title: Inhibition of MMP-9-dependent Degradation of Gelatin, but Not Other MMP-9 Substrates, by the MMP-9 Hemopexin Domain Blades 1 and 4.
abstract: Degradation and remodeling of the extracellular matrix by matrix metalloproteinases (MMPs) plays important roles in normal development, inflammation, and cancer. MMP-9 efficiently degrades the extracellular matrix component gelatin, and the hemopexin domain of MMP-9 (PEX9) inhibits this degradation. To study the molecular basis of this inhibition, we generated GST fusion proteins containing PEX9 or truncated forms corresponding to specific structural blades (B1-B4) of PEX9. GST-PEX9 inhibited MMP-9-driven gelatin proteolysis, measured by gelatin zymography, FITC-gelatin conversion, and DQ-gelatin degradation assays. However, GST-PEX9 did not prevent the degradation of other MMP-9 substrates, such as a fluorogenic peptide, αB crystalline, or nonmuscular actin. Therefore, PEX9 may inhibit gelatin degradation by shielding gelatin and specifically preventing its binding to MMP-9. Accordingly, GST-PEX9 also abolished the degradation of gelatin by MMP-2, confirming that PEX9 is not an MMP-9 antagonist. Moreover, GST-B4 and, to a lesser extent, GST-B1 also inhibited gelatin degradation by MMP-9, indicating that these regions are responsible for the inhibitory activity of PEX9. Accordingly, ELISAs demonstrated that GST-B4 and GST-B1 specifically bound to gelatin. Our results establish new functions of PEX9 attributed to blades B4 and B1 and should help in designing specific inhibitors of gelatin degradation.
authors.affiliation: From the Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain and the Department of Microbiology and Immunology, KULeuven-University of Leuven, Rega Institute for Medical Research, B-3000 Leuven, Belgium.
authors.forename: Estefanía
authors.initials: E
authors.lastname: Ugarte-Berzal
authors.affiliation: the Department of Microbiology and Immunology, KULeuven-University of Leuven, Rega Institute for Medical Research, B-3000 Leuven, Belgium.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Vandooren
authors.affiliation: From the Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain and.
authors.forename: Elvira
authors.initials: E
authors.lastname: Bailón
authors.affiliation: the Department of Microbiology and Immunology, KULeuven-University of Leuven, Rega Institute for Medical Research, B-3000 Leuven, Belgium.
authors.forename: Ghislain
authors.initials: G
authors.lastname: Opdenakker
authors.affiliation: From the Cellular and Molecular Medicine Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain and agarciapardo@cib.csic.es.
authors.forename: Angeles
authors.initials: A
authors.lastname: García-Pardo
mesh_terms: D015153:Blotting, Western; D002448:Cell Adhesion; D005780:Gelatin / Q000378:metabolism*; D006466:Hemopexin / Q000378:metabolism*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects*; D014407:Tumor Cells, Cultured
chemical_list: D005780:Gelatin; D006466:Hemopexin; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9
keywords: PEX9 blades 1 and 4; catalysis; enzyme antagonist; enzyme catalysis; gelatin degradation; hemopexin; inhibition mechanism; inhibitor; matrix metalloproteinase (MMP)