Abstract
Serine proteases stored in neutrophil granules, namely neutrophil elastase, cathepsin G and proteinase 3, have been intensely investigated for several decades. They are known for their role in intracellular killing of pathogens, but are also implicated in the pathobiology of lung diseases, such as emphysema, pulmonary fibrosis or cystic fibrosis. Recently we identified NSP4, the fourth serine protease in neutrophils which has been completely overlooked so far.
For a better understanding of the role of NSP4 during neutrophil immune responses, our objective was to analyze NSP4 with regard to its biosynthesis, storage and extended substrate specificity.
We performed Western blot analyses of subcellular fractions of neutrophils as well as RT-PCR analyses of neutrophil precursors from human bone marrow. To determine the extended specificity of recombinant NSP4, we used a FRET-based optimization strategy. A tailor-made, NSP4-targeting alpha-1-antitrypsin variant was then generated to detect active NSP4.
We identified NSP4 as a novel azurophil granule protein of neutrophils. We further specified the extended NSP4 substrate specificity. Replacing the reactive center loop of α1-antitrypsin by the preferred NSP4 target sequence resulted in an irreversible NSP4-specific inhibitor. This serpin variant was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils.
NSP4 is stored as an already activated enzyme in azurophil granules like the other neutrophil serine proteases. As a secreted and active protease, it potentially also contributes to neutrophil-mediated inflammation and tissue damage in lung diseases.
- © 2014 ERS