Although matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components in vitro, the role of MMPs in the in vivo accumulation of the cells to the site of inflammation in bronchial asthma is still obscure. In this study, we investigated the role of MMPs in the pathogenesis of bronchial asthma, using a murine model of allergic asthma. In this model, we observed the increase of the release of MMP-2 and MMP-9 in bronchoalveolar lavage fluids after Ag inhalation in the mice sensitized with OVA, which was accompanied by the infiltration of lymphocytes and eosinophils. Administration of tissue inhibitor of metalloproteinase-2 to airways inhibited the Ag-induced infiltration of lymphocytes and eosinophils to airway wall and lumen, reduced Ag-induced airway hyperresponsiveness, and increased the numbers of eosinophils and lymphocytes in peripheral blood. The inhibition of cellular infiltration to airway lumen was observed also with tissue inhibitor of metalloproteinase-1 and a synthetic matrix metalloproteinase inhibitor. These data suggest that MMPs, especially MMP-2 and MMP-9, are crucial for the infiltration of inflammatory cells and the induction of airway hyperresponsiveness, which are pathophysiologic features of bronchial asthma, and further raise the possibility of the inhibition of MMPs as a therapeutic strategy of bronchial asthma.

Human pro-matrix metalloproteinase 3 (proMMP-3) lacking the N-terminal 34 amino acids and the C-terminal hemopexin-like domain was expressed in E. coli and used to investigate the process of proenzyme activation and its interaction with an endogenous inhibitor TIMP-1 during activation. The truncated precursor was purified from the E. coli extract in the presence of 5mM EGTA. The active 23.5 kDa form was generated simply by exposure to Ca2+ and Zn2+ but not either by Ca2+ alone or by Zn2+ alone. The rate of MMP-3(deltaC) formation was concentration dependent, indicating that autoactivation is a bimolecular reaction. The truncated precursor was able to interact with the N-terminal domain of TIMP-1 without losing the 48 residue-long propeptide. However, upon a longer incubation, the propeptide was slowly processed, indicating that the association of the N-terminally truncated proMMP-3 with TIMP-1 is weaker than that of the fully activated MMP-3 and TIMP-1. These results indicate that the expression of MMP activities is regulated by endogenous inhibitor TIMPs during their activation processes which provide an additional control mechanism of extracellular matrix breakdown.

Type IV collagen α1‐α6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture Syndrome, an autoimmune disorder, and Alport Syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C‐terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, α‐1‐like and α2‐like, and suggested that vertebrate α3/α4 sequences evolved before α1/α2 and α5/α6. About one fifth of NC1 residues were identified to confer either the α1 or α2 group‐specificity. These residues accumulate opposite Charge in subdomain B of α1 (positive) and α2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic β‐strands and one short α‐helix with a significant hydrophobic moment. The existence of opposite charges in the a‐helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport Syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP‐2, MMP‐3) toward a peptide Substrate, though not to [14C]‐gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying Proteinase inhibitor function.

In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin 1) with preparations of BAG-75 led to inactivation of proteolytic function, e.g., an inability to fragment 125I-labeled BAG-75 added subsequently. MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations. Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of <10 kDa. The fate of MMP-3 incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals. Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM. Autolysis was maximal in the presence of 150 microg/ml hydroxyapatite where MMP-3 was only partially bound to crystals. However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 microg/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.

