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Processing of mutated proinsulin with tetrabasic cleavage sites to mature insulin in non-endocrine nell lines

Research Project

Project/Area Number 04454554
Research Category

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

Allocation TypeSingle-year Grants
Research Field 内分泌・代謝学
Research InstitutionGunma University

Principal Investigator

TAKEUCHI Toshiyuki  Gunma Univ., Inst.for Mol.& Cell.Regulation, Professor, 生体調節研究所・遺伝子発現分野, 教授 (00109977)

Co-Investigator(Kenkyū-buntansha) TATEMOTO Kazuhiko  Gunma Univ., Inst.for Mol.& Cell.Regulation, Professor, 生体調節研究所・分子調節分野, 教授 (60240694)
Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1992: ¥5,300,000 (Direct Cost: ¥5,300,000)
KeywordsProcessing / Proinsulin / Insulin / Furin / カルボキシペプチダーゼ
Research Abstract

Insulin is produced in the pancreatic beta-cells as a precursor proinsulin that comprises of the three peptides linked by two pairs of basic residues in the following order : B chain-Arg-Arg-C peptide-Lys-Arg-A chain. C peptide is cleaved off from proinsulin at the dibasic residues adjacent to C peptide during its transport through the trans-Golgi networks to immature type secretory vesicles. The conversion from proinsulin to insulin is a unique function of endocrine cells. A number of propeptide hormone cDNAs, including a human proinsulin cDNA,have been introduced into both endocrine and non-endocrine cells, and those expressed in endocrine cells were generally processed correctly, while others expressed in non-endocrine cells were secreted constitutively as non-cleaved propeptides. However, inability of non-endocrine cells to convert proinsulin to insulin does not mean their inability to process other propeptides to smaller bioactive peptides. Non-endocrine cells including fibroblast … More s, hepatocytes, and lymphocytes produce biologically inactive propeptides and convert them to bioactive peptides by cleaving a unique consensus sequence -Arg^<-4>-X^<-3>-Lys/Arg^<-2>-Arg^<-1>*X^<+1>-. This sequence is well cleaved by the subtilisin-like endoprotease furin that is characteristic of non-endocrine cells. Thus, we created a mutant proinsulin DNA where peptide structure was comprized of B and A chains linked to C peptide by a pair of tetrabasic residues in the following order : B chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A chain. When mutant insulin was expressed, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium. Both the native and mutant proinsulins were expressed in the following four cell lines : a monkey kidney-derived cell line COS-7, a Chinese hamster ovary-derived cell line CHO,a human liver cancer-derived cell line HepG2 and a mouse fibroblast-like cell line NIH3T3. We used these cell lines because they contain different quantities of furin mRNA,ranking as follows : NIH3T3>HepG2>COS>CHO.When mutant insulin was expressed in these cells, the conversion of proinsulin to mature insulin was approximately 85% in NIH3T3,70% in HepG2,60% in COS,and 50% in CHO.The conversion correlated well with the furin expression in each cell line as measured by the density of its Northern blot band. Moreover, in CHO,the cell line with the lowest furin expression, co-expression of mutant proinsulin with furin resulted in complete conversion of proinsulin to mature insulin. The insulin produced in COS cells presented an identical biological activity to a synthetic human insulin by the capability of incorporating 3-O-[^3H] methyl-D-glucose into adipocytes. Less

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (22 results)

All Other

All Publications (22 results)

