title: Immunochemical analysis of the subcommissural organ-Reissner's fiber complex using antibodies against alkylated and deglycosylated glycoproteins of the bovine Reissner's fiber.
abstract: 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.
authors.affiliation: Instituto de Histología y Patología, Universidad Austral de Chile, Valdivia, Chile.
authors.forename: F
authors.initials: F
authors.lastname: Nualart
authors.forename: E M
authors.initials: EM
authors.lastname: Rodríguez
mesh_terms: 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*
chemical_list: D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D002556:Cerebrospinal Fluid Proteins; D000939:Epitopes; D006023:Glycoproteins