title: NSP4 is stored in azurophil granules and released by activated neutrophils as active endoprotease with restricted specificity.
abstract: 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.
authors.affiliation: Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Member of the German Center for Lung Research, 81377 Munich, Germany.
authors.forename: Natascha C
authors.initials: NC
authors.lastname: Perera
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Wiesmüller
authors.forename: Maria Torp
authors.initials: MT
authors.lastname: Larsen
authors.forename: Beate
authors.initials: B
authors.lastname: Schacher
authors.forename: Peter
authors.initials: P
authors.lastname: Eickholz
authors.forename: Niels
authors.initials: N
authors.lastname: Borregaard
authors.forename: Dieter E
authors.initials: DE
authors.lastname: Jenne
mesh_terms: D000293:Adolescent; D000595:Amino Acid Sequence; D015153:Blotting, Western; D020789:Cathepsin C / Q000235:genetics; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer; D006242:Haptoglobins / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D018375:Neutrophil Activation / Q000502:physiology*; D009504:Neutrophils / Q000201:enzymology*; D010214:Papillon-Lefevre Disease / Q000201:enzymology* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006242:Haptoglobins; D020789:Cathepsin C; D012697:Serine Endopeptidases