Journal Article

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

Min, Kwan-Sik, Choi, Seung-Hee, Kang, Han-Ju, Kang, Myung-Hwa, Byambaragchaa, Munkhzaya, Lee, So-Yun

2021

Overview

Background Equine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and β-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: eCGβ/αΔ56, substitution of Asn 56 of α-subunit with Gln; eCGβ-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the β-subunit; eCGβ-D/αΔ56, 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-eCGβ/α and rec-eCGβ/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGβ-D/α and eCGβ-D/αΔ56, 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-eCGβ/α, rec-eCGβ/αΔ56 and rec-eCG β-D/α 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-eCGβ/αΔ56 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 α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGβ-D/α exhibited markedly downregulated LH-like and FSH-like activities. Conclusions Rec-eCGβ/α 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 α-subunit and/or by the O-linked glycosylation sites of the eCG β-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Analysis / Animals / Applied Microbiology / bioactive properties / Biochemical Engineering / Biological activity / Biomedical Engineering/Biotechnology / Biotechnology / cAMP responses / Cell culture / Chemistry / Chemistry and Materials Science / CHO Cells / CHO-suspension cells / Chorionic gonadotropin / Chorionic Gonadotropin - metabolism / Cricetinae / Cricetulus / Cricetulus griseus / culture media / equine chorionic gonadotropin / Follicle-stimulating hormone / follicle-stimulating hormone receptors / Follicles / Genetic Engineering / Glycoproteins / Glycosylation / Glycosylation sites / Gonadotropins / Horses / Luteinizing hormone / Luteinizing Hormone - metabolism / Molecular weight / Mutants / N-glycanase / Observations / Ovulation / peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase / Peptides / Pituitary (anterior) / Plant Breeding/Biotechnology / Proteins / Rat FSH receptor / Rat LH/CG receptor / Rats / Receptors / Receptors, FSH - metabolism / Receptors, LH - genetics / Receptors, LH - metabolism / Recombinant-equine chorionic gonadotropin / Roles / secretion / Signal Transduction / Transfection