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

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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