Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter January 12, 2016

New insights into the substrate specificity of macrophage elastase MMP-12

  • Anne-Sophie Lamort , Rodolphe Gravier , Anni Laffitte , Luiz Juliano , Marie-Louise Zani and Thierry Moreau EMAIL logo
From the journal Biological Chemistry


Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence Pro-X-X↓Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.


Mass spectrometry analyses were performed by the ‘Plateforme de Spectrométrie de Masse et Protéomique’ of the Center for Molecular Biophysics in Orléans, France. N-terminal amino acid sequence analyses were performed by the ‘Plateforme Protéomique Pissaro’ at the University of Rouen, France. ASL was supported by a PhD fellowship from the Conseil Régional Centre-Val de Loire. RG was awarded with a post-doctoral fellowship from the Conseil Régional Centre-Val de Loire and the programme Investissement d’Avenir Grant Agreement Labex Mab’Improve (ANR-10-LABX-53). The English text was edited by Dr. Owen Parkes.


Banda, M.J., Clark, E.J., and Werb, Z. (1980). Limited proteolysis by macrophage elastase inactivates human a1-proteinase inhibitor. J. Exp. Med. 152, 1563–1570.10.1084/jem.152.6.1563Search in Google Scholar

Banda, M.J., Clark, E.J., Sinha, S., and Travis, J. (1987). Interaction of mouse macrophage elastase with native and oxidized human a1-proteinase inhibitor. J. Clin. Invest. 79, 1314–1317.10.1172/JCI112955Search in Google Scholar

Bertini, I., Calderone, V., Fragai, M., Luchinat, C., Maletta, M., and Yeo, K.J. (2006). Snapshots of the reaction mechanism of matrix metalloproteinases. Angew Chem. Int. Ed. 45, 7952–7955.10.1002/anie.200603100Search in Google Scholar

Bertini, I., Calderone, V., Fragai, M., Jaiswal, R., Luchinat, C., Melikian, M., Mylonas, E., and Svergun, D.I. (2008). Evidence of reciprocal reorientation of the catalytic and hemopexin-like domains of full-length MMP-12. J. Am. Chem. Soc. 130, 7011–7021.10.1021/ja710491ySearch in Google Scholar

Bhaskaran, R., Palmier, M.O., Lauer-Fields, J.L., Fields, G.B., and Van Doren, S.R. (2008). MMP-12 catalytic domain recognizes triple helical peptide models of collagen V with exosites and high activity. J. Biol. Chem. 283, 21779–21788.10.1074/jbc.M709966200Search in Google Scholar

Cadene, M. and Chait, B.T. (2000). A robust, detergent-friendly method for mass spectrometric analysis of integral membrane proteins. Anal. Chem. 72, 5655–5658.10.1021/ac000811lSearch in Google Scholar

Chagas, J.R., Juliano, L., and Prado, E.S. (1991). Intramolecularly quenched fluorogenic tetrapeptide substrates for tissue and plasma kallikreins. Anal. Biochem. 192, 419–425.10.1016/0003-2697(91)90558-BSearch in Google Scholar

Chen, E.I., Kridel, S.J., Howard, E.W., Li, W., Godzik, A., and Smith, J.W. (2002). A unique substrate recognition profile for matrix metalloproteinase-2. J. Biol. Chem. 277, 4485–4491.10.1074/jbc.M109469200Search in Google Scholar PubMed

Chen, E.I., Li, W., Godzik, A., Howard, E.W., and Smith, J.W. (2003). A residue in the S2 subsite controls substrate selectivity of matrix metalloproteinase-2 and matrix metalloproteinase-9. J. Biol. Chem. 278, 17158–17163.10.1074/jbc.M210324200Search in Google Scholar PubMed

Cobos-Correa, A., Trojanek, J.B., Diemer, S., Mall, M.A., and Schultz, C. (2009). Membrane-bound FRET probe visualizes MMP12 activity in pulmonary inflammation. Nat. Chem. Biol. 5, 628–630.10.1038/nchembio.196Search in Google Scholar PubMed

Curci, J.A., Liao, S., Huffman, M.D., Shapiro, S.D., and Thompson, R.W. (1998). Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms. J. Clin. Invest. 102, 1900–1910.10.1172/JCI2182Search in Google Scholar PubMed PubMed Central

Dean, R.A., Cox, J.H., Bellac, C.L., Doucet, A., Starr, A.E., and Overall, C.M. (2008). Macrophage-specific metalloelastase (MMP-12) truncates and inactivates ELR+ CXC chemokines and generates CCL2, -7, -8, and -13 antagonists: potential role of the macrophage in terminating polymorphonuclear leukocyte influx. Blood 112, 3455–3464.10.1182/blood-2007-12-129080Search in Google Scholar PubMed

