Proinsulin cleaved by furin is processed to chromatographically mature insulin by carboxypeptidases in nonneuroendocrine cells.
Proinsulin is converted to mature insulin by two reactions, cleavage by the prohormone convertases PC2 and PC3, and removal of basic residues by carboxypeptidase H. These reactions are performed in the secretory granules of pancreatic beta cells. When we replaced the processing sites of proinsulin with furin-cleavable sites, the three nonneuroendocrine cell lines Hep G2, CHO, and NIH/3T3 produced insulin with the same size as synthetic human insulin. Although the three cell lines expressed different quantities of carboxypeptidase H mRNA, the cytosol fractions of the cells exhibited similar levels of carboxypeptidase activity, suggesting that additional carboxypeptidases were active. The insulins resulting from the three cell lines were eluted as a single peak on a cation-exchange chromatography column, indicating that proinsulin can be maturated to insulin even in nonneuroendocrine cells.
Nishigori
T
T
Department of Molecular Medicine, Gunma University, Maebashi, Japan.
Yanagita
M
M
Takeuchi
T
T
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D045683:Furin; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000096:biosynthesis*; D007515:Islets of Langerhans / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D011384:Proinsulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000235:genetics
D016895:Culture Media, Serum-Free; D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011384:Proinsulin; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D013381:Subtilisins; D045683:Furin