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Analysis of hCG and LH active site by using molecular biological method

Research Project

Project/Area Number 02454383
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Obstetrics and gynecology
Research InstitutionOsaka University

Principal Investigator

TANIZAWA Osamu  Osaka University Medical School Department of Obstetrics and Gynecology, 医学部, 教授 (30028443)

Co-Investigator(Kenkyū-buntansha) KOYAMA Masayasu  Osaka University Medical School Department of Obstetrics and Gynecology, 医学部, 助手 (00183351)
SAJI Fumitaka  Osaka University Medical School Department of Obstetrics and Gynecology, 医学部, 講師 (90093418)
Project Period (FY) 1990 – 1991
Project Status Completed (Fiscal Year 1991)
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)
Keywordschorionic / Human chorionic Gonadotropin / thyroid Stimulating Hormone / Alpha Subunit / Beta Subunit / CAGY Region / Point Mutation / Site-directed Mutagenesis / ヒト絨毛ゴナドトロピン / 黄体ホルモン / 遺伝子組換 / 試験管内転写 / 卵母細胞微量RNA注入
Research Abstract

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 … More 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

Report

(3 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] T.Kameda,M.Koyama et al: "Localizatoin of Three Subtypes of Fc_γ Receptors in human Placenta by Immunohistochemical Analysis" Placenta. 12. 15-16 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M.Koyama,F.Saji et al: "Differential mRNA expression of three distinct classes of Fc_γ receptor at the feto-maternal interface" Journal of Reproductive Immunology. 20. 103-113 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] C.Azuma,F.Saji et al: "The Gene Expressions of Macrophage Colony-stimulating Factor(MCSF) and MCSF Receptor in the Human Myometrium During Pregnancy:Rdgulation by Sex Steroid Hormones" J.Steroid Biochem.Molec.Biol.39. 883-888 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M.Koyama,F.Saji et al: "Probabilistic assessment of the HLA sharing of recurrent spontaneous abortion couples in the Japanese population" Tissue Antigens. 37. 211-217 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T.Kimura,C.Azuma et al: "The Biological Effects of Macrophage-colony stimulating Factor Induced by Progestin on Growth and Differentiation of Endometriall Adenocarcinoma Cells" Int.J.Cancer. 40. 229-233 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] C.Azuma,F.Saji et al: "Application of Gene Amplification by Polymerase Chain Reaction to Genetic Analysis of Molar Mitochondrial DNA:The Detection of Anuclea Empty Ovum as the Cause of Complete Mole" Gynecologic Oncology. 40. 29-33 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] C. Azuma, F. Saji et al: "Application of Gene Amplification by Polymerase Chain Reaction to Genetic Analysis of Molar Mitochondrial DNA : The Detection of A nuclear Empty Ovum as the Cause of Complete Mole" Gynecologic Oncology. 40. 29-33 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Kimura, C. Azuma et al: "The Biological Effects of Macrophage-colony-stimulating Factor Induced by Progestin on Growth and Differentiation of Endometrial Adenocarcinoma Cells" Int. J. Cancer. 40. 229-233 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Koyama, F. Saji et al: "Differential mRNA expression of three distinct classes of Fcgamma receptor at the fete-maternal interface" Journal of Reproductive Immunology. 20. 103-113 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] C. Azuma, F. Saji et al: "The Gene Expressions of Macrophage Colony-stimulating Factor (MCSF) and MCSF Receptor in the Human Myometrium During Pregnancy : Regulation by Sex Steroid Hormones" J. Steroid Biochem. Molec. Biol.39. 883-888 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Kameda, M. Koyama et al: "Localization of Three Subtypes of Fcgamma Receptors in Human Placenta by Immunohistochemical Analysis" Placenta. 12. 15-16 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Koyama, F. Saji et al: "Probabilistic assessment of the HLA sharing of recurrent spontaneous abortion couples in the Japanese population" Tissue Antigens. 37. 211-217 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] C. Azuma, K. Miki et al: "Site-specific mutagenesis of human chorionic gonadotrophin (hCG) -beta subunit : influence of mutation on hCG production" Journal of Molecular Endocrinology. 5. 97-102 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T.Kameda,M.Koyama et al: "Localization of Three Subtypes of Fcr Receptors in Human Placenta by Immunohistochemical Analysis" Placenta. 12. 15-16 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] M.Koyama,F.Saji et al: "Differential mRNA expression of three distinct classes of Fcr receptor at the fetoーmaternal interface" Journal of Reproductive Immunology. 20. 103-113 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] C.Azuma,F.Saji et al: "The Gene Expressions of Macrophage Colonyーstimulating Factor(MCSF) and MCSF Receptor in the Human Myometrium During Pregnancy:Regulation by Sex Steroid Hormones" J.Steroid Biochem.Molec.Biol.39. 883-888 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] M.Koyama,F.Saji et al: "Probabilistic assessment of the HLA sharing of recurrent spontaneous abortion couples in the Japanese population" Tissue Antigens. 37. 211-217 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] T.Kimura,C.Azuma et al: "The Biological Effects of Macrophageーcolonyーstimulating Factor Induced by Progestin on Growth and Differetiation of Endometrial Adenocarcinoma Cells" Int.J.Cancer. 40. 229-233 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] C.Azuma,F.Saji et al: "Application of Gene Amplification by Polymerase Chain Reaction to Genetic Analysis of Molar Mitochondrial DNA:The Detection of Anuclear Empth Ovum as the Cause of Complete Mole" Gynecologic Oncology. 40. 29-33 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Chihiro Azuma: "Siteーspecific mutagenesis of human shorionic gonadotropin(hCG)ーsubunit:influence o fmutation hCG" J.Molecular ndocrinology. 5. 97-102 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Y.Tokugawa: "Analysis of siteーdirected mutagenesis of hCG subunit" J.Reprod.Immunol.suppl.211 (1989)

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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