Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.35 (matrix metalloproteinase 9)
 2,207 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have reported that SV40-transformed human lung fibroblasts secrete a 92-kDa metalloprotease which is not detectable in the parental cell line IMR-90. We now present the complete structure of this enzyme along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes. A similar, perhaps identical, enzyme can be released by polymorphonuclear cells. The preproenzyme is synthesized as a polypeptide of predicted Mr 78,426 containing a 19 amino-acid-long signal peptide and secreted as a single 92,000 glycosylated proenzyme. The purified proenzyme complexes noncovalently with the tissue inhibitor of metalloproteases (TIMP) and can be activated by organomercurials. Activation with phenylmercuric chloride results in removal of 73 amino acids from the NH2 terminus of the proenzyme, yielding an active form capable of digesting native types IV and V collagen. The in vitro substrate specificity of the enzyme using these substrates was indistinguishable from that of the 72-kDa type IV collagenase. The 92-kDa type IV collagenase consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-1, and a unique 54-amino-acid-long proline-rich domain homologous to the alpha 2 chain of type V collagen.
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PMID:SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. 255 98

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.
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PMID:Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain. 821 28

Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases-1 (rTIMP-1) and wild-type and mutant human collagenase type I (rMMP-1) proteins in SF9 cells by the baculovirus expression system. Wild-type MMP-1, as well as the MMP-1 mutant lacking the C-terminal hemopexin-like domain [des-(248-450)-MMP-1], exhibit enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4-aminophenylmercuric acetate. Enzyme activity of both proteins can be inhibited by addition of rTIMP. Deletion of the complete active-site [des-(161-228)-MMP-1] within the catalytic domain, or mutation of a single His residue of the Zn2+ binding domain (His199), generates stable forms of MMP-1 proteins which are unable to digest collagen type I or beta-casein. In addition to co-immunoprecipitation analysis, we have established a rapid and sensitive ELISA assay using immobilized rTIMP to determine the structural requirements of MMP-1 to form complexes with its inhibitor. Only the activated and not the latent forms of wild-type and C-terminal mutant des-(248-450)-MMP-1 proteins are able to form complexes with TIMP. Neither mutation of His199, nor deletion mutants des-(161-228)-MMP-1 and des-(161-228/248-450)-MMP-1, interact with TIMP. This demonstrates that the C-terminal hemopexin domain of MMP-1, in contrast to the corresponding regions of gelatinase A and gelatinase B, does not interact with TIMP-1. In summary, we have shown that the integrity of the catalytic domain of MMP-1 and its ability to bind Zn2+ is absolutely required for complex formation with TIMP-1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1.
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PMID:The catalytic domain of activated collagenase I (MMP-1) is absolutely required for interaction with its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1). 906 49

We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of stromelysin revealed that residues 568-631 of gelatinase A were required for rapid association with TIMP-3. The N-terminal domain of gelatinase B alone also showed slower association with TIMP-3, again implying significant C-domain interactions. The isolation of complexes between TIMP-3 and progelatinases A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proenzymes. We analyzed the effect of various polyanions on the inhibitory activity of TIMP-3 in our soluble assay. The association rate was increased by dextran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matrix, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity.
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PMID:Human tissue inhibitor of metalloproteinases 3 interacts with both the N- and C-terminal domains of gelatinases A and B. Regulation by polyanions. 1019 61

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75-90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.
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PMID:The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties. 1101 Sep 69

The inherited osteolyses or 'vanishing bone' syndromes are a group of rare disorders of unknown etiology characterized by destruction and resorption of affected bones. The multicentric osteolyses are notable for interphalangeal joint erosions that mimic severe juvenile rheumatoid arthritis (OMIMs 166300, 259600, 259610 and 277950). We recently described an autosomal recessive form of multicentric osteolysis with carpal and tarsal resorption, crippling arthritic changes, marked osteoporosis, palmar and plantar subcutaneous nodules and distinctive facies in a number of consanguineous Saudi Arabian families. We localized the disease gene to 16q12-21 by using members of these families for a genome-wide search for homozygous-by-descent microsatellite markers. Haplotype analysis narrowed the critical region to a 1.2-cM region that spans the gene encoding MMP-2 (gelatinase A, collagenase type IV; (ref. 3). We detected no MMP2 enzymatic activity in the serum or fibroblasts of affected family members. We identified two family-specific homoallelic MMP2 mutations: R101H and Y244X. The nonsense mutation effects a deletion of the substrate-binding and catalytic sites and the fibronectin type II-like and hemopexin/TIMP2 binding domains. Based on molecular modeling, the missense mutation disrupts hydrogen bond formation within the highly conserved prodomain adjacent to the catalytic zinc ion.
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PMID:Mutation of the matrix metalloproteinase 2 gene (MMP2) causes a multicentric osteolysis and arthritis syndrome. 1143 82

