Abstract
The activity of neutrophil elastase (NE) is tightly contained by serpins with a1-antitrypsin (a1PI) as the main extracellular inhibitor. Disruption of the protease inhibitor balance as seen in patients with a1PI deficiency or experimentally by intratracheal instillation of neutrophil elastase is associated with the development of lung emphysema.
Characterization of the murine homolog of NE revealed a self-cleavage site near its S1 pocket between the Ala188-Gly189 bond resulting in a two-chain form of NE (tc-NE). In this study we studied the impact of tc-NE on the protease inhibitor balance.
We successfully removed this self-cleavage site by two mutations which changed Gln187 to Lys and Ala188 to Gly. Differences between the stable single-chain (sc-NE) and the two-chain form of NE (tc-NE) were then determined by enzyme kinetics and inhibitor studies. Western blot analysis was used to identify the naturally occurring form of NE. Finally, sc- and tc-NE were applied to mice.
We found that the reactive center loop of a1PI was cleaved by the tc-NE more slowly as compared with sc-NE. In line with this observation, inhibition and covalent complex formation of tc-NE and a1PI was significantly impaired. Small molecule inhibitors also displayed a decline in their affinity for tc-NE. The sc-NE as well as the tc-NE was found in murine and human PMNs. Both forms of NE were able to induce pathological changes in mice.
In conclusion tc-NE maintained its pathogenic properties, but was less susceptible to inhibition by a1PI. Local release of tc-NE from dying neutrophils and during frustrated phagocytosis most likely increases tissue damage and should be considered for the development of more efficient anti-elastase therapies.
- © 2014 ERS