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NSP4 is stored in azurophil granules and released by activated neutrophils as active endoprotease with restricted specificity

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  • Natascha C Perera
  • Karl-Heinz Wiesmüller
  • Maria Torp Larsen
  • Beate Schacher
  • Peter Eickholz
  • Niels Borregaard
  • Dieter E Jenne
Vis graf over relationer
Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer-based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1', P2', P3, P4, and P2 positions were tested. This enabled us to construct an α1-proteinase inhibitor variant (Ile-Lys-Pro-Arg-/-Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefèvre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils.
OriginalsprogEngelsk
TidsskriftJournal of immunology (Baltimore, Md. : 1950)
Vol/bind191
Udgave nummer5
Sider (fra-til)2700-7
Antal sider8
ISSN0022-1767
DOI
StatusUdgivet - 1 sep. 2013

ID: 42570912