Site-directed mutagenesis of recombinant equine chorionic gonadotropin/luteinizing hormone: differential role of oligosaccharides in luteinizing hormone- and follicle-stimulating hormone-like activities.
Equine chorionic gonadotropin (eCG) consists of highly glycosylated alpha- and beta-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 alpha-subunit and O-linked oligosaccharides at the carboxyl-terminal peptide (CTP) of the beta-subunit, two mutant eCGs, in which Asn 56 of the alpha-subunit was replaced with Gln (eCG alpha 56/beta) or CTP was deleted (eCG alpha/ beta-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 eCG alpha 56/beta was greatly reduced, whereas that of eCG alpha/beta-CTP was unaffected, demonstrating that the oligosaccharide at Asn 56 of the alpha-subunit of eCG plays an indispensable role in LH-like activity. Interestingly, the FSH-like activity of eCG alpha 56/beta was increased markedly in comparison with the wild type, and that of eCG alpha/beta-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 alpha-subunit Asn 56 or CTP-associated O-linked oligosaccharides.
Min
K S
KS
Laboratory of Cellular Biochemistry, Veterinary Medical Science/Animal Resource Science, University of Tokyo, Japan.
Hattori
N
N
Aikawa
J
J
Shiota
K
K
Ogawa
T
T
D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000032:analysis; D005260:Female; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics*; D006031:Glycosylation; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006736:Horses; D007985:Leydig Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007986:Luteinizing Hormone / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013739:Testosterone / Q000032:analysis
D006063:Chorionic Gonadotropin; D017931:DNA Primers; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013739:Testosterone; D004958:Estradiol; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone