title: High affinity binding of latent matrix metalloproteinase-9 to the alpha2(IV) chain of collagen IV.
abstract: Association of matrix metalloproteinases (MMPs) with the cell surface and with areas of cell-matrix contacts is critical for extracellular matrix degradation. Previously, we showed the surface association of pro-MMP-9 in human breast epithelial MCF10A cells. Here, we have characterized the binding parameters of pro-MMP-9 and show that the enzyme binds with high affinity (Kd approximately 22 nM) to MCF10A cells and other cell lines. Binding of pro-MMP-9 to MCF10A cells does not result in zymogen activation and is not followed by ligand internalization, even after complex formation with tissue inhibitor of metalloproteinase-1 (TIMP-1). A 190-kDa cell surface protein was identified by ligand blot analysis and affinity purification with immobilized pro-MMP-9. Microsequencing and immunoblot analysis revealed that the 190-kDa protein is the alpha2(IV) chain of collagen IV. Specific pro-MMP-9 surface binding was competed with purified alpha2(IV) and was significantly reduced after treatment of the cells with active MMP-9 before the binding assay since alpha2(IV) is hydrolyzed by MMP-9. A pro-MMP-9.TIMP-1 complex and MMP-9 bind to alpha2(IV), suggesting that neither the C-terminal nor the N-terminal domain of the enzyme is directly involved in alpha2(IV) binding. The closely related pro-MMP-2 exhibits a weaker affinity for alpha2(IV) compared with that of pro-MMP-9, suggesting that sites other than the gelatin-binding domain may be involved in the binding of alpha2(IV) to pro-MMP-9. Although pro-MMP-9 forms a complex with alpha2(IV), the proenzyme does not bind to triple-helical collagen IV. These studies suggest a unique interaction between pro-MMP-9 and alpha2(IV) that may play a role in targeting the zymogen to cell-matrix contacts and in the degradation of the collagen IV network.
authors.affiliation: Department of Pathology and the Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Olson
authors.forename: M
authors.initials: M
authors.lastname: Toth
authors.forename: D C
authors.initials: DC
authors.lastname: Gervasi
authors.forename: Y
authors.initials: Y
authors.lastname: Sado
authors.forename: Y
authors.initials: Y
authors.lastname: Ninomiya
authors.forename: R
authors.initials: R
authors.lastname: Fridman
mesh_terms: D000818:Animals; D001485:Basement Membrane / Q000201:enzymology / Q000378:metabolism; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002846:Chromatography, Affinity; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D017364:Collagenases / Q000302:isolation & purification / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D005109:Extracellular Matrix / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D020780:Matrix Metalloproteinase 9; D051379:Mice; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D004792:Enzyme Precursors; D007457:Iodine Radioisotopes; D003094:Collagen; D017364:Collagenases; D020780:Matrix Metalloproteinase 9