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NSP4, an elastase-related protease in human neutrophils with arginine specificity.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2012 Apr 17; Vol. 109 (16), pp. 6229-34. Date of Electronic Publication: 2012 Apr 02. - Publication Year :
- 2012
-
Abstract
- Neutrophil serine proteases (NSPs) in cytoplasmic granules of neutrophils are regarded as important antimicrobial defense weapons after engulfment and exposure of pathogens to the content of primary granules. Despite intensive studies on neutrophils during the last three decades, only three active serine proteases, neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3) have been identified in these short-lived cells. Here, we report on the identification of a fourth serine protease (NSP4) with 39% identity to NE and PR3, but arginine specificity, yet sharing features like propeptide processing by dipeptidyl peptidase I, storage, and release as an active enzyme with the three active proteases. We established monoclonal antibodies against NSP4, excluded cross-reactivity to human granzymes, NE, CG, PR3, and azurocidin, and screened for NSP4 protein expression in various human tissues and blood leukocyte populations. Only granulocyte precursors and neutrophil populations from peripheral blood were positive. The content of NSP4 in neutrophil lysates, however, was about 20-fold lower compared with CG. Upon neutrophil activation, NSP4 was released into the supernatant. Profiling its specificity with peptide libraries from Escherichia coli revealed a preference for arginine in P1; it cleaved Tyr-Arg-Phe-Arg-AMC and Ala-Pro-Nva-thiobenzyl esters. NSP4 was inhibited by α(1)-proteinase inhibitor (α(1)-antitrypsin), C1 inhibitor, and most efficiently by antithrombin-heparin, but not by elafin, secretory leukocyte protease inhibitor, α(1)-antichymotrypsin, and monocyte-neutrophil elastase inhibitor. Functional specialization and preferred natural substrates of NSP4 remain to be determined to understand the biological interplay of all four NSPs during neutrophil responses.
- Subjects :
- Amino Acid Sequence
Antibodies, Monoclonal immunology
Antibody Specificity immunology
Antithrombins pharmacology
Blotting, Western
Cathepsin C metabolism
Cells, Cultured
HEK293 Cells
Heparin pharmacology
Humans
Molecular Sequence Data
Neutrophils cytology
Pancreatic Elastase antagonists & inhibitors
Pancreatic Elastase genetics
Proteinase Inhibitory Proteins, Secretory pharmacology
Proteolysis
Sequence Homology, Amino Acid
Serine Endopeptidases genetics
Serine Endopeptidases immunology
Serine Proteinase Inhibitors pharmacology
alpha 1-Antitrypsin pharmacology
Arginine metabolism
Neutrophils enzymology
Pancreatic Elastase metabolism
Serine Endopeptidases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1091-6490
- Volume :
- 109
- Issue :
- 16
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 22474388
- Full Text :
- https://doi.org/10.1073/pnas.1200470109