title: Antibody binding to the beta-subunit of deglycosylated chorionic gonadotropin converts the antagonist to an agonist.
abstract: The murine Leydig tumor cell line, MLTC-1, has a gonadotropin-responsive adenylate cyclase system. Binding of human chorionic gonadotropin (hCG) stimulates the accumulation of cyclic AMP in these cells. Chemically deglycosylated hCG (DG-hCG) is an antagonist that binds with high affinity to the gonadotropin receptor, but fails to stimulate adenylate cyclase. This antagonism can be reversed if the binding of DG-hCG is followed by treatment of the DG-hCG-receptor complex with antibodies against hCG. Polyclonal antibodies against DG-hCG were raised in rabbits. These antibodies were strongly cross-reactive with hCG, bound to both the alpha- and beta-subunits of hCG and DG-hCG, and reversed the antagonism of DG-hCG. The antiserum was divided into two fractions by affinity chromatography on hCG-Sepharose. The fraction that was not retained reacted only with DG-hCG (DG-hCG antibodies) and, on Western blots, bound to both the alpha- and beta-subunits of DG-hCG. DG-hCG antibodies did not reverse the antagonism of DG-hCG. However, using 125I-protein A, we were able to detect binding of these antibodies to the cell surface DG-hCG-receptor complex. The fraction of antibodies retained by the affinity column reacted with both DG-hCG and hCG (DG-hCG/hCG antibodies). On Western blots, DG-hCG/hCG antibodies bound to the beta-subunit, but only weakly to the alpha-subunit of both hCG and DG-hCG. These antibodies also bound to the cell surface DG-hCG-receptor complex. In addition, DG-hCG/hCG antibodies were able reverse the antagonism of DG-hCG. Reversal of DG-hCG antagonism by the whole antiserum was blocked by the beta- but not the alpha-subunit of hCG. Polyclonal antiserum against the beta- but not the alpha-subunit of hCG reversed the antagonism of DG-hCG. From these results, we conclude that antibody binding to specific determinants common to both native and deglycosylated beta-subunit reverses the antagonism of DG-hCG. In addition, antibodies directed against unique determinants on the deglycosylated beta-subunit are not capable of reversing the antagonism of DG-hCG.
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001667:Binding, Competitive; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D007700:Kinetics; D051379:Mice; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D011967:Receptors, Gonadotropin / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; C034923:HCG, deglycosylated; D010446:Peptide Fragments; D011967:Receptors, Gonadotropin; D000262:Adenylyl Cyclases