Purification and characterization of a gelatinolytic matrix metalloproteinase from the skeletal muscle of grass carp (Ctenopharyngodon idellus).

A gelatinolytic matrix metalloproteinase (gMMP) from grass carp skeletal muscle was purified by 30-70% ammonium sulphate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, and affinity on gelatin-sepharose. The molecular weight of the proteinase as estimated by SDS-PAGE was 70 kDa under non-reducing conditions. The enzyme revealed high activity from 30 to 50 °C, and the gelatin hydrolysing activity was investigated at a slightly alkaline pH range using gelatin as substrate. Metalloproteinase inhibitor EDTA completely suppressed the gelatinolytic activity, while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca(2+) was essential for the gelatinolytic activity. Further, peptide mass fingerprinting obtained four fragments with 45 amino acid residues, which were highly identical to MMP-2 from fish species. The gMMP could effectively hydrolyse type I collagen even at 4 °C, suggesting its involvement in the texture softening of fish muscle during the post-mortem stage.

Identification and characterization of an amphioxus matrix metalloproteinase homolog BbMMPL2 responding to bacteria challenge.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases mainly involved in extracellular matrix (ECM) degradation. We have cloned and identified BbMMPL2 as homolog of MMPs from adult amphioxus. Recombinant BbMMPL2 proteins underwent self-processing during refolding in vitro. The final ~23 kDa polypeptide displayed proteolytic activity against ECM components like casein, gelatin, collagen IV and fibrinogen, but not laminin, fibronectin or α1-PI. This activity could be inhibited by GM6001 and TIMP-1/2. In addition, real-time RT-PCR analysis revealed that BbMMPL2 expressed in all issues/organs in adult amphioxus we tested. Its transcription was significantly up-regulated 12 h post immune challenge by Escherichia coli in epidermis and hepatic diverticulum but only slightly increased by Staphyloccocus aureus in epidermis. Furthermore, recombinant BbMMPL2-EGFP expressed in 293T and NIH/3T3 cells showed aggregation in cytoplasm and induced cell death. Our results provided new evidence that MMP was involved in immune response which could be conserved through evolution.

Production and characterization of a novel extracellular metalloproteinase by a newly isolated moderate halophile, Halobacillus sp. LY6.

A moderately halophilic bacterium LY6 with high proteolytic activity was isolated. Biochemical and physiological characterization, along with 16S rDNA sequence analysis placed the isolate in the genus Halobacillus. The salinity of the culture medium strongly influenced the proteinase production of LY6. Maximum enzyme production was observed in the medium containing 5% Na(2)SO(4) or 10% NaCl. Proteinase production was synchronized with bacterial growth and reached a maximum level during the mid-stationary phase. Enzyme purification was carried out by a simple approach including a combination of ammonium sulfate precipitation and Sephacryl S-100 gel filtration chromatography. SDS-PAGE and gelatin zymography analysis revealed it was a monomer with high molecular weight of 69 kDa. Optimal proteinase activity was obtained at pH 10.0, 40°C, and 10% NaCl. It was high active over broad temperature (30-80°C), pH (6.0-12.0), and NaCl concentration (0-25%) ranges, indicating its thermostable, alkali-stable, and halotolerant nature. Moreover, the enzyme activity was markedly enhanced by Ca(2+) and Cu(2+), but strongly inhibited by EDTA, PAO, and DEPC, indicating that it probably was a metalloproteinase with cysteine and histidine residues located in its active site.

Expression and activity profiling of selected cysteine cathepsins and matrix metalloproteinases in synovial fluids from patients with rheumatoid arthritis and osteoarthritis.

Cysteine cathepsins and matrix metalloproteases are considered to play important roles in the development of arthritic diseases. Their accumulation in synovial fluid of primarily rheumatoid arthritis patients is also well documented. However, a detailed comparison between the protease levels and activities between rheumatoid arthritis samples and osteoarthritis samples has never been made. Here, we report that both cysteine cathepsins B and S and matrix metalloproteases-1, -3 and -13 are detected in patient synovial fluid samples with significantly higher levels detected in rheumatoid arthritis patients. Among the proteases, cathepsin S was found to be significantly elevated, consistent with its critical role in the immune response. These results suggest that cysteine cathepsins have a major role in inflammation at least in rheumatoid arthritis. In addition to proteases, interleukin-6 was detected at significant levels in most samples, suggesting that proinflammatory cytokines might be in-volved in the stimulation of expression of these proteases during inflammation.

