Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone

Differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities

Kwan Sik Min, Naka Hattori, Jun Ichi Aikawa, Kunio Shiota, Tomoya Ogawa

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 α-subunit Asn 56 or CTP-associated O-linked oligosaccharides.

Original languageEnglish
Pages (from-to)585-593
Number of pages9
JournalEndocrine Journal
Volume43
Issue number5
Publication statusPublished - 1996
Externally publishedYes

Fingerprint

Equine Gonadotropins
Follicle Stimulating Hormone
Chorionic Gonadotropin
Luteinizing Hormone
Site-Directed Mutagenesis
Oligosaccharides
Peptides
Aromatase
CHO Cells
Leydig Cells
Granulosa Cells
Cricetulus
Gonadotropins
Biological Assay
Horses
Testosterone
Ovary
Glycoproteins
Hormones

Keywords

  • LH- and FSH-like activities
  • Recombinant eCG
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Endocrinology

Cite this

Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone : Differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities. / Min, Kwan Sik; Hattori, Naka; Aikawa, Jun Ichi; Shiota, Kunio; Ogawa, Tomoya.

In: Endocrine Journal, Vol. 43, No. 5, 1996, p. 585-593.

Research output: Contribution to journalArticle

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abstract = "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 α-subunit Asn 56 or CTP-associated O-linked oligosaccharides.",
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AU - Ogawa, Tomoya

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AB - 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 α-subunit Asn 56 or CTP-associated O-linked oligosaccharides.

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