Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

Background Equine chorionic gonadotropin (eCG), which comprises highly glycosylated alpha-subunit and beta-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCG beta/alpha Delta 56, substitution of Asn(56) of alpha-subunit with Gln; eCG beta-D/alpha, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the beta-subunit; eCG beta-D/alpha Delta 56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR). Results Both rec-eCG beta/alpha and rec-eCG beta/alpha Delta 56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCG beta-D/alpha and eCG beta-D/alpha Delta 56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200-250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCG beta/alpha, rec-eCG beta/alpha Delta 56 and rec-eCG beta-D/alpha were in the ranges of 40-45, 37-42, and 34-36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5-10 kDa. Rec-eCG beta/alpha Delta 56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn(56) of the alpha-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCG beta-D/alpha exhibited markedly downregulated LH-like and FSH-like activities. Conclusions Rec-eCG beta/alpha exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn(56) of the eCG alpha-subunit and/or by the O-linked glycosylation sites of the eCG beta-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.