Identification and characterization of human MT5-MMP, a new membrane-bound activator of progelatinase a overexpressed in brain tumors.

A cDNA encoding a new member of the membrane-type (MT) matrix metalloproteinase (MMP) family has been identified and cloned from a human brain cDNA library. The isolated cDNA encodes a polypeptide of 645 amino acids that displays a similar domain organization as other MMPs, including a predomain with the activation locus, a zinc-binding site, and a hemopexin domain. The deduced amino acid sequence contains a COOH-terminal extension, rich in hydrophobic residues and similar in size to the equivalent domains identified in MT-MMPs. Immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA revealed that the encoded protein is localized in the plasma membrane. On the basis of these features, this novel human MMP has been called MT5-MMP because it represents the fifth member of the MT-MMP subfamily of MMPs. Fluorescent in situ hybridization experiments showed that the human MT5-MMP gene (MMP-24) maps to 20q11.2, a region frequently amplified in tumors from diverse sources. Northern blot analysis demonstrated that MT5-MMP is predominantly expressed in brain, kidney, pancreas, and lung. In addition, MT5-MMP transcripts were detected at high levels compared to normal brain tissue in a series of brain tumors, including astrocytomas and glioblastomas. The catalytic domain of MT5-MMP, produced in Escherichia coli as a fusion protein with glutathione S-transferase, exhibits a potent proteolytic activity against progelatinase A, leading to the generation of the Mr 62,000 active form of this enzyme. These data suggest that MT5-MMP may contribute to the activation of progelatinase A in tumor tissues, in which it is overexpressed, thereby facilitating tumor progression.

The helping hand of collagenase-3 (MMP-13): 2.7 A crystal structure of its C-terminal haemopexin-like domain.

Collagenase-3 (MMP-13) is a matrix metalloproteinase involved in human breast cancer pathology and in arthritic processes. The crystal structure of its C-terminal haemopexin-like domain has been solved by molecular replacement and refined to an R-value of 0.195 using data to 2.7 A resolution. This structure reveals a disk-like shape. The chain is folded into a beta-propeller structure of pseudo 4-fold symmetry, with the four propeller blades arranged around a funnel-like tunnel. This central tunnel tube harbours four ions assigned as two calcium and two chloride ions. The C-terminal domain of collagenase-3 has a similar structure to the equivalent domain of gelatinase A and fibroblast collagenase 1; however, its detailed structure and surface charge pattern has a somewhat greater similarity to the latter, in agreement with the subgrouping of MMP-13 with the collagenase subfamily of MMPs. It is proposed that several small structural differences may act together to confer the characteristic binding and cleavage specificities of collagenases for triple-helical substrates, probably in co-operation with a fitting interdomain linker.

Recognition and catabolism of synthetic heterotrimeric collagen peptides by matrix metalloproteinases.

BACKGROUND: The general consensus is that interstitial collagens are digested by collagenases and denatured collagen by gelatinases, although processing of fibrillar and acetic-acid-soluble collagen by gelatinase A has also been reported. One of the main difficulties in studying the mechanism of action of these matrix metalloproteinases (MMPs) derives from the physicochemical properties of the natural triple-helical collagen, which makes it difficult to handle. RESULTS: Synthetic heterotrimeric collagenous peptides that contain the collagenase cleavage site of human collagen type I and differ in the thermal stability of the triple-helical fold were used to mimic natural collagen and gelatin, respectively. Results from digestion of these substrates by fibroblast and neutrophil collagenases (MMP-1 and MMP-8), as well as by gelatinase A (MMP-2), confirmed that the two classes of enzymes operate within the context of strong conformational dependency of the substrates. It was also found that gelatinases and collagenases exhibit two distinct proteolytic mechanisms: gelatinase digests the gelatin-like heterotrimer rapidly in individual steps with intermediate releases of partially processed substrate into the medium, whereas collagenases degrade the triple-helical heterotrimer by trapping it until scission through all three alpha chains is achieved. CONCLUSIONS: The results confirm the usefulness of synthetic heterotrimeric collagenous peptides in the folded and unfolded state as mimics of the natural substrates collagen and gelatin, respectively, to gain a better a insight into the proteolytic mechanisms of matrix metalloproteinases.

Modified platelet deposition on matrix metalloproteinase 13 digested collagen I.