A bacterial expression system for the inhibitory N-terminal domain of human tissue inhibitor of metalloproteinases 1 (N-TIMP-1) (Huang, W., Suzuki, K., Nagase, H., Arumugam, S., Van Doren, S. R., and Brew, K. (1996) FEBS Lett. 384, 155–161) has been used to produce 20 single- and double-site mutants that probe the roles of different residues in its inhibitory action on metalloproteinases. Mutations that produce the largest increases in theKi for a C-terminally truncated form of stromelysin 1, MMP-3(ΔC), but do not disturb the conformation involve substitutions of residues that are located in a ridge that is centered around the disulfide bond between Cys1 and Cys70. Specific residues that have a large influence on activity include Cys1, Thr2, Met66, Val69, and Cys70. Of the mutations introduced, the greatest functional disturbances, reflected inKi increases of 2–4 orders of magnitude, are generated by changes that disrupt the Cys1–Cys70 disulfide bond and by substitution of Ala for Thr2. Most mutations that perturb the interaction with MMP-3 have parallel effects on the affinity of N-TIMP-1 for MMP-1 (interstitial collagenase) and MMP-2 (gelatinase A). However, the Thr2 to Ala mutation produces an inhibitor that is 17-fold more effective against MMP-3 than MMP-1, suggesting that it is feasible to engineer TIMP-1 variants that are more specifically targeted to selected matrix metalloproteinases. The reactive site identified by these studies is a structurally constrained but elongated region of TIMP that can fit the matrix metalloproteinase substrate-binding site.

Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs) by forming a 1:1 stoichiometric complex, but the inhibition mechanism of these inhibitors is not known. Here we have investigated the reactive site of TIMP-1 by its proteinase susceptibility before and after forming a complex with MMP-3 (stromelysin 1). When TIMP-1 was allowed to react with human neutrophil elastase, its inhibitory activity was destroyed. This resulted from cleavage of the Val69-Cys70 bond. However, cleavage of this bond by neutrophil elastase was prevented when TIMP-1 formed a complex with the catalytic domain of MMP-3, and full TIMP-1 activity was restored after dissociation of the complex at pH 3.0 in the presence of EDTA. These results indicate that the region around Val69 closely associates with an active MMP. The three-dimensional structure of the N-terminal domain of TIMP-2 elucidated by NMR studies [Williamson, Martorell, Carr, Murphy, Docherty, Freedman and Feeney (1994) Biochemistry 33, 11745-11759] reveals that Val69 and Cys70 form part of an extended ridge that also includes the N-terminal section of the inhibitor. This region is probably involved in the interaction with the catalytic domains of MMPs.

Matrix metalloproteinases (MMPs), also termed "matrixins", constitute a family of zinc metalloendopeptidases that participate in breakdown of extracellular matrix macromolecules (Woessner, 1991). These enzymes are considered to play an important role in many biological processes such as in reproduction, embryogenesis, tissue resorption, and in the control of cell behavior. Overproduction of matrixins is associated with various connective tissue diseases such as arthritis, periodontitis, glomerulonephritis, tissue ulceration as well as being connected with tumor cell invasion and metastasis (Woessner, 1991; Birkedal-Hansen et al., 1993).

Methods are described for producing an active amino‐terminal domain of tissue inhibitor of metalloproteinases‐1 (N‐TIMP‐1) from inactive protein expressed as inclusion bodies in E. coli. Yields exceed 20 mg per litre of bacterial culture. Activity measurements, CD spectroscopy and NMR spectroscopy of the 15N‐labeled protein show that it is fully active, homogeneous in conformation and suitable for high‐resolution structural analysis. The affinity of N‐TIMP‐1 for matrix metalloproteinases 1, 2 and 3 is 6–8‐fold less than that of the recombinant full‐length protein, indicating that deletion of the C‐terminal domain reduces the free energy of interaction by < 10%.

Histological studies have previously demonstrated an association between mast-cell activation/degranulation and areas of connective-tissue lysis in vivo; in addition, mast-cell extracts have been shown to activate latent forms of collagenase and stromelysin. In the present study we have examined the potential roles of rat mast-cell proteinase (RMCP) I and RMCP II as activators of the precursors of matrix metalloproteinase (MMP)-1 (interstitial collagenase), MMP-2 (gelatinase A) and MMP-3 (stromelysin 1). Both RMCPs I and II activated proMMP-3 by converting the 57 kDa precursor into a 45 kDa polypeptide. The N-terminal amino acid of 45 kDa MMP-3 activated by RMCP II was identified as Phe83. By contrast, only RMCP II activated the 52 kDa proMMP-1 by converting it into a 41 kDa protein and generating the new N-termini, namely Gln80 and Val82. The collagenolytic activity which resulted from this cleavage was only 35% of the full activity, but this could not be augmented by subsequent treatment with MMP-3, the latter being a crucial enzyme for the generation of the fully active MMP-1 with Phe81 at the N-terminus, in conjunction with other serine proteinases. Thus RMCP II activates proMMP-1 via a mechanism different from that reported for the stepwise processing by combinations of other trypsin-like enzymes and MMP-3. ProMMP-2 (pro-gelatinase A) was not activated by either RMCP I or RMCP II, despite processing to smaller products.

