Site-Directed Mutagenesis of Recombinant Equine Chorionic Gonadotropin/Luteinizing Hormone

[摘要] References(45)Cited-By(7)Equine chorionic gonadotropin (eCG) consists of highly glycosylated α- and β-subunits and belongs to the glycoprotein hormone family that includes LH and FSH. eCG is a unique member of the gonadotropin family because it elicits response characteristics of both FSH and LH in other species than the horse. To determine the biological role of the N-linked oligosaccharide at Asn 56 of the α-subunit and O-linked oligosaccharides at the carboxyl-terminal peptide (CTP) of the β-subunit, two mutant eCGs, in which Asn 56 of the α-subunit was replaced with Gln (eCGα56/β) or CTP was deleted (eCGα/β-CTP), were produced by site-directed mutagenesis and transfecting chinese hamster ovary (CHO-K1) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The wild type eCG showed similar LH- and FSH-like activities to native eCG in the in vitro bioassays. The LH-like activity of eCGα56/β was greatly reduced, whereas that of eCGα/β-CTP was unaffected, demonstrating that the oligosaccharide at Asn 56 of the α-subunit of eCG plays an indispensable role in LH-like activity. Interestingly, the FSH-like activity of Ecgα56/β was increased markedly in comparison with the wild type, and that of eCGα/β-CTP was also considerably increased. These data indicate that the dual activities of eCG, LH- and FSH-like activities, could be separated by removal of the N-linked oligosaccharide on the a-subunit Asn 56 or CTP-associated O-linked oligosaccharides.

[学科分类] 内分泌与代谢学

[关键词] Recombinant eCG;Site-directed mutagenesis;LH- and FSH-like activities