BACKGROUND: Atherothrombosis underlies acute coronary syndromes, including unstable angina and acute myocardial infarction. Within the unstable plaque, monocytes express collagenolytic matrix metalloproteinases (MMPs), including MMP-13, which degrades fibrous collagen. Following rupture, vessel wall components including degraded collagen are exposed to circulating platelets. Platelet receptors then mediate the recruitment and activation of platelets to form a thrombus, blocking blood flow and resulting in myocardial infarction and sudden death. OBJECTIVES: Here we aim to provide information on the effects of collagen degradation on platelet adhesion and thrombus formation. METHODS: Using increasing concentrations of MMP-13, we induced progressive degradation of fibrous and monomeric collagen I, visualized by electrophoresis, and then investigated the capacity of the resulting fragments to support static platelet adhesion and thrombus formation in whole flowing blood. RESULTS: Both integrin and glycoprotein VI-dependent interactions with fibrous collagen underpin high levels of platelet adhesion under both conditions, with little obvious effect of MMP-13 treatment. Static platelet adhesion to monomeric collagen was strongly α2ß1-dependent regardless of degradation status. Under flow conditions, partially degraded monomeric collagen supported increased thrombus deposition at 10 µg mL(-1) MMP-13, falling close to background when collagen degradation was complete (100 µg mL(-1) MMP-13). CONCLUSIONS: New binding activities come into play after partial digestion of collagen monomers, and net platelet-reactivity through all axes is abolished as degradation becomes more complete.

Relating matrix metalloproteinase structure to function: why the "hemopexin" domain?

Matrix metalloproteinases are thought to be key players in the remodelling activity of cells associated with both physiological and pathological processes. They share a relatively conserved structure with a number of identifiable modules linked to their specific functions. The structure of the individual domains of a number of matrix metalloproteinases have now been elucidated. Here we review these data in the light of complementary studies on the behaviour of these enzymes in biological systems. In particular we focus on the C-terminal hemopexin-like domain which has intriguingly specific roles in individual matrix metalloproteinases.

Inactivation of human plasma alpha 1-proteinase inhibitor by human PMN leucocyte collagenase.

Highly purified human polymorphonuclear leucocyte collagenase cleaved human alpha-1-proteinase inhibitor (alpha 1-PI) at the carboxyl site of Phe352 (P7). The inhibitor was thereby rapidly inactivated and generated a primary degradation product as shown by reverse-phase HPLC and N-terminal sequencing. Prolonged incubation of the modified inhibitor with polymorphonuclear leucocyte collagenase led to the generation of a secondary degradation product with additional cleavage at the carboxyl site of Pro357 (P2).

Inactivation of human plasma C1-inhibitor by human PMN leucocyte matrix metalloproteinases.

Highly purified human polymorphonuclear (PMN) leucocyte matrix metalloproteinases, collagenase and gelatinase, cleaved human plasma C1-inhibitor at the carboxyl site of Ala439 (P6). This led to a concomitant loss of C1-inhibitor activity. An additional cleavage site, at the carboxyl site of Ser441 (P4), was observed during PMN leucocyte gelatinase-induced inactivation, and a minor fragment of the plasma C1-inhibitor was generated.

Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase).

Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl2 and was investigated by kinetic analysis and N-terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp15-Leu16, Glu40-Met41, Leu52-Leu53 and Ala74-Met75 peptide bonds. The PRCGVPD sequence motif (residues Pro78-Asp84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C-terminal cleavages. The Glu666-Leu667, Ala506-Glu507 and Ala398-Leu399 bonds were hydrolysed successively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys497, Cys653, Cys683. Cleavage of the first C-terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin-like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.

Specific collagenolysis by gelatinase A, MMP-2, is determined by the hemopexin domain and not the fibronectin-like domain.

In view of the essential role of the hemopexin domain of the traditional interstitial collagenases, MMP-1, -8, -13 and MT1-MMP (MMP-14), in determining specific collagen cleavage we have studied the function of this domain in MMP-2, relative to that of the fibronectin-like domain that promotes gelatinolysis. Although the fibronectin-like domain promotes avid binding to collagen, our data demonstrate that the catalytic and hemopexin domains of MMP-2 are sufficient to effect the critical step in cleavage of rat type I collagen into 3/4 and 1/4 fragments. The mechanism of MMP-2 cleavage of collagen proceeds in two phases, the first resembling that of the interstitial collagenases, followed by gelatinolysis, promoted by the fibronectin-like domain.

Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis.

Analysis of the hinge region of neutrophil collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme. The mutagenesis of the amino acid residues in the P1' position of the two autoproteolytically cleaved peptide bonds (Leu243 and Ile248) to Ala showed that the mutant enzymes were more resistant to autoproteolysis. However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala239-Ile240 peptide bond and the half-life of the active enzyme was increased by 50%. In contrast, mutagenesis of Pro247 --> Ala (P1 of the minor cleavage site Pro247-Ile248) lead to increased susceptibility of the enzyme to autoproteolysis. However, when the other P1 position Gly242 was altered to Ala no effect on stability was observed. The analysis of the ability of the mutant active enzymes to hydrolyse 14C-type I collagen was assessed and our results demonstrate that the hinge sequence motif of neutrophil collagenase is important for collagenolytic activity. The alteration of the Gly242-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro247 sequence motif to Gly242-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro247 showed that the collagenolytic activity was reduced by 68.4%. In addition, mutagenesis of the downstream sequence motif Pro247-Thr-Gly-Pro-Ser-Thr-Pro-Lys-Pro258 to Pro247-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro258 had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%. When the Pro residues in the hinge motif (Pro247, Pro250, Pro253 and Pro256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme.

