Kiyoshi Miyai

Loss of biological activity of human chorionic gonadotropin (hCG) by the amino acid substitution on the “CMGCC” region of the α-subunit

In order to study the bioactive sites of the glycoprotein hormones, we have prepared five point mutants on the CMGCC (Cys28-Met29-Gly30-Cys31-Cys32) region of the human alpha-subunit by using site-directed mutagenesis. Each mutant human chorionic gonadotropin (hCG) agr; cDNA and a wild-type hCG beta cDNA were transcribed by T3 RNA polymerase, and the mixture of the hCG alpha mRNA and hCG beta mRNA was microinjected into Xenopus laevis oocytes. All five mutant hCGs produced in oocyte culture supernatants were detected as immunoreactive forms by enzyme immunoassay. In contrast, four mutants (Cys28-->Tyr28, Gly30-->Arg30, Ala30, Asp30) were devoid of biological activity in vitro bioassay using the production of testosterone with mouse Leydig cells. These results indicate that the CMGCC region in the alpha-subunit, particularly the cysteine residue at position 28 and the glycine residue at position 30, plays an important role in the biosynthesis of glycoprotein hormones.