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

The enzyme responsible for the metalloproteinase activity which cleaves the Glu143-Leu144 bond of (pro)urokinase has been isolated from the conditioned medium of cultured normal human kidney cells. Using S-Sepharose and Cibacron Blue-agarose chromatography, then C-4 reversed phase high pressure liquid chromatography, a protein of about 20,000 Da was isolated. Through an identical amino-terminal sequence, the protein was shown to be the matrix metalloproteinase previously referred to in the literature as "pump-1" (putative metalloproteinase). When aprotinin was added during the course of the purification, the major species isolated was the zymogen form (28,000 Da) of pump-1. Pump-1 has been shown to efficiently cleave the susceptible bond of both pro-urokinase (single-chain) and active (two-chain) urokinase and thereby produce the corresponding low molecular weight forms. The amino-terminal sequences of the A and B chains of low molecular weight urokinase prepared by action of pump-1 on recombinant high molecular weight urokinase are identical to those of the low molecular weight urokinase isolated from human kidney cell culture. Since the reaction of urokinase with this metalloproteinase results in separation of its serine proteinase region from the domain which mediates binding to the urokinase receptor, it may be of importance in the regulation of the functional activity of the plasminogen activator in cellular processes.
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PMID:The matrix metalloproteinase pump-1 catalyzes formation of low molecular weight (pro)urokinase in cultures of normal human kidney cells. 162 80

The gene for PUMP (putative metalloproteinase), a human matrix metalloproteinase, was synthesized by a PCR-based method. The DNA fragment of 546 bases containing the PUMP gene was generated by overlap extension of six long oligonucleotides (length ranging from 101 to 116 bases) and subsequent amplification by two short terminal oligonucleotide primers (length from 20 to 48 bases) in one pot without using restriction and ligation enzymes. The synthetic gene was cloned into a T7 expression vector in two ways to express PUMP as a non-fusion protein. Both constructs showed high level expression in E. coli.
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PMID:Gene synthesis and expression in E. coli for pump, a human matrix metalloproteinase. 163 63

Interleukin 1 (IL-1) mediates many cellular functions, but the signal transduction mechanisms of its actions are not clearly understood. Here, we have examined the exact participation of cAMP in the IL-1-induced production of the precursors of matrix metalloproteinase (MMPs) and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP) in human uterine cervical fibroblasts. IL-1 significantly augmented the production of proMMP-1 (vertebrate procollagenase), proMMP-3 (prostromelysin), and TIMP without detectable changes in the intracellular level of cAMP. Dibutyryl cAMP (Bt2cAMP) and the cAMP elevating agent (forskolin) did not replace IL-1 as MMP inducers. On the contrary, the IL-1-mediated induction of proMMP-1 and proMMP-3 was significantly suppressed by treatment of the cells with Bt2cAMP, forskolin, or theophylline. The suppressive effect of Bt2cAMP on the IL-1-induced production of proMMP-1 and -3 was not due to the inhibition of zymogen secretion, but resulted from the decrease in the steady-state levels of proMMP-1 and proMMP-3 mRNAs. In contrast, Bt2cAMP slightly enhanced the IL-1-induced production of TIMP. The synthesis of proMMP-2 (72-kDa progelatinase/type IV procollagenase) was not altered by IL-1 and/or Bt2cAMP. These results suggest, first, that induction of proMMP-1 and -3 synthesis may share similar transduction pathways but they are distinct from those for proMMP-2 and TIMP synthesis and, second, that cAMP does not function as a second messenger in the MMPs' induction upon IL-1 stimulation in human uterine cervical fibroblasts. Thus, it is further suggested that the system that increases the intracellular cAMP level may be involved in negative regulation of proMMP-1 and -3 production.
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PMID:Cyclic adenosine 3',5'-monophosphate suppresses interleukin 1-induced synthesis of matrix metalloproteinases but not of tissue inhibitor of metalloproteinases in human uterine cervical fibroblasts. 165 5