Isolation and characterization of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) from human rheumatoid synovial fluid.

The tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non-covalent gelatinase/TIMP complexes. TIMP-1 migrated as a single polypeptide with Mr 28,500 on SDS-PAGE, while the Mr of TIMP-2 was 21,000. The inhibitory activity was stable under heat and acid pH. N-terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP-1 and TIMP-2. This is the first demonstration that TIMP-1 and TIMP-2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting.

Catalytic activities of membrane-type 6 matrix metalloproteinase (MMP25).

This study describes the biochemical characterisation of the catalytic domain of membrane-type 6 matrix metalloproteinase (MT6-MMP, MMP25, leukolysin). Its activity towards synthetic peptide substrates, components of the extracellular matrix and inhibitors of MMPs was studied and compared with MT1-MMP, MT4-MMP and stromelysin-1. We have found that MT6-MMP is closer in function to stromelysin-1 than MT1 and MT4-MMP in terms of substrate and inhibitor specificity, being able to cleave type-IV collagen, gelatin, fibronectin and fibrin. However, it differs from stromelysin-1 and MT1-MMP in its inability to cleave laminin-I, and unlike stromelysin-1 cannot activate progelatinase B. Our findings suggest that MT6-MMP could play a role in cellular migration and invasion of the extracellular matrix and basement membranes and its activity may be tightly regulated by all members of the TIMP family.

Degradation of cartilage aggrecan by collagenase-3 (MMP-13).

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.

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3.

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.

TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3.

TNF-alpha converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-alpha to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-alpha secretion is involved.

Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP).

Matrix metalloproteinase (MMP)-19 and MMP-20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP-19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP-20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP-19 and MMP-20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well-described MMP cleavage site between residues Asn(341) and Phe(342), as shown by Western blotting using neo-epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP-19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP-20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.

Phosphinic pseudo-tripeptides as potent inhibitors of matrix metalloproteinases: a structure-activity study.

Several phosphinic pseudo-tripeptides of general formula R-XaaPsi(PO(2)-CH(2))Xaa'-Yaa'-NH(2) were synthesized and evaluated for their in vitro activities to inhibit stromelysin-3, gelatinases A and B, membrane type-1 matrix metalloproteinase, collagenases 1 and 2, and matrilysin. With the exception of collagenase-1 and matrilysin, phosphinic pseudo-tripeptides behave as highly potent inhibitors of matrix metalloproteinases, provided they contain in P(1)' position an unusual long aryl-alkyl substituent. Study of structure-activity relationships regarding the influence of the R and Xaa' substituents in this series may contribute to the design of inhibitors able to block only a few members of the matrix metalloproteinase family.

Mechanisms for pro matrix metalloproteinase activation.

The activation of pro matrix metalloproteinases (MMPs) by sequential proteolysis of the propeptide blocking the active site cleft is regarded as one of the key levels of regulation of these proteinases. Potential physiological mechanisms including cell-associated plasmin generation by urokinase-like plasminogen activator, or the action of cell surface MT1-MMPs appear to be involved in the initiation of cascades of pro MMP activation. Gelatinase A, collagenase 3 and gelatinase B may be activated by MT-MMP based mechanisms, as evidenced by both biochemical and cell based studies. Hence the regulation of MT-MMPs themselves becomes critical to the determination of MMP activity. This includes activation, assembly at the cell surfaces as TIMP-2 complexes and subsequent inactivation by proteolysis or TIMP inhibition.

Evaluation of some newer matrix metalloproteinases.

Recombinant protein expression techniques have been utilized to facilitate the biochemical and cell biological characterization of human matrix metalloproteinases (MMPs). The importance of the membrane type 1 MMP (MMP 14) in the regulation of pericellular proteolysis, either directly or through the activation of MMP-2, MMP-9, and MMP-13 has been identified. Studies on an in vitro chondrocyte-like cell and an in vivo cartilage repair model indicated that such MT1 MMP-regulated activation cascades are physiologically feasible. MMP19 shows a limited sequence identity with other MMPs and may represent a novel subclass. However, analysis of the recombinant protein identified a number of biochemical properties typical of the MMP family.

Some properties of latent collagenase from human synovial fluid.

Latent collagenase has been isolated in pure form from the rheumatoid synovial fluid. The final preparation, activated by trypsin, yielded a collagenase of specific activity 2,227 units/mg. Electrophoresis in sodium dodecyl sulfate polyacrylamide gels revealed a protein doublet of 54 and 50 kDa. Trypsin or HgCl2 activation resulted in disappearance of the doublet and emergence of a new doublet of 47 and 43 kDa. The latent collagenase could also be activated by leucocyte cathepsin G or plasmin. Neither the latent nor the active collagenase from synovial fluid showed any cross-reactivity with the antibodies against leucocyte collagenase. The trypsin activated collagenase degraded collagen type I, II, III giving typical cleavage products but did not degrade type IV and V collagen.