Immunochemical analysis of the subcommissural organ-Reissner's fiber complex using antibodies against alkylated and deglycosylated glycoproteins of the bovine Reissner's fiber.
Reissner's fiber (RF) has been isolated, solubilized, and used to raise polyclonal antibodies. The present investigation has been designed: (1) to obtain antibodies against RF-glycoproteins in their native form (anti-RF-BI), after irreversible denaturation by alkylation (anti-RF-A), and after alkylation and deglycosylation by using endoglycosidase F (anti-RF-DE); (2) to use these antisera for a comparative immunocytochemical study of the subcommissural organ (SCO)-RF complex; (3) to establish the molecular mass of the deglycosylated RF-glycoproteins. Anti-RF-BI reacts with the SCO of all the species investigated. Anti-RF-A and anti-RF-DE only react with bovine SCO and RF. The three antisera stain the same bands in immunoblots of extracts of bovine SCO and RF, but anti-RF-A and anti-RF-DE reveal additional bands. The epitope common to all species reacting with anti-RF-BI is thus probably conformational in nature and associated with the integrity of the disulfide bonds. The lack of antibodies against conserved sequential epitopes in anti-RF-A does not support previous assumptions on the conserved nature of the SCO secretion. After deglycosylation by using endoglycosidase F, the RF-glycoproteins present a reduction in their molecular mass ranging between 10% and 25%. The three larger compounds (450, 300, and 230 kDa) lose their affinity for Limax flavus agglutinin (affinity = sialic acid), whereas the 190-kDa compound (170 kDa after deglycosylation) keeps its affinity for this lectin suggesting that it has N-linked and O-linked carbohydrate moieties, the three larger proteins probably having only N-linked carbohydrates.
Nualart
F
F
Instituto de Histología y Patología, Universidad Austral de Chile, Valdivia, Chile.
Rodríguez
E M
EM
D000478:Alkylation; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001923:Brain Chemistry / Q000502:physiology; D002417:Cattle; D002457:Cell Extracts / Q000737:chemistry / Q000276:immunology; D002556:Cerebrospinal Fluid Proteins / Q000032:analysis* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology* / Q000378:metabolism; D006031:Glycosylation; D015151:Immunoblotting; D007150:Immunohistochemistry; D008970:Molecular Weight; D009412:Nerve Fibers / Q000737:chemistry*; D013116:Spinal Cord / Q000737:chemistry; D013351:Subcommissural Organ / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D002556:Cerebrospinal Fluid Proteins; D000939:Epitopes; D006023:Glycoproteins