Twenty-five surgical specimens of malignant human prostate, 3 lymph nodes with metastatic prostate carcinoma, 11 normal human prostates, as well as 3 human prostate cell lines (DU-145, PC3 and LNCaP) were examined for the expression of the human matrix metalloproteinase-7 gene (MMP-7) from the human collagenase family (originally called PUMP-1 for putative metalloproteinase-1) [Quantin et al. (1989) Biochemistry 28:5327-5334; Muller et al. (1988) Biochem J 253:187-192; Matrisian and Bowden (1990) Semin Cancer Biol 1:107-115]. Northern blots were prepared using total RNA extracted from 18 prostate adenocarcinomas, 2 lymph nodes with metastatic prostate carcinoma and 11 normal human prostates. When the northern blots were hybridized with a 32P-labeled MMP-7 cDNA probe, a 1.2-kb mRNA was detected in 14 out of 18 prostate adenocarcinomas, 1 out of 2 metastatic lymph nodes, and 3 out of 11 normal prostates. The 3 human prostate cell lines did not show any evidence of the MMP-7 transcript. In situ hybridization was conducted to localize the MMP-7 mRNA to individual cells using a 35S-labeled MMP-7 cRNA. In situ hybridization was carried out on snap-frozen tissue sections of 7 prostate adenocarcinomas and 3 lymph nodes containing metastatic prostate adenocarcinoma using the same tissues previously probed by northern analysis as well as new samples. In situ hybridization revealed that the MMP-7 gene was expressed in the epithelial cells of primary prostate adenocarcinoma as well as in invasive and metastatic cells. MMP-7 expression was also seen focally in some dysplastic glands but not in stroma. Additional northern blot analysis was performed using probes to human type-IV collagenase, type-I collagenase and stromelysin I in human prostate adenocarcinoma as well as normal prostate tissue. Our results indicated that 6 out of 10 adenocarcinoma samples and none of the 4 normal samples were positive for type-IV collagenase transcripts. Tissue samples were also examined for the expression of type-I collagenase (9 adenocarcinomas and 4 normal) and stromelysin I (13 adenocarcinomas) by northern analysis. None of the tissues was found to express the transcripts of interest at detectable levels. These data suggest that certain metalloproteinases are present in prostatic adenocarcinoma and may play a role in invasion and metastasis.
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PMID:Expression of metalloproteinase genes in human prostate cancer. 184 60

Human promatrilysin (matrix metalloproteinase-7) has been produced in Escherichia coli as an N-terminal fusion protein with ubiquitin. The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric acetate. Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells. Following activation, matrilysin was purified to > 95% homogeneity using a Sepharose-Pro-Leu-Gly-NHOH affinity column. The matrilysin purified by this procedure is indistinguishable from the enzyme purified from CHO cells with respect to the kinetic parameters for hydrolysis of a peptide substrate and the ability to obtain diffraction quality crystals in the presence of an inhibitor of the enzyme. Additionally, to facilitate detailed kinetic analyses of matrilysin, a new fluorogenic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (Dnp, dinitrophenyl) has been synthesized. This peptide is the best substrate developed for matrilysin thus far with Km and kcat values of 26 microM and 5.0 s-1, respectively.
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PMID:Purification of human matrilysin produced in Escherichia coli and characterization using a new optimized fluorogenic peptide substrate. 750 60

