| Co-Investigator(Kenkyū-buntansha) |
ISHIKAWA Hidekazu Gunma Univ, School of Medicine Dept. of Dermatology Professor, 医学部・皮膚科, 教授 (70008233)
KOGURE Kimitaka Gunma Univ, School of Medicine The 1stDept. of Surgery Lecturer, 医学部・第1外科, 講師 (80143220)
HORIUCHI Ryuya Gunma Univ. Inst. of Endocrinol. Pharmacology Division Associate Professor, 内分泌研究所・薬学部門, 助教授 (90008342)
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| Budget Amount *help |
¥9,300,000 (Direct Cost: ¥9,300,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1989: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Research Abstract |
Most of peptide hormones are produced as a propeptide and converted to a biologically active peptide during their transport from the Golgi apparatus to secretory granules. The conversion from propeptide to biologically active peptide 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 propeptides to biologically active peptides does not mean their inability to process propeptides to active peptides. Non-endocrine cells including fibroblasts, 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^<-2>@*X^<+1
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>-. Thus, we took proinsulin as a model propeptide for its expression in non-endocrine cells, and constructed 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, while the native proinsulin structure was B chain-Arg-Arg-C peptide-Lys-Arg-A chain. Both mutant and native proinsulin were expressed in a monkey kidney derived cell line, COS-7 cells that possess only a constitutive secretory pathway and is thought not to process native proinsulin to mature insulin. When mutant insulin was expressed, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium. Moreover, mutant proinsulin was completely converted to mature one by co-expressing it with the subtilisin-like endoprotease, furin. The insulin produced in COS cells presented an identical biological activity to a synthetic human insulin by the capability of incorporating 3-O-[ ^3] methyl-D-glucose into adipocytes. We demonstrated that the mutated proinsulin with a pair of tetrabasic residues at the processing sites can be converted to fully bioactive insulin in a non-endocrine cell line, COS-7 cells. In the future this type of a mutant proinsulin DNA construct may be utilized for a hybrid type artificial islet or for gene therapy. Less
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