C-terminal peptide (hCTP) of human chorionic gonadotropin enhances in vivo biological activity of recombinant Japanese eel follicle-stimulating hormone and luteinizing hormone produced in FreeStyle 293-F cell lines

Abstract

Gonadotropins (Gths), follicle-stimulating hormone (Fsh), and luteinizing hormone (Lh) play central roles in the reproductive biology of vertebrates. In this study, recombinant single-chain Japanese eel Gths (rGth: rFsh and rLh), and recombinant chimeric Gths (rGth-hCTPs: rFsh-hCTP and rLh-hCTP; rGth-eCTPs: rFsh-eCTP and rLh-eCTP) with an extra O-glycosylation site (either a C-terminal peptide of human (hCTP) or equine (eCTP) chorionic gonadotropin), which are known to prolong the half-life of glycoprotein were produced in HEK293 cells and highly purified. Lectin blot analyses demonstrated that all these recombinant Gths contained N-glycans of the high mannose and complex types. In contrast, only rGth-hCTPs and rGth-eCTPs possessed highly sialylated O-linked oligosaccharides. Further analyses of glycans by liquid chromatography-mass spectrometry suggested that the species, amount, and degree of sialylation of N-glycans were comparable among recombinant Fshs and recombinant Lhs, while the amount of O-glycans with sialic acids in rGth-hCTPs was higher than that in the corresponding rGth-eCTPs. The serum levels of recombinant Gths in male eels significantly increased 12-24 h after a single injection of the Gths. The levels of rGth-hCTPs tended to be higher than those of the corresponding rGths and rGth-eCTPs throughout the experimental period, coinciding with the serum fluctuations of 11-ketotestosterone (11KT). The long-term treatment of male eels with these recombinant Gths also revealed the superiority of rGth-hCTPs in assisted reproduction; thus, the serum levels of 11KT and gonadosomatic indices in eels treated with rGth-hCTPs were higher than those in eels treated with the corresponding rGths and rGth-eCTPs. The induction of the entire process of spermatogenesis was only histologically observed in rGth-hCTPs-treated eels. These findings strongly suggest that hCTP enhances the in vivo biological activity of recombinant Japanese eel Gths due to the high abundance of O-linked glycans with sialylated antennae.

Keywords: Japanese eel; N-linked glycan; O-linked glycan; Recombinant follicle-stimulating hormone; Recombinant luteinizing hormone; Spermatogenesis.