Demedts, I.K., Morel-Montero, A., Lebecque, S., Pacheco, Y., Cataldo, D., Joos, G.F., Pauwels, R.A., and Brusselle, G.G. (2006). Elevated MMP-12 protein levels in induced sputum from patients with COPD. Thorax 61, 196–201.10.1136/thx.2005.042432Search in Google Scholar PubMed PubMed Central

Deng, S.J., Bickett, D.M., Mitchell, J.L., Lambert, M.H., Blackburn, R.K., Carter, H.L., 3rd, Neugebauer, J., Pahel, G., Weiner, M.P., and Moss, M.L. (2000). Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. J. Biol. Chem. 275, 31422–31427.10.1074/jbc.M004538200Search in Google Scholar PubMed

Devel, L., Rogakos, V., David, A., Makaritis, A., Beau, F., Cuniasse, P., Yiotakis, A., and Dive, V. (2006). Development of selective inhibitors and substrate of matrix metalloproteinase-12. J. Biol. Chem. 281, 11152–11160.10.1074/jbc.M600222200Search in Google Scholar PubMed

Finlay, G.A., O’Driscoll, L.R., Russell, K.J., D’Arcy, E.M., Masterson, J.B., FitzGerald, M.X., and O’Connor, C.M. (1997). Matrix metalloproteinase expression and production by alveolar macrophages in emphysema. Am. J. Respir. Crit. Care Med. 156, 240–247.10.1164/ajrccm.156.1.9612018Search in Google Scholar PubMed

Fu, J.Y., Lyga, A., Shi, H., Blue, M.L., Dixon, B., and Chen, D. (2001). Cloning, expression, purification, and characterization of rat MMP-12. Protein Expr. Purif. 21, 268–274.10.1006/prep.2000.1376Search in Google Scholar PubMed

Gabant, G. and Cadene, M. (2008). Mass spectrometry of full-length integral membrane proteins to define functionally relevant structural features. Methods 46, 54–61.10.1016/j.ymeth.2008.10.021Search in Google Scholar PubMed

Gronski, T.J., Jr., Martin, R.L., Kobayashi, D.K., Walsh, B.C., Holman, M.C., Huber, M., Van Wart, H.E., and Shapiro, S.D. (1997). Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase. J. Biol. Chem. 272, 12189–12194.10.1074/jbc.272.18.12189Search in Google Scholar PubMed

Haq, I., Lowrey, G.E., Kalsheker, N., and Johnson, S.R. (2011). Matrix metalloproteinase-12 (MMP-12) SNP affects MMP activity, lung macrophage infiltration and protects against emphysema in COPD. Thorax 66, 970–976.10.1136/thx.2011.159087Search in Google Scholar PubMed

Hautamaki, R.D., Kobayashi, D.K., Senior, R.M., and Shapiro, S.D. (1997). Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277, 2002–2004.10.1126/science.277.5334.2002Search in Google Scholar

Heinz, A., Jung, M.C., Duca, L., Sippl, W., Taddese, S., Ihling, C., Rusciani, A., Jahreis, G., Weiss, A.S., Neubert, R.H., and Schmelzer, C.E. (2010). Degradation of tropoelastin by matrix metalloproteinases – cleavage site specificities and release of matrikines. FEBS J. 277, 1939–1956.10.1111/j.1742-4658.2010.07616.xSearch in Google Scholar

Hirata, I.Y., Cezari, M.H.S., Nakaie, C.R., Boschcov, P., Ito, A.S., Juliano, M., and Juliano, L. (1994). Internally quenched fluorogenic protease substrates: solid-phase synthesis and fluorescent spectroscopy of peptides containing ortho-aminobenzoyl-dinitrophenyl groups as donor-acceptor pairs. Lett. Pept. Sci. 1, 299–308.10.1007/BF00119771Search in Google Scholar

Houghton, A.M., Quintero, P.A., Perkins, D.L., Kobayashi, D.K., Kelley, D.G., Marconcini, L.A., Mecham, R.P., Senior, R.M., and Shapiro, S.D. (2006). Elastin fragments drive disease progression in a murine model of emphysema. J. Clin. Invest. 116, 753–759.10.1172/JCI25617Search in Google Scholar

Hunninghake, G.M., Cho, M.H., Tesfaigzi, Y., Soto-Quiros, M.E., Avila, L., Lasky-Su, J., Stidley, C., Melen, E., Soderhall, C., Hallberg, J., et al. (2009). MMP12, lung function, and COPD in high-risk populations. N. Engl. J. Med. 361, 2599–2608.10.1056/NEJMoa0904006Search in Google Scholar