The gelatinases are a subgroup of the matrix metalloproteinase family. The interaction of their C-terminal hemopexin-like domain with a tissue inhibitor of metalloproteinases (TIMP) is a major part of the regulatory mechanisms of gelatinases. To investigate the interaction of the hemopexin-like domain of gelatinase B (92-Pex) and TIMP-1, we expressed the individual domain in Pichia pastoris. The active refolded domain was purified by ion exchange chromatography and gel filtration. We investigated the formation of the 92-Pex/TIMP-1 complex by surface plasmon resonance (SPR). The dissociation constant Kd was calculated to be 0.86 nM. Analogous to the complex of the hemopexin-like domain of gelatinase A and TIMP-2 (Olson, M. W. et al., 1997), the binding curves of the 92-Pex/TIMP-1 complex were best fitted with a monophasic model.
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PMID:Kinetic analysis of the binding of hemopexin-like domain of gelatinase B cloned and expressed in Pichia pastoris to tissue inhibitor of metalloproteinases-1. 1470 83

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.
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PMID:A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains. 1713 15

The procoagulatory serine protease, thrombin, is known to induce invasion and metastasis in various cancers, but the mechanisms by which it promotes tumorigenesis are poorly understood. Because the 92-kDa gelatinase (MMP-9) is a known mediator of tumor cell invasion, we sought to determine whether and how thrombin regulates MMP-9. The thrombin receptor, PAR-1, and MMP-9 are expressed in osteosarcomas, as determined by immunohistochemistry. Stimulation of U2-OS osteosarcoma cells with thrombin and a thrombin receptor-activating peptide induced pro-MMP-9 secretion as well as cell surface-associated pro-MMP-9 expression and proteolytic activity. This was paralleled by an increase in MMP-9 mRNA and MMP-9 promoter activity. Thrombin-induced invasion of U2-OS cells through Matrigel was mediated by the phosphatidylinositol 3-kinase signaling pathway and could be inhibited with an MMP-9 antibody. The stimulation of MMP-9 by thrombin was paralleled by an increase in beta1-integrin mRNA and beta1-integrin expression on the cell surface, which was also mediated by phosphatidylinositol 3-kinase and was required for invasion. Thrombin activation induced and co-localized both beta1-integrin and pro-MMP-9 on the cell membrane, as evidenced by co-immunoprecipitation, confocal microscopy, and a protein binding assay. The thrombin-mediated association of these two proteins, as well as thrombin-mediated invasion of U2-OS cells, could be blocked with a cyclic peptide and with an antibody preventing binding of the MMP-9 hemopexin domain to beta1-integrin. These results suggest that thrombin induces expression and association of beta1-integrin with MMP-9 and that the cell surface localization of the protease by the integrin promotes tumor cell invasion.
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PMID:Thrombin induces tumor invasion through the induction and association of matrix metalloproteinase-9 and beta1-integrin on the cell surface. 1804 60

The extracellular domain of beta-amyloid precursor protein (APP) contains an inhibitor against matrix metalloproteinase-2 (MMP-2, gelatinase A). Our previous study ( Higashi, S. and Miyazaki, K. (2003) J Biol Chem 278, 14020-14028 ) demonstrated that the inhibitor is localized within the ISYGN-DALMP sequence of APP, and a synthetic decapeptide containing this sequence (named APP-derived inhibitory peptide, APP-IP) selectively inhibits the activity of MMP-2. To determine the region of interaction that correlates with the selective inhibition, we constructed various MMP-2 mutants. An MMP-2 mutant, which had the hemopexin-like domain and three fibronectin-like type II domains of MMP-2 deleted, and native MMP-2 showed similar affinities for APP-IP, suggesting that only the catalytic domain of MMP-2 is essential for the interaction. Studies of chimeric proteases, consisting of various parts of the MMP-2 catalytic domain and those of MMP-7 (matrilysin) or MMP-9 (gelatinase B), further revealed that Ala(88) and Gly(94) in the non-prime side and Tyr(145) and Thr(146) in the prime side of the substrate-binding cleft of MMP-2 contribute separately to the selective inhibition. Replacement of the amino acid residue at position 94 of a chimeric MMP mutant affected its interaction with the C-terminal Pro(10) of APP-IP, whereas that of residues 145-148 affected the interaction with Tyr(3) of the inhibitor, suggesting that the N to C direction of APP-IP relative to the substrate-binding cleft of MMP is analogous to that of propeptide in proMMP, and opposite to that of substrate. When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor.
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PMID:Identification of amino acid residues of the matrix metalloproteinase-2 essential for its selective inhibition by beta-amyloid precursor protein-derived inhibitor. 1823 79


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