 kDa were recognized following SDS-PAGE of detergent-solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N-glycosidase F (EC 3.2.2.18) converted the 45.5-50-kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N-Glycosidase F treatment also converted the 69-kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32-kDa species. Treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5-kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O-glycosidase (endo-alpha-N-acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for alpha 2,6-linked sialic acid) was confined to the higher molecular mass species of the 45.5-kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue-specific posttranslational modifications, particularly differences in sialylation of antigen-bearing polypeptides.
5A11/HT7抗原是免疫球蛋白超基因家族的成员之一，在视网膜发育过程中参与异型细胞间的相互作用。从用Nonidet P - 40溶解的视网膜膜中免疫纯化得到的5A11抗原，经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）测定有两个组分，一个是45.5 kDa的双峰，另一个是69 kDa的多肽。在用去污剂溶解的肝脏、肾脏和红细胞膜蛋白进行SDS - PAGE后，可识别出46 - 50 kDa的免疫反应条带。用N - 糖苷酶F（EC 3.2.2.18）处理后，所有组织中45.5 - 50 kDa的免疫反应性多肽都转变为32 kDa，这表明观察到的分子量差异是由于糖基化不同所致。N - 糖苷酶F处理还使视网膜中的69 kDa形式转变为46 kDa，这表明其多肽核心与32 kDa的物种不同。用内切β - N - 乙酰氨基葡萄糖苷酶H（EC 3.2.1.96）处理后，由于高甘露糖或杂合寡糖的水解以及对内切β - N - 乙酰氨基葡萄糖苷酶H消化有抗性的复杂寡糖未被水解，导致电泳迁移率适度增加。去糖基化后仍保留免疫反应性。分子量的大部分差异可归因于唾液酸化的变化。视网膜中45.5 kDa双峰的较高分子量物种以及其他组织的多肽对神经氨酸酶（EC 3.2.1.18）和O - 糖苷酶（内切α - N - 乙酰半乳糖胺酶；EC 3.2.1.97）消化敏感。接骨木树皮凝集素（对α2,6 - 连接的唾液酸具有特异性）标记仅限于45.5 kDa双峰的较高分子量物种，并且在上皮来源的抗原中比神经视网膜来源的抗原中显著更强。在鸡视网膜的石蜡切片中，接骨木树皮凝集素染色仅限于视网膜色素上皮、光感受器细胞和双极细胞，而穆勒细胞（其携带大部分5A11抗原）无染色。这些结果表明存在组织特异性的翻译后修饰，特别是含抗原多肽的唾液酸化差异。
