title: Osteopontin is proteolytically processed by matrix metalloproteinase 9.
abstract: Osteopontin is robustly upregulated following myocardial infarction (MI), which suggests that it has an important role in post-MI remodeling of the left ventricle (LV). Osteopontin deletion results in increased LV dilation and worsened cardiac function. Thus, osteopontin exerts protective effects post-MI, but the mechanisms have yet to be defined. Matrix metalloproteinases (MMPs) regulate LV remodeling post-MI, and osteopontin is a known substrate for MMP-2, -3, -7, and -9, although the cleavage sites have not been mapped. Osteopontin-derived peptides can exert distinct biological functions that may depend on their cleavage sites. We mapped the MMP-9 cleavage sites via LC-MS/MS analysis using label-free and N-terminal labeling methods, and compared them with those of MMP-2, -3, and -7. Each MMP yielded a unique cleavage profile with few overlapping cleavage sites. Using synthetic peptides, we validated 3 sites for MMP-9 cleavage at amino acid positions 151-152, 193-194, and 195-196. Four peptides were synthesized based on the upstream- and downstream-generated fragments and were tested for biological activity in isolated cardiac fibroblasts. Two peptides increased cardiac fibroblast migration rates post-wounding (p < 0.05 compared with the negative control). Our study highlights the importance of osteopontin processing, and confirms that different cleavage sites generate osteopontin peptides with distinct biological functions.
authors.affiliation: a San Antonio Cardiovascular Proteomics Center.|b Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505, USA.|c Research Service, G.V. (Sonny) Montgomery Veterans Affairs Medical Center, Jackson, Mississippi, USA.
authors.forename: Merry L
authors.initials: ML
authors.lastname: Lindsey
authors.affiliation: a San Antonio Cardiovascular Proteomics Center.|b Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505, USA.
authors.forename: Fouad A
authors.initials: FA
authors.lastname: Zouein
authors.affiliation: a San Antonio Cardiovascular Proteomics Center.|b Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tian
authors.affiliation: a San Antonio Cardiovascular Proteomics Center.|b Mississippi Center for Heart Research, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505, USA.
authors.forename: Rugmani
authors.initials: R
authors.lastname: Padmanabhan Iyer
authors.affiliation: a San Antonio Cardiovascular Proteomics Center.|d East Carolina University, Department of Physiology, Greenville, North Carolina, USA.
authors.forename: Lisandra E
authors.initials: LE
authors.lastname: de Castro Brás
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005347:Fibroblasts / Q000187:drug effects; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D032383:Myocytes, Cardiac / Q000187:drug effects; D053495:Osteopontin / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D059748:Proteolysis; D011994:Recombinant Proteins; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D020257:Ventricular Remodeling / Q000502:physiology
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D053495:Osteopontin; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse
keywords: MMP-9; MPM-9; cardiac; cardiaque; cleavage sites; fibroblastes; fibroblasts; infarctus du myocarde; mass spectrometry; matrix metalloproteinases; myocardial infarction; métalloprotéinases matricielles; osteopontin; ostéopontine; peptides; proteomics; protéomique; sites de clivage; spectométrie de masse