Carbohydrate influences the immunogenic and antigenic characteristics of the ZP3 macromolecule (Mr 55 000) of the pig zona pellucida.
The glycoprotein molecule ZP3 of the pig zona pellucida (Mr 55 000) was deglycosylated by trifluoromethane-sulphonic acid. While gas chromatography established that deglycosylation resulted in greater than 91% carbohydrate removal, deglycosylated ZP3 (DG-ZP3) still retained significant immunogenic potential as shown by its ability to elicit antibody production in the rabbit. Several lines of evidence, however, demonstrated that deglycosylation produced significant modifications in the ZP3 macromolecule which profoundly altered its antigenicity: 125I-radiolabelling procedures using chloramine-T consistently resulted in 125I-labelled ZP3 with a higher specific activity than 125I-labelled DG-ZP3; in competitive binding radioimmunoassays (RIAs) using an 125I-labelled-ZP3 vs anti-ZP3 system, unlabelled DG-ZP3 successfully displaced ZP3 in a dose-related manner; in direct binding RIAs comparing the reaction of labelled ZP3 and DG-ZP3 versus 5 different zona antisera, higher titres were consistently achieved with 125I-labelled ZP3; as compared to ZP3, 2-dimensional immunoelectrophoresis using DG-ZP3 as antigen yielded significantly modified precipitin arc patterns; with electrophoretic blotting procedures, antisera to ZP3 or DG-ZP3 cross-reacted with the heterologous antigen; treatment of pig zonae with anti-DG-ZP3 serum produced a dense precipitation layer on the zona surface. When evaluated collectively, these results provide grounds for an important role for protein and carbohydrate in establishing the immunological characteristics of the Mr 55 000 macromolecule of the pig zona pellucida.
D000818:Animals; D000941:Antigens / Q000032:analysis*; D002241:Carbohydrates / Q000032:analysis / Q000502:physiology*; D004527:Egg Proteins; D005260:Female; D006023:Glycoproteins / Q000032:analysis / Q000276:immunology*; D007123:Immunoelectrophoresis, Two-Dimensional; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008698:Mesylates / Q000494:pharmacology; D010063:Ovum / Q000276:immunology*; D011817:Rabbits; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface; D015044:Zona Pellucida / Q000187:drug effects / Q000276:immunology*; D000072216:Zona Pellucida Glycoproteins
D000941:Antigens; D002241:Carbohydrates; D004527:Egg Proteins; D006023:Glycoproteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008698:Mesylates; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; C012077:trifluoromethanesulfonic acid