Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.23 (MMP)
4,246 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.
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PMID:The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B. 132 52

We have developed a monoclonal antibody AF-28 that specifically recognizes a neo-epitope on polypeptides with N-terminal FFGVG ... sequences. This sequence is found at the N-terminus of aggrecan fragments that have been digested with matrix metalloproteinases (MMPs). By immunoblotting, monoclonal antibody AF-28 specifically detected G2 fragments derived from an aggrecan G1-G2 substrate digested with stromelysin, collagenase, gelatinase and matrilysin, but failed to detect G2 fragments obtained from elastase, trypsin or cathepsin B digests. Undigested G1-G2 was not detected. In addition, AF-28 antibody detected fragments derived from whole aggrecan and this detection did not require prior treatment with chondroitinase or keratanase. Competition experiments confirmed that peptides containing internal ... FFGVG ... sequences were not detected by the antibody, while native MMP-digested aggrecan fragments and a synthetic 32-mer peptide with FFGVG ... N-termini were equally competitive on a molar basis. An FFGVG 5-mer, and an FGVGGEEDI9-mer which lacked the N-terminal phenylalanine residue, were 50 times and 230 times respectively less competitive than the FFGVG ... 32-mer. Two fragments from the interglobular domain, F342-F373 and F342-D441, that are predicted products of G1-G2 digestion by neutrophil collagenase but have not previously been detected, could be detected with AF-28. The epitope recognized by AF-28 was also detected in human synovial fluids by Western blot analysis. A broad band of 100-200 kDa was detected in some patients and a dominant band of 40-60 kDa was found in two patients. The size of this small fragment corresponds with that seen for the porcine F342-E373 product and may represent the natural physiological product of aggrecan cleaved in vivo at both the MMP site (... DIPEN341 decreases F342FGVG ...) and the aggrecanase site (... ITEGE373 decreases A374RGSVI ...).
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PMID:Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids. 754 17

The actions of human recombinant stromelysins-1 and -2, collagenase, gelatinases A and B and matrilysin on neonatal human proteoglycan aggregates were examined. With the exception of gelatinase B, aggrecan was degraded extensively by most metalloproteinases studied, whereas link protein showed only limited proteolysis. Sequencing studies of modified link protein components revealed that stromelysins-1 and -2, gelatinases A and B and collagenase cleaved specifically between His16 and Ile17, and matrilysin, stromelysin-2 and gelatinase A cleaved between Leu25 and Leu26. Cleavage at the former bond generated a link protein component with the same N-terminus as that isolated from newborn human cartilage. Based on previously determined in situ cleavage sites it is evident that matrix metalloproteinases are not solely responsible for the accumulation of link protein degradation products in adult human cartilage, indicating that additional proteolytic agents are involved in the normal catabolism of human cartilage matrix.
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PMID:Matrix metalloproteinases cleave at two distinct sites on human cartilage link protein. 769 69

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

Human pro-MMP-3 (pro-matrix metalloproteinase-3) was purified from three sources: articular cartilage and conditioned media from synovial fibroblasts and Chinese hamster ovary cells expressing recombinant pro-MMP-3. All three preparations reacted with two monoclonal antibodies specific for human fibroblast pro-MMP-3. Each preparation of active MMP-3 possessed properties identical to those previously reported for the cartilage acid metalloproteinase (MMP-6; Azzo and Woessner, J. F., Jr. (1986) J. Biol. Chem. 261, 5434-5441): an acid pH optimum of 5.3-5.5 for digestion of cartilage aggrecan; digestion of oxidized insulin B-chain at Ala14-Leu15 and Tyr16-Leu17 in a ratio of 3:1; and heat stability at neutral pH. Further characterization of MMP-3 establishes that the acid pH optimum for cartilage aggrecan is not due to substrate denaturation since the same optimum is found by viscosity assay, by SDS-polyacrylamide gel electrophoresis assay of G1 domain, and by digestion of aggrecan in fresh cartilage fragments in vitro. Fibronectin was also digested optimally at pH 5.5 and NH2-terminal sequence analysis revealed no pH change in a major proteolytic site of cleavage at the Pro689-Leu690 bond. The specificity constant kcat/Km is maximal at pH 5.5 as determined in a quenched fluorescence peptide assay. This is due to an increase in kcat at pH 5.5 without any substantial effect on Km. The affinity of MMP-3 for calcium is decreased about 10-fold at pH 5.3 compared to neutral pH. Finally, the neutral cartilage metalloproteinase is identified as 72-kDa pro-MMP-2 based on M(r), specificity of insulin B-chain cleavage, and reactivity with a specific polyclonal antibody to human MMP-2.
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PMID:Matrix metalloproteinase-3 (stromelysin-1). Identification as the cartilage acid metalloprotease and effect of pH on catalytic properties and calcium affinity. 840 46

