Background Genetic variability in MMP-12 is associated with COPD; the matrix Metalloproteinase (MMP)-12 knockout (KO) mouse is resistant to emphysema despite cigarette smoke exposure, strongly implicating MMP-12 in COPD pathogenesis. However, the complete MMP-12 substrate profile (degradome) in COPD remains unknown. Terminal amine isobaric labelling of substrates (TAILS) is a novel proteomic technique allowing identification of a protease degradome on an organism-wide scale. Identification of the MMP-12 degradome will lead to novel drugs, desperately needed in COPD.
Objectives To identify the MMP-12 degradome in COPD by comparing cigarette smoke exposed MMP-12 KO and wildtype (WT) controls by TAILS and validating these targets against the human COPD sputum proteome during exacerbations and stable disease.
Methods C57BL/6J MMP-12 KO and WT mice (n = 4) were exposed to cigarette smoke and airways sampled by bronchoalveolar lavage (BAL). BAL fluid was analysed by TAILS, high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Matched COPD exacerbation and stable disease sputum samples (n = 9) were analysed by TAILS, HPLC and MS/MS.
Results The following new MMP-12 targets in the COPD mouse model were identified: alpha-2-HS glycoprotein, anti-thrombin III, clusterin, complement C3, complement C4b, complement factor H-related protein-1, hemopexin, serotransferrin and serum albumin, alpha-2-macroglobulin, beta-1, 4-galactosyltransferase 2, transmembrane protease 7, DEP domain-containing mTOR-interacting protein, kininogen-1, tumour necrosis factor ligand superfamily member 11. Of these, alpha-2-HS-glycoprotein, anti-thrombin III, complement factors C3 and C4B, hemopexin and serum albumin were identified in both exacerbation and stable COPD human sputum. Furthermore, 1,116 peptides were identified in COPD exacerbation and stable disease sputum, grouped into the following categories: cell adhesion/migration, complement system, acute phase response, extracellular matrix structure/function, anti-microbicidal activity, cytoskeletal function/remodelling, carbohydrate metabolism, oxidoreductase activity, cell death regulation/DNA synthesis/repair, immune response, protease activity, protease inhibition and ATP synthesis/function.
Conclusion This study identifies the MMP-12 degradome in COPD and provides the most comprehensive analysis of the proteome of COPD sputum at exacerbation and stable disease. It suggests a role for MMP-12 in complement regulation and haemostasis in COPD. Thus an important peptide library has been unravelled, providing an ideal tool in developing drugs and understanding COPD pathogenesis.