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Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus.

ABSTRACT: Proinsulin is converted to insulin by the two endoproteases PC2 and PC3. For complete conversion to insulin, cleavage must occur at both the B-chain/C-peptide and C-peptide/A-chain junctions of proinsulin. Studies in vitro have shown the specificity of PC3 for the B-chain/C-peptide junction and that of PC2 for the C-peptide/A-chain junction. In contrast, studies in vivo have suggested that the proinsulin cleavage substrate specificity of these two endoproteases might be more complex. We have now used recombinant adenovirus to overexpress PC2 or PC3 in the rat insulinoma cell line INS. These cells convert proinsulin more slowly than primary pancreatic beta-cells, possibly reflecting their lower levels of PC3. Infection of INS cells with the corresponding recombinant adenovirus led to 5-10-fold and 20-40-fold increases in PC2 and PC3 expression respectively. Recombinant adenovirus is thus an effective tool for expressing proteins at high levels in slow-growing INS cells. Overexpression of either PC2 or PC3 in INS cells led to a striking acceleration of conversion of proinsulin to insulin and to a decreased accumulation of the conversion intermediate des-64.65-split proinsulin (cleaved only at the A-chain/C-peptide junction). There was no detectable accumulation of des-31.32-split proinsulin (cleaved only at the B-chain /C-peptide junction) after overexpression of either enzyme. Taken together, the data indicate that when expressed at high levels, both PC2 and PC3 seem to be able to cleave proinsulin at both its junctions in vivo.

SUBMITTER: Irminger JC

PROVIDER: S-EPMC1217891 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Levels of the conversion endoproteases PC1 (PC3) and PC2 distinguish between insulin-producing pancreatic islet beta cells and non-beta cells.

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Gene expression changes in response to doxycyline-induced expression of proinsulin (C96Y)-GFP in an INS-1 insulinoma cell line

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2010-06-24 | GSE22537 | GEO

Gene expression changes in response to doxycyline-induced expression of proinsulin (C96Y)-GFP in an INS-1 insulinoma cell line

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Pax6 regulates the proglucagon processing enzyme PC2 and its chaperone 7B2.

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Expression data from rat insulinoma INS-1 cells under glucotoxic condition

Project description:Glucotoxicity of INS-1 cells stimulation by chronic high glucose. We used microarrays to detail the global programme of gene expression underlying cellularization and identified distinct classes of up and down-regulated genes during this process.

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Role of Proinsulin Self-Association in Mutant <i>INS</i> Gene-Induced Diabetes of Youth.

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Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2.

Project description:Human prohormone convertase PC2 was expressed in Xenopus oocytes and its properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236]. Recombinant PC2 had the same substrate specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.

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