Enzymatic deglycosylation of the subunits of chorionic gonadotropin. Effects on formation of tertiary structure and biological activity.
Both the O- and N-linked oligosaccharide moieties of the subunits of the placental glycoprotein hormone, human choriogonadotropin (hCG), are removed by treatment with a mixture of glycosidases produced by Streptococcus (Diplococcus) pneumoniae. The resulting deglycosylated subunits recombine with their native counterparts in good yield, and the reassociated hormones bind to gonadotropin receptors equally as well as the untreated hormone. Stimulation of steroidogenesis by the deglycosylated alpha-native beta recombinant, however, was markedly less than the stimulation by unmodified hCG both in terms of relative potency (0.10-0.15) and the maximal amount of steroid (40-50%) produced. The native alpha-deglycosylated beta recombinant produced a maximum level of steroid production of 80-90% that of control hCG although its relative potency had decreased approximately 4-fold. The data are in accord with results by others in which either hCG or lutropin was partially deglycosylated by treatment with anhydrous hydrofluoric acid. In addition, the effects of deglycosylation on the ability of each subunit to refold after reduction of their disulfide bonds was studied. Of particular interest is that, after deglycosylation, the beta subunit can correctly refold to a significant degree, in contrast to several unsuccessful attempts to demonstrate correct refolding of the unmodified beta subunit of either lutropin or hCG. Alpha subunit, as measured by a conformation sensitive radioimmunoassay, refolds with equal facility both before and after deglycosylation.
D000818:Animals; D001681:Biological Assay; D002241:Carbohydrates / Q000032:analysis; D006063:Chorionic Gonadotropin / Q000378:metabolism / Q000494:pharmacology; D006026:Glycoside Hydrolases; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D009844:Oligosaccharides / Q000032:analysis*; D011487:Protein Conformation; D011489:Protein Denaturation; D011869:Radioligand Assay; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D011974:Receptors, LH; D013329:Structure-Activity Relationship; D013739:Testosterone / Q000096:biosynthesis
D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D046911:Macromolecular Substances; D009844:Oligosaccharides; D011956:Receptors, Cell Surface; D011974:Receptors, LH; D013739:Testosterone; D006026:Glycoside Hydrolases