Purification of matrix metalloproteinases by column chromatography.

Matrix metalloproteinases (MMPs) are zinc endopeptidases composed of 23 members in humans, which belong to a subfamily of the metzincin superfamily. They play important roles in many pathophysiological events including development, organogenesis, angiogenesis, tissue remodeling and destruction, and cancer cell proliferation and progression by degradation of extracellular matrix (ECM) and non-ECM proteins and interaction with various molecules. Here, we present standard protocols for purification of native proMMPs (proMMP-1, -2, -3, -7, -9 and -10) and recombinant MT1-MMP (MMP-14) using conventional column chromatography. Purification steps comprise the initial common step [diethylaminoethyl (DEAE)-cellulose, Green A Dyematrex gel and gelatin-Sepharose columns], the second step for removal of nontarget proMMPs by immunoaffinity columns (anti-MMP-1 and/or anti-MMP-3 IgG-Sepharose columns) and the final step for further purification (IgG-Sepharose, DEAE-cellulose, Zn2+-chelate-Sepharose and/or gel filtration columns). Purified proMMPs and MMP are functionally active and suitable for biochemical analyses. The basic protocol for the purification from culture media takes approximately 7-10 d.

Characterization of matrix metalloproteinase expressed by human embryonic kidney cells.

There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3-MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.

Characterization of a novel metalloproteinase in Duvernoy's gland of Rhabdophis tigrinus tigrinus.

During the characterization of hemorrhagic factor in venom of Rhabdophis tigrinus tigrinus, so-called Yamakagashi in Japan, one of the Colubridae family, a novel metalloproteinase with molecular weight of 38 kDa in the Duvernoy's gland of Yamakagashi was identified by gelatin zymography and by monitoring its proteolytic activity using a fluorescence peptide substrate, MOCAc-PLGLA2pr(Dnp)AR-NH2, which was developed for measuring the well-known matrix metalloproteinase (MMP) activity. After purification by gel filtration HPLC and/or column switch HPLC system consisting of an affinity column, which was immobilized with a synthetic BS-10 peptide (MQKPRCGVPD) originating from propeptide domain of MMP-7 and a reversed-phase column, the N-terminal amino acid sequence of the 38 kDa metalloproteinase was identified as FNTFPGDLK which shared a high homology to Xenopus MMP-9. The 38 kDa metalloproteinase required Zn2+ and Ca2+ ions for its proteolytic activity. In addition, the proteolytic activity was almost completely inhibited by BS-10, a MMP inhibitor, but not by the serine proteinase inhibitors, cysteine proteinase inhibitors and aspartic proteinase inhibitors. Together these results demonstrated that the 38 kDa proteinase is a novel snake verom metalloproteinase (SVMP) containing HExGHxxGxxH motif which possesses high affinity to the BS-10 peptide, into its molecule, and the enzymatic properties are closed to that of MMPs. Based on the results obtained in the present study, we concluded that the 38 kDa metalloproteinase is a novel metalloproteinase whose activity may be regulated by the cysteine switch mechanism, and could be classified as one of the matrix metalloproteinases rather than snake venom metalloproteinases.

Filter sterilization of highly infectious samples to prevent false negative analysis of matrix metalloproteinase activity.

Matrix metalloproteinases (MMPs) are implicated in the immunopathology of numerous infectious diseases. High risk samples such as those generated after infection with Mycobacterium tuberculosis require filter sterilization for safe analysis of MMP concentrations. Here, we report that commercial filter membranes may cause artefacts by binding MMPs. Anopore 0.2 microM membrane filtration reduced MMP-1 concentrations to undetectable levels by zymography and Western blotting. Polypropylene 0.45 microM filtration removed some MMP-1, while Polysulphone, Durapore and Bio-inert 0.2 microM membranes did not remove MMP-1. Anopore filtration also removed all MMP-7 and -9 activity, suggesting that the conserved MMP catalytic domain binds the membrane. This study demonstrates the importance of selecting the appropriate filter in MMP analysis to avoid incorrectly excluding MMP involvement in infection-related immunopathology.

