X-ray absorption studies of human matrix metalloproteinase-2 (MMP-2) bound to a highly selective mechanism-based inhibitor. comparison with the latent and active forms of the enzyme.

Malignant tumors express high levels of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metastasis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been implicated in malignant processes, and thus, they have been a target for drugs designed to block their activity. Therefore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full-length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of MMP-2 in complex with SB-3CT, a recently reported novel mechanism-based synthetic inhibitor that was designed to be highly selective in gelatinases. It is shown that SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme.

Tissue inhibitor of metalloproteinase (TIMP)-2 acts synergistically with synthetic matrix metalloproteinase (MMP) inhibitors but not with TIMP-4 to enhance the (Membrane type 1)-MMP-dependent activation of pro-MMP-2.

The membrane-type 1 matrix metalloproteinase (MT1-MMP) has been shown to be a key enzyme in tumor angiogenesis and metastasis. MT1-MMP hydrolyzes a variety of extracellular matrix components and is a physiological activator of pro-MMP-2, another MMP involved in malignancy. Pro-MMP-2 activation by MT1-MMP involves the formation of an MT1-MMP.tissue inhibitors of metalloproteinases 2 (TIMP-2). pro-MMP-2 complex on the cell surface that promotes the hydrolysis of pro-MMP-2 by a neighboring TIMP-2-free MT1-MMP. The MT1-MMP. TIMP-2 complex also serves to reduce the intermolecular autocatalytic turnover of MT1-MMP, resulting in accumulation of active MT1-MMP (57 kDa) on the cell surface. Evidence shown here in Timp2-null cells demonstrates that pro-MMP-2 activation by MT1-MMP requires TIMP-2. In contrast, a C-terminally deleted TIMP-2 (Delta-TIMP-2), unable to form ternary complex, had no effect. However, Delta-TIMP-2 and certain synthetic MMP inhibitors, which inhibit MT1-MMP autocatalysis, can act synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP. In contrast, TIMP-4, an efficient MT1-MMP inhibitor, had no synergistic effect. These studies suggest that under certain conditions the pericellular activity of MT1-MMP in the presence of TIMP-2 can be modulated by synthetic and natural (TIMP-4) MMP inhibitors.

Binding of active (57 kDa) membrane type 1-matrix metalloproteinase (MT1-MMP) to tissue inhibitor of metalloproteinase (TIMP)-2 regulates MT1-MMP processing and pro-MMP-2 activation.

Previous studies have shown that membrane type 1-matrix metalloproteinase (MT1-MMP) (MMP-14) initiates pro-MMP-2 activation in a process that is tightly regulated by the level of tissue inhibitor of metalloproteinase (TIMP)-2. However, given the difficulty in modulating TIMP-2 levels, the direct effect of TIMP-2 on MT1-MMP processing and on pro-MMP-2 activation in a cellular system could not be established. Here, recombinant vaccinia viruses encoding full-length MT1-MMP or TIMP-2 were used to express MT1-MMP alone or in combination with various levels of TIMP-2 in mammalian cells. We show that TIMP-2 regulates the amount of active MT1-MMP (57 kDa) on the cell surface whereas in the absence of TIMP-2 MT1-MMP undergoes autocatalysis to a 44-kDa form, which displays a N terminus starting at Gly(285) and hence lacks the entire catalytic domain. Neither pro-MT1-MMP (N terminus Ser(24)) nor the 44-kDa form bound TIMP-2. In contrast, active MT1-MMP (N terminus Tyr(112)) formed a complex with TIMP-2 suggesting that regulation of MT1-MMP processing is mediated by a complex of TIMP-2 with the active enzyme. Consistently, TIMP-2 enhanced the activation of pro-MMP-2 by MT1-MMP. Thus, under controlled conditions, TIMP-2 may act as a positive regulator of MT1-MMP activity by promoting the availability of active MT1-MMP on the cell surface and consequently, may support pericellular proteolysis.

Characterization of the monomeric and dimeric forms of latent and active matrix metalloproteinase-9. Differential rates for activation by stromelysin 1.

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K(m) values in the nanomolar range and relative low k(cat) values (1.9 x 10(-3) and 4.1 x 10(-4) s(-1), for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1.

Matrix metalloproteinase-9 expression in bladder washes from bladder cancer patients predicts pathological stage and grade.

The matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and MMP-9) have been associated with tumor cell invasion and metastasis in many human cancers. Here we examined the expression of proMMP-2 (gelatinase A) and proMMP-9 (gelatinase B) proteins in the cellular component of bladder washes obtained from 65 patients. Twenty-six patients had active bladder cancer, 24 had a history of bladder cancer but no evidence of active disease at the time of cystoscopy (recurrence-free), and 15 patients had lesions other than bladder cancer (controls). The results were correlated with the cytological findings of the bladder wash and the histopathological results of the tumor resection when performed. In patients with active transitional cell carcinoma of the bladder, 71 and 38% had expression and overexpression of the latent form of MMP-9 (proMMP-9), respectively. In contrast, neither latent nor active MMP-2 could be detected in any of the samples examined, regardless of tumor status. Overexpression of proMMP-9 correlated with higher grade (P = 0.003) and pathological stage (P = 0.04) of disease in the active bladder cancer group. No significant gelatinase expression was detected in the recurrence-free and control cases. Compared with urine cytology, proMMP-9 expression had an overall higher sensitivity for bladder cancer identification (71 versus 54%, P = 0.11). Detection of proMMP-9 in bladder washes may be a novel approach for the identification of patients with more aggressive forms of bladder cancer.

High affinity binding of latent matrix metalloproteinase-9 to the alpha2(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 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.

Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2.

The dissociation constants (Kd) of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 for the active and latent forms of matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies. SPR analysis shows biphasic kinetics with high (nM) and low (microM) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- and 82-kDa species), respectively. In contrast, binding data of TIMP-2 to an MMP-2 45-kDa active form lacking the C-terminal domain and to an MMP-2 C-terminal domain (CTD) fragment displays monophasic kinetics with Kd values of 315 and 60 nM, respectively. This suggests that the CTD contains the high affinity binding site, whereas the catalytic domain contains the low affinity site. Also, binding of TIMP-2 to pro-MMP-2 is stronger at both the high and low affinity sites than the corresponding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the importance of the N-terminal prodomain. In addition, the Kd value of TIMP-1 for the MMP-2 62-kDa species is 28. 6 nM at the high affinity site, yet neither the MMP-2 45-kDa species nor the CTD interacts with TIMP-1. Enzyme inhibition studies demonstrate that TIMPs are slow binding inhibitors with monophasic inhibition kinetics. This suggests that a single binding event results in enzyme inhibition. The kinetic parameters for the onset of inhibition are fast (kon approximately 10(5) M-1 s-1) with slow off rates (koff approximately 10(-3) s-1). The inhibition constants (Ki) are in the 10(-7)-10(-9) M range and correlate with the values determined by SPR.

Phorbol ester-induced cell surface association of matrix metalloproteinase-9 in human MCF10A breast epithelial cells.

Cell surface association of extracellular matrix (ECM)-degrading enzymes has been suggested to facilitate proteolysis of ECM in areas of cell-matrix contacts and to be crucial for the process of tumor cell invasion. Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family of endopeptidases that has been shown to play a critical role in hydrolysis of ECM components and has been localized on the surface of tumor cells. However, the nature of the cell surface association of MMP-9 is unknown. Here, we report the cell surface association of MMP-9 in human breast epithelial MCF10A cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Surface biotinylation and immunoprecipitation with anti-MMP-9 antibodies revealed the presence of two MMP-9 forms (M(r) 92,000 and 85,000) on the surface of TPA-treated MCF10A cells, whereas in the media, only the M(r) 92,000 form was detected, mostly in complex with TIMP-1, a specific MMP-9 inhibitor. The MMP-9 forms were also found in purified plasma membranes of TPA-treated cells. In contrast, the plasma membranes contained little or no TIMP-1. The surface-bound MMP-9 forms were recognized by an antibody to the NH2-terminal prodomain, indicating that both represent latent enzymes. Pulse-chase analysis and endoglycosidase H digestion of surface-biotinylated MMP-9 forms demonstrated that the M(r) 85,000 species was endoglycosidase H sensitive, suggesting targeting of the precursor form of MMP-9 to the cell surface. These studies demonstrate a specific cell surface association of MMP-9 in response to TPA that may help to localize TIMP-1-free enzyme on the surface of breast epithelial cells.

Carbohydrate-mediated regulation of matrix metalloproteinase-2 activation in normal human fibroblasts and fibrosarcoma cells.

Matrix metalloproteinase-2 (MMP-2) is activated on the cell surface by membrane type 1-MMP (MT1-MMP). Activation of proMMP-2 is induced in vitro by concanavalin A (ConA). The regulation of proMMP-2 activation is, however, not yet fully understood. We investigated the effect of plant lectins, carbohydrates and inhibitors of the cytoskeleton on proMMP-2 activation in normal (HLF1) and malignant fibroblast (HT1080) cells. Native ConA induced proMMP-2 activation in both cell types while dimeric succinyl-ConA had no effect, suggesting that receptor clustering is involved in activation. Wheat germ agglutinin (WGA) also induced proMMP-2 activation. N-acetyl-D-glucosamine (GlcNac) inhibited the effects of ConA and WGA while mannose only inhibited ConA-induced proMMP-2 activation. Mannose also inhibited the expression of MT1-MMP mRNA induced by ConA. Cytochalasin B and colchicine had no effect on the ConA induction of proMMP-2 activation. These studies help to define some of the cellular and molecular mechanisms for the induction of proMMP-2 activation.