Research paper The action of neutrophil serine proteases on elastin and its precursor Andrea Heinz a , Michael C. Jung a , Günther Jahreis b , Anthony Rusciani c , Laurent Duca c , Laurent Debelle c , Anthony S. Weiss d , Reinhard H.H. Neubert a , Christian E.H. Schmelzer a, * a Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Faculty of Natural Sciences I, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany b Max Planck Research Unit for Enzymology of Protein Folding, Halle (Saale), Germany c Laboratoire Signalisation et Récepteurs Matriciels (SiRMa), UMR CNRS 6237, Université de Reims Champagne Ardenne, Faculté des Sciences, Reims, France d School of Molecular Bioscience, University of Sydney, NSW, Australia article info Article history: Received 15 May 2011 Accepted 12 October 2011 Available online 20 October 2011 Keywords: Cathepsin G Human leukocyte elastase Proteinase 3 Mass spectrometry GxxPG abstract This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric tech- niques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tro- poelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P 1 , which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P 1 in tropoelastin, whereas Lys was not identified at P 1 in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P 2 and P 4 0 . With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction The neutrophil serine proteases (NSPs) human leukocyte elas- tase (HLE), proteinase 3 (PR3) and cathepsin G (CG) are members of the chymotrypsin family, which is characterized by the presence of a catalytic triad consisting of His57, Asp102 and Ser195 (chymo- trypsin numbering) [1]. NSPs play fundamental roles in a variety of physiological processes including defense against infections, degradation of extracellular matrix (ECM) components during inflammation processes, tissue remodeling, wound healing, coag- ulation and fibrinolysis. Moreover, NSPs have been identified to be important regulators of the innate immune response by controlling cellular signaling through proteolytic activation or inactivation of cytokines (interleukins) and specific cellular receptors [1]. HLE, PR3 and CG are secreted by human polymorphonuclear neutrophils and are stored in their active forms in azurophilic granules from which they are released in response to inflammation processes. All three NSPs rapidly hydrolyze various components of the ECM and plasma proteins and are furthermore involved in the degradation of ingested pathogens [1]. HLE (EC 3.4.21.37), which is also referred to as neutrophil elastase, is a highly cationic glyco- protein. It is the major protease involved in ECM degradation mediated by neutrophils and catalyzes the hydrolysis of a variety of matrix macromolecules including elastin, collagens, fibronectin and laminins, in addition to plasma proteins such as immuno- globulins and tumor necrosis factor-a [2]. CG (EC 3.4.21.20) shows 37% sequence homology with HLE and cleaves a variety of substrates including elastin, type IV collagen, fibronectin, laminins Abbreviations: ACN, acetonitrile; CG, cathepsin G (accession number P08311); EBP, elastin-binding protein (accession number P16278-2); ECM, extracellular matrix; EDP, elastin-derived peptide; ELISA, enzyme-linked immunosorbent assay; FA, formic acid; HPLC, high performance liquid chromatography; HLE, human leukocyte elastase (accession number P08246); MALDI, matrix-assisted laser/ desorption ionization; MS, mass spectrometry; NSP, neutrophil serine protease; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PR3, proteinase 3 (accession number P24158); QqTOF, quadrupole time-of-flight with collision cell; TFA, trifluoroacetic acid; Tris, 2-amino-2-(hydroxymethyl)-1,3- propanediol. * Corresponding author. Tel.: þ49 345 5525215; fax: þ49 345 5527292. E-mail address: schmelzer@pharmazie.uni-halle.de (C.E.H. Schmelzer). Contents lists available at SciVerse ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.10.006 Biochimie 94 (2012) 192e202