en
Scientific article
English

Processing of proinsulin by furin, PC2, and PC3 in (co) transfected COS (monkey kidney) cells

Published inDiabetes, vol. 44, no. 9, p. 1075-1080
Publication date1995
Abstract

The enzymology of proinsulin conversion was studied in COS cells by cotransfection of three species of proinsulin and each of three conversion endoproteases (furin, PC2, and PC3). In addition to the parts of basic residues linking the B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65), which were present in all three proinsulins studied, human proinsulin presents a P4 basic residue (four residues NH2-terminal to the point of cleavage) only at the former junction (Lys29) and rat proinsulin II only at the latter (Arg62). Human proinsulin Arg62 (prepared by site-directed mutagenesis of human proinsulin) contains a P4 basic residue at both junctions. Transfected cells were incubated for four successive 2-h periods. The media were pooled, and pro-insulin, conversion intermediates, and insulin were separated by reverse-phase high-performance liquid chromatography to monitor conversion activity. There was little conversion of any proinsulin in COS cells without cotransfection of an exogenous endoprotease. When furin or PC3 was cotransfected with any of the three proinsulins, there was extensive processing, with insulin as the major conversion product. PC2, by contrast, failed to cleave human proinsulin but was able to cleave both human proinsulin Arg62 and rat proinsulin II. Cleavage by PC2 of these proinsulins was predominantly at the C-peptide-A-chain junction, generating the conversion intermediate des-64,65-split proinsulin as the major product and only very small amounts of insulin itself.

Keywords
  • Amino Acid Sequence
  • Animals
  • Arginine
  • C-Peptide/ metabolism
  • Cell Line
  • Cercopithecus aethiops
  • Chromatography, High Pressure Liquid
  • Endopeptidases/ metabolism
  • Furin
  • Humans
  • Insulin/analysis/metabolism
  • Kidney
  • Lysine
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Proinsulin/ metabolism
  • Protein Processing, Post-Translational
  • Rats
  • Recombinant Proteins/metabolism
  • Substrate Specificity
  • Subtilisins/ metabolism
  • Transfection
Citation (ISO format)
VOLLENWEIDER, F. et al. Processing of proinsulin by furin, PC2, and PC3 in (co) transfected COS (monkey kidney) cells. In: Diabetes, 1995, vol. 44, n° 9, p. 1075–1080. doi: 10.2337/diabetes.44.9.1075
Identifiers
ISSN of the journal0012-1797
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