Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken

Publisher Website
Google Scholar
Abstract
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 Ftreatment also converted the 69‐kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32‐kDa species. Treatment with endo‐β‐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‐β‐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‐α‐N‐acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for α2, 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.