  • [Publications] N.Hayashi,T.Kayo,K.Sugano and T.Takeuchi.: "Production of bioactive gastrin from the non-endocrine cell lines CHO and COS-7." FEBS Lett.337. 27-32 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Y.C.Liu,M.Kawagishi,T.Mikayama,Y.Inagaki,T.Takeuchi and H.Ohashi.: "Processing of a fusion protein by endoprotease in COS-7 cells for the secretion of mature peptide by using a novel expression vector." Proc.Natl.Acad.Sci.USA. 90. 8857-8961 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] C.J.Dickinson,T.Takeuchi,Y.J.Guo,B.T.Stadler and T.Yamada.: "Expression and processing of prohormones in nonendocrine cells." Am.J.Physiol.264. G553-G560 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Yanagita,H.Hoshino,K.Nakayama and T.Takeuchi.: "Processing of mutated proinsulin with tetrabasic cleavage sites to mature insulin reflects the expression of furin in nonendocrine cell lines." Endocrinology. 133. 639-644 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Yasuda,K.Inoue,H.Shibata,T.Takeuchi,Y.Eto,et al.: "Existence of Activin-A in A- and D-Cells of rat pancreatic islet." Endocrinology. 133. 624-630 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Yanagita,K.Nakayama and T.Takeuchi.: "Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line,COS-7." FEBS Lett.311. 55-59 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 竹内利行: "プロセシングとアミド化:生化学的特徴とその機能的反映 Vol.12" 病理と臨床, 1409-1415 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 竹内利行: "臨床遺伝医学VI-遺伝子治療と予防" 糖尿病の遺伝子治療(in press), (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Yanagita, K.Nakayama, and T.Takeuchi: "Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7" FEBS Lett.311. 55-59 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] C.J.Dickinson, T.Takeuchi, Y-I.Guo, B.T.Stadler, and T.Yamada: "Expression and processing of prohormones in nonendocrine cells" Am.J.Physiol.27. G553-G560 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Yasuda, K.Inoue, H.Shibata, T.Takeuchi, Y.Eto, Y.Hasegawa, N.Sekine, Y.Totsuka, T.Mine, E.Ogata, and I.Kojima: "Existence of activin-A in A- and D-cells of rat pancreatic islets" Endocrinol.133. 624-630 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Yanagita, H.Hoshino, K.Nakayama, and T.Takeuchi: "Processing of mutated proinsulin with tetrabasic cleavage sites to mature insulin reflects the expression of furin in nonendocrine cell lines" Endocrinol.133. 639-644 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Y-C.Liu, M.Kawagishi, T.Mikayama, Y.Inagaki, T.Takeuchi, H.Ohashi: "Processing to a fusion protein by endoprotease in COS-7 cells for the secretion of mature peptide by using a novel expression vector" Proc.Natl.Acad.Sci.USA. 90. 8957-8961 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] N.Hayashi, T.Kayo, K.Sugano and T.Takeuchi: "Production of bioactive gastrin from the nonendocrine cell lines CHO and COS-7" FEBS Lett.337. 27-32 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] N.Hayashi,T.Kayo,K.Sugano,T.Takeuchi.: "Production of bioactive gastrin from the non-endocrine cell lines CHO and COS-7." FEBS Lett.337. 27-32 (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] Y.C.Liu.,M.Kawagishi,T.Mikayama,Y.Inagaki,T.Takeuchi,H.Ohashi.: "Processing of a fusion protein by endoprotease in COS-7 cells for the secretion of mature peptide by using a novel expression vector." Proc.Natl.Acad.Sci.USA. 90. 8957-8961 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] H.Yasuda,T.Mine,H.Shibata,Y.Eto,Y.Hasegawa,T.Takeuchi,et al.: "Activin A:An autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes." J.Clin.Invest. 92. 1491-1496 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Yanagita,H.Hoshino,K.Nakayama,T.Takeuchi.: "Processing of mutated proinsulin with tetrabasic cleavage sites to mature insulin reflects the expression of furin in nonendocrine cell lines." Endocrinology. 133. 639-644 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] H.Yasuda,K.Inoue,H.Shibata,T.Takeuchi,Y.Eto,et al.: "Existence of Activin-A in A-and D-Cells of rat pancreatic islet." Endocrinology. 133. 624-630 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] C.J.Dickinson,T.Takeuchi,Y.J.Guo,B.T.Stadler,T.Yamada.: "Expression and processing of prohormones in nonendocrine cells." Am.J.Physiol.264. G553-G560 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Yanagita M,Nakayama K,Takeuchi T,: "Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line,COS-7" FEBS Letters. 311. 55-59 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Dickinson C.J,Takeuchi T,et.al.: "Expression and processing of prohormones in non-endocrine cells" American Journal of Physiology. (1993)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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