|Budget Amount *help
¥7,000,000 (Direct Cost : ¥7,000,000)
Fiscal Year 1991 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1990 : ¥5,300,000 (Direct Cost : ¥5,300,000)
The heterodimeric glycoprotein hormones, human chorionic gonadotropin(hCG), lutenizing hormone(LH), thyroid stimulating hormone(FSH)and follicles stimulating hormone(FSH), consist of two non-covalently linked subunits, the alpha and beta subunits. The beta subunit is specific for each hormone and is responsible for the biological specificity, but the beta subunits of different hormones show some degree of structural homology. The CAGY(cysteine-alanine-glycine-tyrosine)region is one of the amino acid sequences that is homologous in different beta subunits and is highly conserved between species. In the present study, site-specific in-vitro mutagenesis was used to change three individual nucleotides in the center of the CAGY region of the hCG-beta subunit, and the effect of these mutations on hCG production was determined by in-vitro translation in Xenopus laevis oocytes. Three mutated cDNAs, MI(Ala->Ser), M2(Gly->Arg), and M3(Gly->Asp)were prepared. Oocytes treated with a mixture of nor
mal alpha and mutant beta mRNAs(M2 and M3)produced as little hCG as negative control whereas oocytes treated with non-nal alpha MRNA and Ml mutant MRNA produced the same amount of hCG as the positive control. These results indicate that the CAGY region, particularly the glycine residue at position 36 in the beta subunit, is essential for the production of immunoreactive fiCG. This finding is consistent with previous studies showing that this region is necessary for the biological activity of human TSH.
The alpha subunit is shared commonly with the all four hormones. To investigate the role of CAGY region on the interaction between alpha and beta subunits, we further analyzed the stereo-coaguration of hCG molecule. By the three dimensional analyses of these hormones, the important regions Of ^<28>Cys- ^<29>Met- ^<30>Gly- ^<31>Cys- ^<32>Cys- ^<33>phe- ^<34>Ser- ^<35>Arg- ^<36>Ala- ^<37>Tyr were presumed. We therefore introduced several point mutations into native alpha CDNA by using polymerase chain reaction method. As a result, 6 mutants of alpha subunit, MI( ^<28>Cys->Tyr), M2( ^<29>Met->Arg), M3(^<30>Gly->Arg), M4( ^<30>GIy->Ala), M5( ^<30>Gly->Asp), and M6( ^<37>Tyr->Asp)were generated. Messenger RNAs of mutated or native alpha subunit CDNA were transcribed in vitro and were co-injected into oocytes with MRNA of native beta subunit. The characterizations of the produced mutated hCG molecules were currently underway. Less