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The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 x 107 M-1 ⋅ s-1, which is ~100-fold higher than that observed with wt-SerpinB1 and compares with that of al-protease inhibitor (α1-PI) toward HNE. SerpinB1 (S/ DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1 (S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1 (S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1 (S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis. [ABSTRACT FROM AUTHOR]

Abstract: Understanding the differences between murine (m) and human (h) proteinase 3 (PR3) and neutrophil elastase (NE) is crucial for the interpretation of in vivo studies of inflammatory processes. We built structural models of mPR3 and mNE and analyzed their surface properties. We performed molecular dynamics (MD) simulations on several enzyme–peptide complexes to investigate their interaction patterns. The analysis of trajectories confirms that murine and human complexes have different interaction patterns with peptidic substrates. We provide a map of the binding sites of the murine proteases and suggest sequence motifs that we predict to be specific for mPR3 or mNE. [Copyright &y& Elsevier]