Abstract
Alpha 1-antitrypsin (A1AT) is a serine protease inhibitor that mainly inhibits neutrophil elastase in the lungs. A variant of A1AT at the P1 position with methionine 358 to arginine (A1AT-Pittsburgh) is a rapid inhibitor of thrombin with greatly diminished anti-elastase activity. The P2 residue (position 357) of A1AT-Pittsburgh has been shown to play an important role in interactions with thrombin and kallikrein, but the role of P2 residue in wild-type A1AT has largely been unraveled. Here, we investigated the effects of P2 proline substitutions in wild-type A1AT on interactions with porcine pancreatic elastase (PPE) and human neutrophil elastase (HNE). The mutant A1AT proteins (P357A, P357D, P357K, P357L, P357N, P357S, and P357W) were less efficient than the wild-type A1AT at inhibiting PPE and HNE. Among the mutants, P357D did not form a complex with PPE, whereas P357L, P357N, and P357W showed significantly reduced complex formation with PPE. Surprisingly, mass spectrometry analysis revealed that P357D had two cleavage sites after the P9 alanine and the P3 isoleucine residues. Our results indicate that the size and negative charge of the R group of the P2 residue influence the interaction with elastases. Specifically, the negative charge at the P2 residue is disfavored and the resulting conformational changes in the reactive center loop upon interaction with PPE lead to cleavage at new sites. Overall, the results of this study demonstrate a previously unknown role for P2 residue in determining inhibitory specificity of A1AT.
Highlights
Serine protease inhibitors comprise a structurally similar superfamily of proteins that are distributed among animals, plants, viruses, and bacteria, and have evolved to regulate the activity of serine proteases, cysteine proteases, and metalloproteases [1]
We investigated the effects of P2 proline substitutions in human WT Alpha 1-antitrypsin (A1AT) on the interaction with porcine pancreatic elastase (PPE) and human neutrophil elastase (HNE)
The inhibitory activities of WT and seven A1AT mutants were evaluated against two elastases, PPE and HNE
Summary
Serine protease inhibitors (serpins) comprise a structurally similar superfamily of proteins that are distributed among animals, plants, viruses, and bacteria, and have evolved to regulate the activity of serine proteases, cysteine proteases, and metalloproteases [1]. The reactive center loop, either fully exposed above the serpin molecule (Fig 1a, the structure of human α1-antitrypsin (A1AT)) or partially inserted into the central β-sheets, serves as bait to the proteases, which cleave the peptide bond at P1-P10. A natural variant of human A1AT at the P1 position with methionine 358 to arginine ( called A1AT-Pittsburgh) that causes bleeding disorders showed loss of inhibitory activity against elastases [13], but was found to inhibit thrombin [13], factor XIa, kallikrein, factor XIIf [14], and activated protein C [15].
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