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

Membrane-type matrix metalloproteinase (MT-MMP), which we have identified recently, is unique in its transmembrane (TM) domain at the C terminus and mediates activation of pro-gelatinase A on the cell surface (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65; Takino, T., Sato, H., Yamamoto, E., and Seiki, M. (1995) Gene (Amst.) 115, 293-298). In addition to MT-MMP, a novel MMP-related cDNA of 2.1 kilobases was isolated from a human placenta cDNA library. The cDNA contains an open reading frame for a new MMP. The deduced protein composed of 604 amino acids was closely related to MT-MMP in the amino acid sequence (66% homology at the catalytic domains) and has a potential TM domain at the C terminus. Monoclonal antibodies raised against the synthetic peptide recognized a 64-kDa protein as the major product in the transfected cells. TIMP-1 fused with the potential TM domain was localized on the cell surface while native TIMP-1 is in the culture medium. Thus, we called the second membrane-type MMP, MT-MMP-2 and renamed MT-MMP, MT-MMP-1. MT-MMP-1 and -2 are thought to form a distinct membrane-type subclass in the MMP family since all the others are secreted as soluble forms. Like MT-MMP-1, expression of MT-MMP-2 induced processing of pro-gelatinase A (68-kDa in gelatin zymography) into the activated form of 62-kDa fragments through a 64-kDa intermediate form. Expression of MT-MMP-2 mRNA was at the highest levels in the brain where MT-MMP-1 was at the lowest level compared to other tissues. MT-MMP-1 and -2 are thought to be utilized for extracellular matrix turnover on the surface of cells under different genetic controls.
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PMID:Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family. 755 40

To explore the role of the matrix metalloproteinase matrilysin (MAT) in normal tissue remodeling, we cloned the murine homologue of MAT from postpartum uterus using RACE polymerase chain reaction and examined its pattern of expression in embryonic, neonatal, and adult mice. The murine coding sequence and the corresponding predicted protein sequence were found to be 75% and 70% identical to the human sequences, respectively, and organization of the six exons comprising the gene is similar to the human gene. Northern analysis and in situ hybridization revealed that MAT is expressed in the normal cycling, pregnant, and postpartum uterus, with levels of expression highest in the involuting uterus at early time points (6 h to 1.5 days postpartum). The mRNA was confined to epithelial cells lining the lumen and some glandular structures. High constitutive levels of MAT transcripts were also detected in the small intestine, where expression was localized to the epithelial Paneth cells at the base of the crypts. Similarly, MAT expression was found in epithelial cells of the efferent ducts, in the initial segment and cauda of the epididymis, and in an extra-hepatic branch of the bile duct. MAT transcripts were detectable only by reverse transcription-polymerase chain reaction in the colon, kidney, lung, skeletal muscle, skin, stomach, juvenile uterus, and normal, lactating, and involuting mammary gland, as was expression primarily late in embryogenesis. Analysis of MAT expression during postnatal development indicated that although MAT is expressed in the juvenile small intestine and reproductive organs, the accumulation of significant levels of MAT mRNA appears to correlate with organ maturation. These results show that MAT expression is restricted to specific organs in the mouse, where the mRNA is produced exclusively by epithelial cells, and suggest that in addition to matrix degradation and remodeling, MAT may play an important role in the differentiated function of these organs.
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PMID:The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse. 757 99

The initiation of the angiogenic process requires a locally confined and time-limited proteolysis of the basement membrane (BM) components at the site of new vessel sprout. Gelatinase A, a member of the matrix metalloproteinase family, degrades BM type IV collagen and is involved in the BM breakdown by migrating tumor cells and endothelial cells (EC). Gelatinase A is synthesized as latent proenzyme and must be activated in order to express its proteolytic activity. A plasma membrane-dependent mechanism of activation has been described for several tumor and transformed cells lines. In the present study, we show that latent (72 kD) and mature (62-59 kD) forms of gelatinase A are present in EC membrane fraction from Triton X-114 extract while only latent form is found in the cytosolic fraction. The incubation of EC membrane fraction with exogenous latent gelatinase A resulted in a significant activation giving rise to 62-59 kD mature forms. 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong potentiator of angiogenesis in vitro and in vivo, increases the amount of both latent and activated forms of gelatinase A in EC membrane fraction as well as the ability of this latter fraction to activate exogenous latent gelatinase A. We show that the mRNA transcript coding for the membrane-integrated MMP, the MT-MMP, previously described as a potential gelatinase A activator in invasive tumor cells is also expressed in vascular EC and is regulated through a TPA sensitive process. This enzyme may be responsible for membrane-dependent gelatinase A activation in normal vascular EC and may therefore be a determinant in the control of BM proteolysis during angiogenesis.
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PMID:Plasma membrane-dependent activation of gelatinase A in human vascular endothelial cells. 759 26


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