Kalupov, T., Brillard-Bourdet, M., Dade, S., Serrano, H., Wartelle, J., Guyot, N., Juliano, L., Moreau, T., Belaaouaj, A., and Gauthier, F. (2009). Structural characterization of mouse neutrophil serine proteases and identification of their substrate specificities: relevance to mouse models of human inflammatory diseases. J. Biol. Chem. 284, 34084–34091.10.1074/jbc.M109.042903Search in Google Scholar

Kaynar, M. and Shapiro, S.D. (2012). Matrix Metallopeptidase-12/Macrophage elastase. In: Handbook of Proteolytic Enzymes, N. D. Rawlings and G. S. Salvesen, eds. (London: Academic Press), pp. 800–804.Search in Google Scholar

Kis-Toth, K., Bacskai, I., Gogolak, P., Mazlo, A., Szatmari, I., and Rajnavolgyi, E. (2013). Monocyte-derived dendritic cell subpopulations use different types of matrix metalloproteinases inhibited by GM6001. Immunobiology 218, 1361–1369.10.1016/j.imbio.2013.06.012Search in Google Scholar

Knight, C.G., Willenbrock, F., and Murphy, G. (1992). A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS Lett. 296, 263–266.10.1016/0014-5793(92)80300-6Search in Google Scholar

Korkmaz, B., Attucci, S., Juliano, M.A., Kalupov, T., Jourdan, M.L., Juliano, L., and Gauthier, F. (2008). Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates. Nat. Protoc. 3, 991–1000.10.1038/nprot.2008.63Search in Google Scholar PubMed

Kridel, S.J., Chen, E., Kotra, L.P., Howard, E.W., Mobashery, S., and Smith, J.W. (2001). Substrate hydrolysis by matrix metalloproteinase-9. J. Biol. Chem. 276, 20572–20578.10.1074/jbc.M100900200Search in Google Scholar PubMed

Kridel, S.J., Sawai, H., Ratnikov, B.I., Chen, E.I., Li, W., Godzik, A., Strongin, A.Y., and Smith, J.W. (2002). A unique substrate binding mode discriminates membrane type-1 matrix metalloproteinase from other matrix metalloproteinases. J. Biol. Chem. 277, 23788–23793.10.1074/jbc.M111574200Search in Google Scholar

Lavigne, M.C., Thakker, P., Gunn, J., Wong, A., Miyashiro, J.S., Wasserman, A.M., Wei, S.Q., Pelker, J.W., Kobayashi, M., and Eppihimer, M.J. (2004). Human bronchial epithelial cells express and secrete MMP-12. Biochem. Biophys. Res. Commun. 324, 534–546.10.1016/j.bbrc.2004.09.080Search in Google Scholar

Liu, M., Sun, H., Wang, X., Koike, T., Mishima, H., Ikeda, K., Watanabe, T., Ochiai, N., and Fan, J. (2004). Association of increased expression of macrophage elastase (matrix metalloproteinase 12) with rheumatoid arthritis. Arthritis Rheum. 50, 3112–3117.10.1002/art.20567Search in Google Scholar

London, N., Raveh, B., Cohen, E., Fathi, G., and Schueler-Furman, O. (2011). Rosetta FlexPepDock web server--high resolution modeling of peptide-protein interactions. Nucleic Acids Res. 39, W249–W253.10.1093/nar/gkr431Search in Google Scholar

Marchant, D.J., Bellac, C.L., Moraes, T.J., Wadsworth, S.J., Dufour, A., Butler, G.S., Bilawchuk, L.M., Hendry, R.G., Robertson, A.G., Cheung, C.T., et al. (2014). A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity. Nat. Med. 20, 493–502.10.1038/nm.3508Search in Google Scholar

Matsumoto, S., Kobayashi, T., Katoh, M., Saito, S., Ikeda, Y., Kobori, M., Masuho, Y., and Watanabe, T. (1998). Expression and localization of matrix metalloproteinase-12 in the aorta of cholesterol-fed rabbits: relationship to lesion development. Am. J. Pathol. 153, 109–119.10.1016/S0002-9440(10)65551-4Search in Google Scholar

Molet, S., Belleguic, C., Lena, H., Germain, N., Bertrand, C.P., Shapiro, S.D., Planquois, J.M., Delaval, P., and Lagente, V. (2005). Increase in macrophage elastase (MMP-12) in lungs from patients with chronic obstructive pulmonary disease. Inflamm Res. 54, 31–36.10.1007/s00011-004-1319-4Search in Google Scholar PubMed

Neumann, U., Kubota, H., Frei, K., Ganu, V., and Leppert, D. (2004). Characterization of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluorogenic substrate with increased specificity constants for collagenases and tumor necrosis factor converting enzyme. Anal Biochem. 328, 166–173.10.1016/j.ab.2003.12.035Search in Google Scholar PubMed