Insulin-like growth factor binding protein-3 (IG-FBP-3) is degraded by a Zn(2+)-dependent protease(s) produced by human dermal fibroblasts in vitro (Fowlkes, J. (1994) Endocrine J. 2, 63-68). Initial studies using IG-FBP-3-substrate zymography identified several IGFBP-3-degrading proteases with M(r) 52,000-72,000, which were inhibitable by EDTA and were shifted to lower M(r) species after treatment of conditioned medium with an organomercurial, suggesting that they might represent one or more of the matrix metalloproteinases (MMPs). Immunoblotting of conditioned medium demonstrated the presence of proMMP-1 (52 and 55 kDa), proMMP-3 (58 and 60 kDa), and proMMP-2 (72 kDa) whose molecular masses corresponded identically to those of the IGFBP-3-degrading proteases. Degradation of recombinant human (rh) IGFBP-3 by conditioned media was blocked (> 80% inhibition) by tissue inhibitor of metallo-proteinases-1, a specific inhibitor of all MMPs, while removal of MMPs -1, -2, and -3 from conditioned medium by sequential immunoaffinity and gelatin-Sepharose chromatography resulted in the complete loss of IGFBP-3-degrading proteinase activity. Furthermore, human MMP-1, MMP-3, and to a lesser extent MMP-2 degraded rhIGFBP-3 in vitro. Sequence analysis of rhIGFBP-3 cleavage sites produced by MMP-1, -2, or -3 demonstrated that each cleaved within the mid-region of the binding protein, a domain with little or no homology with the other five cloned IGFBPs. These studies suggest that MMPs, beyond their previously described functions as extracellular degrading enzymes, may also exert effects on cellular growth and proliferation via degradation of IGFBP-3, thus enhancing IGF bioavailability.

The zymogens of matrix metalloproteinase 1 (MMP-1: tissue collagenase), MMP-2 (gelatinase/type IV collagenase) and MMP-3 (stromelysin) were purified from the culture medium of human rheumatoid synovial fibroblasts and the mechanisms of activation of each zymogen by proteinases and 4-aminophenylmercuric acetate (APMA) were studied by kinetic and sequence analyses. The treatment of proMMP-1 (M(r) = 52,000) with proteinases or APMA converted the zymogen to M(r) = 43,000, but it exhibited only 14-25% of the maximal activity. Incubation of a partially active MMP-1 with MMP-3 resulted in rapid, full activation by generating the 41,000-M(r) MMP-1 with Phe81 as the NH2-terminus. MMP-3 directly activated proMMP-1 by cleaving the Gln80-Phe81 bond, but this reaction was extremely slow, indicating that the Gln80-Phe81 bond is not readily available to MMP-3 in the native proMMP-1 molecule. ProMMP-2 (M(r) = 72,000) was activated only by APMA, but not by proteinases. The activation by APMA was rapid and generated an active MMP-2 of M(r) 68,000, but the enzymic activity declined rapidly after activation by autolysis. The NH2-terminal sequence analysis of active MMP-2 indicated that the Asn80-Tyr81 bond was cleaved upon APMA treatment. In contrast, proMMP-3 (M(r) = 57,000) was activated by a variety of proteinases with different specificities. The initial attacks of these proteinases are on a stretch of highly charged groups at the position 34-39 in the propeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Matrix metalloproteinases play a central role in the catabolism of extracellular matrix macromolecules. Here the authors report that giant cell tumor of bone (GCT) produces two matrix metalloproteinases (MMPs) in zymogen form, which have been identified as proMMP-2 (also known as "72-kDa-progelatinase/type IV procollagenase") and proMMP-3 (prostromelysin). Giant cell tumor is known to consist of two major cell populations, multinucleated giant cells and stromal cells. On several passages of the tumor cells in culture, only stromal cells proliferated. These stromal cells produced proMMP-2 but not proMMP-3. Addition of the conditioned medium of primary GCT culture or human macrophage-conditioned medium to the passaged stromal cells induced the production of proMMP-3. The production of proMMP-3 was also induced by interleukin 1 (IL-1), but not by tumor necrosis factor alpha (TNF alpha). ProMMP-1 (tissue procollagenase) was not detected even after treatment with these stimuli. Immunohistochemical studies have demonstrated that multinucleated giant cells in GCT both produce IL-1 and TNF alpha, suggesting that IL-1 secreted by multinucleated giant cells may be responsible for in vivo production of proMMP-3 by the stromal cells. The authors propose that GCT has a self-stimulatory system for the production of matrix-degrading proteinases and that the ability of the passaged stromal cells to synthesize and secrete proMMP-3 with appropriate stimuli may contribute the malignant behavior of GCT.