Matrix metalloproteinase-26 is associated with estrogen-dependent malignancies and targets alpha1-antitrypsin serpin.

Proteases exert control over cell behavior and affect many biological processes by making proteolytic modification of regulatory proteins. The purpose of this paper is to describe novel, important functions of matrix metalloproteinase (MMP)-26. alpha1-Antitrypsin (AAT) is a serpin, the primary function of which is to regulate the activity of neutrophil/leukocyte elastase. Insufficient antiprotease activity because of AAT deficiency in the lungs is a contributing factor to early-onset emphysema. We recently discovered that AAT is efficiently cleaved by a novel metalloproteinase, MMP-26, which exhibits an unconventional PH(81)CGVPD Cys switch motif and is autocatalytically activated in cells and tissues. An elevated expression of MMP-26 in macrophages and polymorphonuclear leukocytes supports the functional role of MMP-26 in the AAT cleavage and inflammation. We have demonstrated a direct functional link of MMP-26 expression with an estrogen dependency and confirmed the presence of the estrogen-response element in the MMP-26 promoter. Immunostaining of tumor cell lines and biopsy specimen microarrays confirmed the existence of the inverse correlations of MMP-26 and AAT in cells/tissues. An expression of MMP-26 in the estrogen-dependent neoplasms is likely to contribute to the inactivation of AAT, to the follow-up liberation of the Ser protease activity, and because of these biochemical events, to promote matrix destruction and malignant progression. In summary, we hypothesize that MMP-26, by cleaving and inactivating the AAT serpin, operates as a unique functional link that regulates a coordinated interplay between Ser and metalloproteinases in estrogen-dependent neoplasms.

Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are critical for development, wound healing, and for the progression of cancer. It is generally accepted that MMPs are secreted in a latent form (proMMP) and are activated only upon removal of their inhibitory propeptides. This report shows that three members of the SIBLING (Small, Integrin-Binding LIgand, N-linked Glycoprotein) family can specifically bind (Kd approximately equal nM) and activate three different MMPs. Binding of SIBLING to their corresponding proMMPs is associated with structural changes as indicated by quenching of intrinsic tryptophan fluorescence, increased susceptibility to plasmin cleavage, and decreased inhibition by specific natural and synthetic inhibitors. Activation includes both making the proMMPs enzymatically active and the reactivation of the TIMP (tissue inhibitors of MMP) inhibited MMPs. Bone sialoprotein specifically binds proMMP-2 and active MMP-2, while osteopontin binds proMMP-3 and active MMP-3, and dentin matrix protein-1 binds proMMP-9 and active MMP-9. Both pro and active MMP-SIBLING complexes are disrupted by the abundant serum protein, complement Factor H, thereby probably limiting SIBLING-mediated activation to regions immediately adjacent to sites of secretion in vivo. These data suggest that the SIBLING family offers an alternative method of controlling the activity of at least three MMPs.

Direct activation of pro-matrix metalloproteinase-2 by leukolysin/membrane-type 6 matrix metalloproteinase/matrix metalloproteinase 25 at the asn(109)-Tyr bond.

