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 alpha(1)-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-Lefevre 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

Effector and Regulatory T Cells Roll at High Shear Stress by Inducible Tether and Sling Formation

“…The seven cell types were isolated, differentiated in vitro, phenotyped by flow cytometry and FACS-sorted to >99% purity, generating enough cells to get high quality RNA-Seq data from three biological replicates (Figure S3 and Table S1). Specific markers for neutrophils and promyelocytes (Perera et al, 2013, Theilgaard-Monch et al, 2005) were confirmed by transcriptomics (Figure S4). As expected, neutrophils and promyelocytes clustered together in the principal component analysis (PCA), but were completely segregated from all types of CD4 T cells (naïve or differentiated) (Figure S5A).…”

Autoprocessing of neutrophil elastase near its active site reduces the efficiency of natural and synthetic elastase inhibitors

“…The expected mouse neutrophil elastase band at 27 kDa and the two bands around 19 kDa are found in wild type, but not in PR3/NE doubledeficient PMN lysate 16 confirming the specificity of the antibodies. Haptoglobin served as a loading control 17 . In contrast to mouse PMNs, human PMNs were lysed in the presence of a biotinylated AAPV-chloromethylketone, an irreversible activity-based inhibitor of both proteinase 3 and neutrophil elastase.…”

Papillon-Lefèvre syndrome patient reveals species-dependent requirements for neutrophil defenses

“…CTSC is expressed mainly in hematopoietic tissues. It is essential for posttranslational trimming of serine proteases stored in myeloid cells, in particular of the neutrophil granule-associated serine proteases: neutrophil elastase (NE), cathepsin G (CTSG), proteinase 3 (PR3) (5), and the recently described neutrophil serine protease 4 (NSP4) (6), but CTSC is also required for activation of granzymes A and B of cytotoxic lymphocytes (5,7) and for activation of mast cell chymases (8). CTSC removes 2 N-terminal amino acids that block the active site of the proteases (5,9).…”