Characterization of tethered equine chorionic gonadotropin and its deglycosylated mutants by ovulation stimulation in mice

Abstract Background To directly assess the biological role of oligosaccharides in recombinant equine chorionic gonadotropin (rec-eCG) functioning, cDNA encoding the full-length eCGβ-subunit was fused with the mature protein part of the α-subunit, and we examined the expression levels of deglycosylated eCG mutants, the ovulation rate for deglycosylated mutants in C57BL/6 mice. Results The characterizations of heterodimeric and tethered mutants were studied following their respective secretions in culture medium, molecular weight and ovulation in vivo. Rec-eCG variants containing mutations at glycosylation sites at Asn82 of the α-subunit (eCGβ/αΔ82) and Asn13 of the β-subunit (eCGβΔ13/α) were not efficiently secreted into the culture medium from transfected cells. Western blot analysis revealed that the rec-eCGβ/α proteins have an approximate broad range of molecular weights of 40–46 kDa. Three rec-eCG mutants—a deglycosylated site at Asn56 of the α-subunit (eCGβ/αΔ56), a deletion of the C-terminal region of the β-subunit (eCGβ-D/α), and the double mutant (eCGβ-D/αΔ56)—turned out to have clearly lower (approximately 4–23 kDa) molecular weights. Protein N-glycosydase F (PNGase F) treatment markedly decreased the molecular weight to approximately 2–10 kDa. Normal oocytes were significantly more abundant in the natural eCG–treated group than in mutant rec-eCG–treated groups. In particular, numbers of nonfuntional oocytes were remarkably lower in all rec-eCG groups. Conclusions Our results indicate that the ovulation rates of oocytes are not affected by the deglycosylated rec-eCGβ/α mutant proteins. There are around 20% non-functional oocytes with natural eCG and only 2% with the rec-eCGs tested. These results provide insight into the molecular mechanisms underlying the production of rec-eCG hormones with excellent bioactivity in vivo.