HIGH-AFFINITY BINDING OF LATENT MATRIX-METALLOPROTEINASE-9 TO THE ALPHA-2(IV) CHAIN OF COLLAGEN-IV

Association of matrix metalloproteinases (MMPs) with the cell surface and with areas of cell-matrix contacts is critical for extracellular m atrix degradation. Previously, we showed the surface association of pr o-MMP-9 in human breast epithelial MCF10A cells. Here, we have charact erized the binding parameters of pro-MMP-9 and show that the enzyme bi nds with high affinity (K-d similar to 22 nM) to MCF10A cells and othe r 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. Micros equencing and immunoblot analysis revealed that the 190-kDa protein is the alpha 2(IV) chain of collagen IV. Specific pro-MMP-9 surface bind ing was competed with purified alpha 2(IV) and was significantly reduc ed after treatment of the cells with active MMP-9 before the binding a ssay since alpha 2(IV) is hydrolyzed by MMP-9. A pro-MMP-9 TIMP-1 comp lex and MMP-9 bind to alpha 2(IV), suggesting that neither the C-termi nal nor the N-terminal domain of the enzyme is directly involved in al pha 2(IV) binding. The closely related pro-MMP-2 exhibits a weaker aff inity for alpha 2(IV) compared with that of pro-MMP-9, suggesting that sites other than the gelatin-binding domain may be involved in the bi nding of alpha 2(IV) to pro-MMP-9. Although pro-MMP-9 forms a complex with alpha 2(IV), the proenzyme does not bind to triple-helical collag en IV. These studies suggest a unique interaction between pro-MMP-9 an d alpha 2(IV) that may play a role in targeting the zymogen to cell-ma trix contacts and in the degradation of the collagen IV network.