Abstract
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.
Acknowledgments:
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.
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