Palmier, M.O. and Van Doren, S.R. (2007). Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect. Anal. Biochem. 371, 43–51.10.1016/j.ab.2007.07.008Search in Google Scholar PubMed PubMed Central

Ratnikov, B.I., Cieplak, P., Gramatikoff, K., Pierce, J., Eroshkin, A., Igarashi, Y., Kazanov, M., Sun, Q., Godzik, A., Osterman, A., et al. (2014). Basis for substrate recognition and distinction by matrix metalloproteinases. Proc. Natl. Acad. Sci. USA 111, E4148–E4155.10.1073/pnas.1406134111Search in Google Scholar PubMed PubMed Central

Rawlings, N.D., Waller, M., Barrett, A.J., and Bateman, A. (2014). MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 42, D503–D509.10.1093/nar/gkt953Search in Google Scholar

Schechter, I. and Berger, A. (1967). On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157–162.10.1016/S0006-291X(67)80055-XSearch in Google Scholar

Schirmer, H., Basso da Silva, L., Teixeira, P.J., Moreira, J.S., Moreira, A.L., and Simon, D. (2009). Matrix metalloproteinase gene polymorphisms: lack of association with chronic obstructive pulmonary disease in a Brazilian population. Genet. Mol. Res. 8, 1028–1034.10.4238/vol8-3gmr596Search in Google Scholar

Shapiro, S.D. (2000). Animal models for chronic obstructive pulmonary disease: age of klotho and marlboro mice. Am. J. Respir. Cell. Mol. Biol. 22, 4–7.10.1165/ajrcmb.22.1.f173Search in Google Scholar

Shapiro, S.D. (2003). Proteolysis in the lung. Eur. Respir. J. 44 (Suppl.), 30s–32s.10.1183/09031936.03.00000903aSearch in Google Scholar

Shapiro, S.D., Kobayashi, D.K., and Ley, T.J. (1993). Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages. J. Biol. Chem. 268, 23824–23829.10.1016/S0021-9258(20)80459-1Search in Google Scholar

Shipley, J.M., Wesselschmidt, R.L., Kobayashi, D.K., Ley, T.J., and Shapiro, S.D. (1996). Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc. Natl. Acad. Sci. USA 93, 3942–3946.10.1073/pnas.93.9.3942Search in Google Scholar PubMed PubMed Central

Taddese, S., Jung, M.C., Ihling, C., Heinz, A., Neubert, R.H., and Schmelzer, C.E. (2009a). MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III. Biochim. Biophys. Acta 1804, 731–739.10.1016/j.bbapap.2009.11.014Search in Google Scholar PubMed

Taddese, S., Weiss, A.S., Jahreis, G., Neubert, R.H., and Schmelzer, C.E. (2009b). In vitro degradation of human tropoelastin by MMP-12 and the generation of matrikines from domain 24. Matrix Biol. 28, 84–91.10.1016/j.matbio.2008.12.002Search in Google Scholar PubMed

Taddese, S., Jung, M.C., Ihling, C., Heinz, A., Neubert, R.H., and Schmelzer, C.E. (2010). MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III. Biochim. Biophys. Acta 1804, 731–739.10.1016/j.bbapap.2009.11.014Search in Google Scholar

Wallace, A.M. and Sandford, A.J. (2002). Genetic polymorphisms of matrix metalloproteinases: functional importance in the development of chronic obstructive pulmonary disease? Am. J. Pharmacogenomics 2, 167–175.10.2165/00129785-200202030-00002Search in Google Scholar

Wang, X., Liang, J., Koike, T., Sun, H., Ichikawa, T., Kitajima, S., Morimoto, M., Shikama, H., Watanabe, T., Sasaguri, Y., et al. (2004). Overexpression of human matrix metalloproteinase-12 enhances the development of inflammatory arthritis in transgenic rabbits. Am. J. Pathol. 165, 1375–1383.10.1016/S0002-9440(10)63395-0Search in Google Scholar

Zhou, H., Wu, Y., Jin, Y., Zhou, J., Zhang, C., Che, L., Jing, J., Chen, Z., Li, W., and Shen, H. (2013). Genetic polymorphism of matrix metalloproteinase family and chronic obstructive pulmonary disease susceptibility: a meta-analysis. Sci. Rep. 3, 2818.10.1038/srep02818Search in Google Scholar PubMed PubMed Central

Supplemental Material:

The online version of this article (DOI: 10.1515/hsz-2015-0254) offers supplementary material, available to authorized users.

Received: 2015-9-24
Accepted: 2016-1-4
Published Online: 2016-1-12
Published in Print: 2016-5-1

©2016 by De Gruyter

Downloaded on 3.12.2023 from
Scroll to top button