Degradation of the large cartilage proteoglycan aggrecan in arthritis involves an unidentified enzyme aggrecanase, and at least one of the matrix metalloproteinases. Proteinase-sensitive cleavage sites in the aggrecan interglobular domain (IGD) have been identified for many of the humman MMPs, as well as for aggrecanase and other proteinases. The major MMP expressed by chondrocytes stimulated with retinoic acid to degrade their matrix is collagenase-3 or MMP-13. Because of its potential role in aggrecan degradation we examined the specificity of MMP-13 for an aggrecan substrate. The results show that MMP-13 cleaves aggrecan in the IGD at the same site (..PEN314-FFG..) identified for other members of the MMP family, and also at a novel site ..VKP384-VFE.. not previously observed for other proteinases.
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PMID:Degradation of cartilage aggrecan by collagenase-3 (MMP-13). 860 31

A series of isothiazolones that inhibit pro-(matrix metallo-proteinase) (proMMP) activation but do not inhibit the active enzyme are effective as cartilage protectants in bovine nasal cartilage organ culture, preventing interleukin-1 (IL-1)-induced proteoglycan (aggrecan) degradation without affecting its synthesis. These compounds were found to bind to prostromelysin (proMMP-3) in a non-dialysable and stoichiometric manner. Preincubation with cartilage-protectant isothiazolones prevented the binding of [14C]iodoacetamide to Cys75 of the MMP-3 propeptide, suggesting that the activity of these compounds involves their binding to the Cys75 of the MMP zymogen. Studies following chymotrypsin activation of proMMP-3 by SDS/PAGE indicated that altered processing of the 57 kDa zymogen to the active form occurred in the presence of compound. The 53 kDa intermediate seen on normal activation was not formed; instead a different intermediate appeared with a molecular mass of approx. 46 kDa. N-terminal sequence analysis indicated that this intermediate was formed by cleavage at the putative 4-aminophenylmercuric acid cleavage site. Importantly the 45 kDa active MMP-3 species formed in the presence of compound was one amino acid residue shorter than the native MMP-3. These results suggest that the inhibition of cartilage proteoglycan degradation by isothiazolones might be due to their ability to bind to the Cys75 in the propeptide region of the MMP zymogen and interfere with its normal activation process.
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PMID:Isothiazolones interfere with normal matrix metalloproteinase activation and inhibit cartilage proteoglycan degradation. 880 28

Soluble proenzyme forms of the catalytic domains of membrane-type matrix metalloproteinases 1 and 2 (MT1-MMP and MT2-MMP) and a form of MT1-MMP containing the catalytic and hemopexin domains were expressed as soluble recombinant proteins. Purified, activated forms of the MT-MMP were shown to degrade fibronectin, tenascin, nidogen, aggrecan and perlecan. Only MT2-MMP showed activity against laminin. MT1-MMP retaining the hemopexin domain was able to specifically cleave native type-I and type-III collagens into the 3/4-1/4 fragments typical of the specific collagenases. The catalytic domain alone did not retain this activity. The MT-MMP did not degrade interleukin-1beta, but, similarly to many other MMP, could process a pro [tumor necrosis factor (TNF) alpha] fusion protein to release mature TNF. However, the latter was subsequently degraded into smaller fragments. These results demonstrate that, in addition to their ability to activate other MMP, such as progelatinase A/proMMP2 and procollagenase-3/proMMP13, MT-MMP degrade a number of extracellular matrix macromolecules. Their location at the surface of cells implies that they could play a significant role in the modulation of cell-matrix interactions.
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PMID:Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases. 946 Dec 98

An aggrecan G1-G2 substrate was used to determine sites within the interglobular domain that were susceptible to cleavage by MT1-MMP. Degradation products were identified by Western blotting with neo-epitope antibodies specific for MMP-derived N- and C-terminal sequences. The results showed that MT1-MMP cleaved at the N341-F342 and D441-L442 bonds, as shown for other MMPs, and also at a site 13 amino acids C-terminal to the N341-F342 site. The G2 product of this additional cleavage was identified by sequence analysis and revealed an N-terminus commencing T355VxxPDVELPLP. The data are consistent with MT1-MMP cleavage at three sites in the aggrecan interglobular domain; one at N342-F342, a second at D441-L442 and a third at Q354-T355.
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PMID:Membrane-type 1 MMP (MMP-14) cleaves at three sites in the aggrecan interglobular domain. 968 35

A method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix.
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PMID:Generation and characterization of aggrecanase. A soluble, cartilage-derived aggrecan-degrading activity. 1003 54


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