Leukolysin/membrane-type 6 matrix metalloproteinase (leukolysin/MT6-MMP), a glycosylphosphatidylinositol-anchored neutrophil matrix metalloproteinase, is also abnormally expressed in brain cancer tissues. Yet, little is known about its role in cancer progression. Here we show that MT6-MMP is capable of activating proMMP-2, an enzyme implicated in tumor invasion and metastasis. Although MT6-MMP is only 10% as active as MT5-MMP in mediating proMMP-2 activation, it generates a higher ratio of mature/intermediate forms of MMP-2 than MT5-MMP. Consistently, purified CAT of MT6-MMP converts proMMP-2 into mostly the mature form. Using the catalytically inactive mutant MMP-2EA (the E404A mutant of proMMP-2), which cannot autocatalytically mature from the intermediate form into the mature one, we show that MT6-MMP cleaves not only the known MT-MMP-processing site at Asn(66)-Leu but also the previously unsuspected Asn(109)-Tyr to yield a fully mature molecule. Despite their difference in mediating proMMP-2 activation in transfected cells, the CAT of MT6-MMP appears to be as efficient as that of MT5-MMP in cleaving proMMP-2EA in buffer, suggesting that its CAT is a strong proMMP-2 activator. Indeed, the CAT of MT6-MMP can partially substitute the CAT of prototypical MT1-MMP in mediating proMMP-2 activation. Taken these facts together, we conclude that MT6-MMP may participate in tumor invasion and metastasis by directly converting proMMP-2 into active form.

Matrix metalloproteinases.

Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods-cell-mediated dissolution of type I collagen fibrils, direct and reverse zymography, enzyme capture based on a-2 macroglubulin and TIMP-1 and -2, and demonstration of crytic thiol groups in metalloproteinase precursors-that are used to characterize the functions of matrix metalloproteinases and their inhibitors.

Molecular mechanism involved in matrix dependent upregulation of matrix metalloproteinases in monocyte/macrophage.

Production of macrophage specific matrix metalloproteinases (MMPs) by monocyte/macrophage (mo/mphi) maintained in vitro on matrix protein substrata has been examined to study the mechanism of matrix protein dependent upregulation of macrophage specific activity. Using specific blocking reagents we have found that interaction of peripheral blood mononuclear cells (PBMC) with extracellular matrix components is crucial for its differentiation to macrophages. Multiwell zymography has shown that production of MMPs was significantly inhibited in cells maintained on fibronectin (FN) pretreated with antibodies to alpha(5), beta(1) integrins and synthetic peptide RGDS. Further, quantification by ELISA showed a significant inhibition in MMP production in cells pretreated with these blocking reagents. Genistein, a non-specific inhibitor of tyrosine kinases, significantly reduced production of MMPs in cells maintained on FN and collagen type IV (COL IV). Immunoblotting analysis has shown that tyrosine phosphorylation occurs in 30 min and two proteins of approximately 115 and approximately 72 kDa are being phosphorylated upon PBMC-FN interaction. These results indicate that integrin mediated downstream signalling involving tyrosine phosphorylation is required for mediating intracellular events associated with differentiation of monocytes to macrophages.

Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro.

The pathogenesis of pseudomonal keratitis was investigated by focusing on induction and activation of matrix metalloproteinases (MMPs) by pseudomonal virulence factors and proinflammatory cytokines. Corneal lesions and MMP induction in vivo were evaluated in rabbit corneas infected with a clinical isolate of Pseudomonas aeruginosa. Effects of pseudomonal virulence factors [elastase, alkaline protease, exotoxin A and lipopolysaccharide (LPS)], tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta on MMP induction and activation were further examined in vitro in rabbit corneal fibroblasts (RCF) and human fibrosarcoma (HT1080) cells using reverse transcriptase-polymerase chain reaction (RT-PCR), zymography and immunoblotting. Corneal ulcers with typical ring abscesses were observed 12-24 h after infection, and MMPs, particularly MMP-9, were upregulated in infected corneas. Pseudomonal elastase caused the most extensive damage to both cell types. RCF treated with pseudomonal exoproteases or LPS expressed and secreted MMP-9. Exotoxin A had no effect on MMP expression. Both IL-1beta and TNF-alpha augmented MMP-9 expression in HT1080 cells. Pseudomonal elastase proteolytically activated MMP-2 and MMP-9 released from the cells. In conclusion, corneal destruction seen with P. aeruginosa infections may result from enhanced expression of MMPs by corneal stromal cells stimulated with pseudomonal exoproteases and proinflammatory cytokines and the proteolytic activation of MMPs by pseudomonal elastase.

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors.

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.