1. We previously reported an endogenous activator of procollagenase from the culture medium of rabbit uterine cervical fibroblasts (Ishibashi et al. (1987) Biochem. J. 241, 527-534).2. Similar activator was also purified and characterized from rabbit synovial fibroblasts (Vater et al. (1983) J. biol. Chem. 258, 9374-9382), but its mode of activation of procollagenase was reported to be different from that of purified activator from uterine cervical fibroblasts.3. Here we report the comparative studies of the two activators of procollagenase and demonstrate that they are identified as matrix metalloproteinase 3 (stromelysin) by their immunological and functional criteria. The specific role of the activator in procollagenase activation is also described.

Two zymogens of matrix metalloproteinases (MMPs), proMMP-1 (tissue procollagenase) and proMMP-3 (prostromelysin) were isolated from the culture medium of human rheumatoid synovial fibroplasts and their activation mechanisms by proteinases and 4-aminophenylmercuric acetate (APMA) were studied by kinetic and sequence analyses. Both zymogens were activated by unique stepwise activation mechanisms through which sequential processing events occur in the propeptide regions. The initial cleavage sites attacked by activator proteinases are located in the middle of the propeptides at Glu33-Lys-Arg-Arg-Asn37 in proMMP-1 and Phe34-Val-Arg-Arg-Lys-Asp39 in proMMP-3. The initial products of proMMP-1 generated by proteinases then undergo further autocleavage of the Thr64-Leu65 bond. The treatment of proMMP-1 and proMMP-3 with APMA results in the intramolecular cleavage of the Val67-Met68 and Glu68-Val69 bonds, respectively. The removal of a portion of propeptides results in conformational changes around the Gln80-Phe81 and His82-Phe83 bonds in respective intermediates of MMP-1 and MMP-3 and render them to rapid specific cleavage by MMP-3 to generate stable, fully active enzymes.

Conference Article| August 01 1991 Substrate specificities and activation mechanisms of matrix metalloproteinases Hideaki Nagase; Hideaki Nagase 1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Yutaka Ogata; Yutaka Ogata 1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Ko Suzuki; Ko Suzuki 1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Jan J. Enghild; Jan J. Enghild *Department of Pathology, Duke University Medical Center, Durham, North Carolina 22710, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Guy Salvesen Guy Salvesen *Department of Pathology, Duke University Medical Center, Durham, North Carolina 22710, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (3): 715–718. https://doi.org/10.1042/bst0190715 Article history Received: April 25 1991 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation Hideaki Nagase, Yutaka Ogata, Ko Suzuki, Jan J. Enghild, Guy Salvesen; Substrate specificities and activation mechanisms of matrix metalloproteinases. Biochem Soc Trans 1 August 1991; 19 (3): 715–718. doi: https://doi.org/10.1042/bst0190715 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: MMP, matrix metalloproteinase, TIMP, tissue inhibitor of metalloproteinases, APMA, 4-aminophenylmercuric acetate This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.