Comparison of the biological and immunological properties of glycosylation deficient human chorionic gonadotropin variants produced by site directed mutagenesis and chemical deglycosylation.
Appropriate glycosylation of gonadotropins is essential for the full expression of their biological activity. In this investigation we have compared the properties of glycosylation deficient human chorionic gonadotropin (hCG) produced by chemical deglycosylation (HF treatment--DG-hCG) or recombinant techniques (site directed mutagenesis). Among the recombinant hCG molecules secreted into the culture medium, the following variants containing selective N-glycosylation deletions at delta alpha 1 or delta alpha 1,2 or in both subunits could not stimulate steroidogenesis in mouse Leydig tumor cells (MA-10 cell line) and in this respect were very similar to DG-hCG. The other variants were fully active like native hCG, but the alpha + delta beta 1,2 recombinant hCG was a partial agonist. In radioimmunoassay with antibodies against native hCG, the DG-hCG as well as all recombinant hCG variants, including the wild type (WT), were similar. However, with antisera against DG-hCG or affinity purified antibodies specific to DG-hCG the alpha/delta 1,2 beta mutant, the WT hormone and native hCG were less active. In this assay mutants containing N-glycosylation deletions in the alpha subunit as well as the delta alpha 1,2/delta beta 1,2, variant showed higher activity. A similar pattern was evident in reaction with a selected monoclonal antibody showing preferential binding of 125I-labeled DG-hCG (antagonist). Affinity purified antibody directed against native hCG conformation was successful in converting DG-hCG and some inactive glycosylation deficient variants into an active form by stimulating progesterone in MA-10 cells. These data suggest that there are similarities as well as subtle differences in conformation of DG-hCG and various recombinant hCG molecules.(ABSTRACT TRUNCATED AT 250 WORDS)
Sairam
M R
MR
Reproduction Research Laboratory, Clinical Research Institute of Montreal, Québec, Canada.
Jiang
L G
LG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000276:immunology* / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000276:immunology*; D011374:Progesterone / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D006063:Chorionic Gonadotropin; D011374:Progesterone