Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7.
The amino acid sequence, Arg-4-X-3-Lys/Arg-2-Arg-1 decreases X+1, is thought to be a consensus processing site for a constitutive secretory pathway in non-endocrine cells. We created a mutant proinsulin DNA with a peptide structure of B chain-Arg-Arg-Lys-Arg-C peptide-Arg-Arg-Lys-Arg-A chain, which compares to the native proinsulin structure of B chain-Arg-Arg-C peptide-Lys-Arg-A chain. When the mutant insulin was expressed in a monkey kidney-derived cell line, COS-7, approximately 60% of the total immunoreactive insulin appeared as mature insulin in the culture medium. This conversion to the mature form was strikingly facilitated by co-expressing the mutant proinsulin with furin, a homologue of the yeast endoprotease, Kex2.
Yanagita
M
M
Division of Molecular Endocrinology, Gunma University, Maebashi, Japan.
Nakayama
K
K
Takeuchi
T
T
D019325:3-O-Methylglucose; D000273:Adipose Tissue / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D001692:Biological Transport; D002460:Cell Line; D016384:Consensus Sequence; D004822:Epididymis / Q000187:drug effects / Q000378:metabolism; D045683:Furin; D007328:Insulin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000078790:Insulin Secretion; D008297:Male; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009928:Organ Specificity; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D013381:Subtilisins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D008757:Methylglucosides; D021382:Protein Sorting Signals; D019325:3-O-Methylglucose; D011384:Proinsulin; D013381:Subtilisins; D045683:Furin