Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments

Abstract

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3-related autoimmune processes investigated in wild-type-, mNE-and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico-chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc-AAPV-pNA and Suc-AAPV-pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by a 1 -antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom-designed hNE inhibitor, Val 15 -aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species-specific assessment of neutrophil protease function and inhibition.

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

“…Also, enzyme inhibitors may exhibit different affinities toward mouse and human NSPs. In support of this, eglin C inhibits mPr3 but not hPr3 and recombinant hNE inhibitor Val 15 aprotinin is a poor inhibitor of mNE (30). Another example is the recently engineered hNE inhibitor, EPI-hNE4.…”

Dysregulation of Autoantigen Genes in ANCA-Associated Vasculitis Involves Alternative Transcripts and New Protein Synthesis

“…Murine and human PR3 also differ in the amino acid sequence and biophysical properties. 26 The differences in the murine Prtn3 gene, alternative transcript, and Pr3 protein compared with the human counterpart may contribute to the difficulty in developing a mouse model for PR3-ANCA disease that is as robust as the MPO-ANCA mouse model. 27 p24 PR3/MBN , encoded by PRTN3-002, could have a unique role in neutrophil activation or proliferation.…”

Mapping of Conformational Epitopes on Human Proteinase 3, the Autoantigen of Wegener’s Granulomatosis

“…Single stable integration of the cDNA constructs into the nucleus was achieved by homologous recombination using the flp-in technology. The flip-in system was faster and more efficient than the conventional transfection protocol (27), and expression from the same nuclear integration site resulted in reproducible stable levels of expression. Constitutively secreted recombinant human and gibbon proPR3 containing an N-terminal S-tag and a C-terminal 6xHis-tag was detected by Western blotting in the culture supernatant using S-protein-HRP (Fig.…”

“…PR3 was detected using sera from WG patients (lanes 2-12) and mAbs (lanes 13, 16-26) to PR3, respectively. As a control, five other granulocyte proteins were tested using human autoantibodies to myeloperoxidase (1) and mAbs to human neutrophil elastase (14), human azurocidin (15), human lactoferrin (27) and human lysozyme (28). Monoclonal mouse anti-hPR3 Abs used are indicated as follows: CLB12.…”