Update The Matrix Metalloproteinases and Their Inhibitors Gillian Murphy and Andrew J. P. Docherty Strangeways Research Laboratory, Cambridge, and Celltech Ltd., Slough, United Kingdom
A number of metalloproteinases that degrade the extracellular matrix of connective tissues and two specific tissue inhibitors of metalloproteinases (TIMPs) have now been isolated, characterized, and cloned. Comparison of the enzyme sequences has allowed the delineation of domain structures, and initial studies have been carried out to assess the contribution of these domains to their biochemical and biologic properties, including activation, inhibition by TIMPs, and matrix binding. Such events represent the major levels of extracellular regulation of metalloproteinase activity, which is thought to be an important aspect of their control. Activation is probably a cell surface phenomenon, involving the plasminogen activator cascade or other membrane-associated mechanisms. The inhibitory action of TIMPs is postulated to be as important in activation as in the subsequent regulation of enzyme degradation of the matrix.
The matrix metalloproteinases (MMPs) are a family of zincand calcium-dependent endopeptidases that have the combined ability to degrade the various components of connective tissue matrices (1). They are synthesized and secreted by connective tissue and some hematopoietic cells and are known to be important in both normal remodeling processes (2) and in the accelerated destruction occurring in many diseases (3, 4). Regulation of the MMPs is stringent, occurring not only at the level of gene expression but extracellularly, after secretion, by the action of activators of the proenzyme forms and of specific inhibitors. The major natural inhibitor, tissue inhibitor ofMMPs (TIMPs), which is produced by the same cells, has been identified in two forms to date. This report will review our newly developing knowledge of the structure of MMPs and TIMPs and the potential contribution of their definable domain structures to their biochemical behavior. One of the major questions confronting biologists is the importance of MMP activation as a regulatory mechanism in vivo. In relation to this, our current understanding of possible activation pathways will be discussed.
Matrix Metalloproteinase Structure Nine MMPs have been identified by cDNA cloning and sequencing. Comparison of these sequences, in conjunction (Received in original form February 24, 1992 and in revised form March 17, 1992) Address correspondence to: Dr. Gillian Murphy, Cell and Molecular Biology Department, Strangeways Research Laboratory, Worts' Causeway, Cambridge CBl 4RN, United Kingdom. Abbreviations: matrix metalloproteinase, MMP; tissue inhibitor of metalloproteinases, TIMP. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 120-125, 1992
with biochemical and immunologic data, have indicated that two forms of collagenase, two gelatinases (type IV collagenases), and two stromelysins occur (Table 1). Three other enzymes, pump (punctuated metalloproteinase, matrilysin), "stromelysin-S," and metallo-elastase have somewhat different sequences and properties (Table 1). The interstitial collagenases are the most specific of the MMPs, cleaving the native helix of the fibrillar collagens (types I, II, and III) at a single locus. One form, MMP1, is produced by most connective tissue cells whereas MMP8 is confined to neutrophil granules (5, 6). The, gelatinases degrade types IV, V, VII, and X collagens and may act synergistically with interstitial collagenases in the degradation of collagens, since they efficiently degrade their denatured gelatin forms. They also degrade elastin. Two separate but similar cDNAs encoding a 72-kD gelatinase A (MMP2) and a 95-kD gelatinase B (MMP9) have been identified to date (Table 1), and the corresponding proteins have been shown to be associated with monocyte/macrophages, as well as many stimulated connective tissue cells (7, 8). The stromelysins 1 (MMP3) and 2 (MMP10) (5, 9) are potentially very important members of the MMP family, having broad pH optima and substrate specificities. They are able to degrade many extracellular matrix proteins, including proteoglycans and laminin, and are also potentiators of collagenase and 95-kD gelatinase activity (10, 11). A more recently discovered MMP, punctuated metalloproteinase (pump, MMP7, matrilysin), appears to be distantly related to the stromelysins but lacks the C-terminal domain (see below and Figure 2). Pump has broad proteolytic activity that includes elastin and proteoglycans (12) and has been shown to be associated with immature monocytes (13). A further putative MMP identified in the stroma of tumors, the trophoblast, and fetal lung fibroblasts by both cloning and immunologic studies has
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Update
TABLE 1
The matrix metalloproteinase family* MMP No.
8
Source
Substrates
Trivial Name
kD
Interstitial collagenase
55
Connective tissue cells
Fibrillar collagens I, II, and III; collagen X; gelatin; proteoglycans
Neutrophil collagenase
75
Neutrophils
Fibrillar collagens, gelatin, proteoglycans
Connective tissue cells, macrophages
Proteoglycan; cross-link: regions of collagens II, IV, and IX; collagens X and XI; procollagens; fibronectin; laminin; gelatin; collagenase; gelatinase B
3
Stromelysin-l
57
10
Stromelysin-2
57
Macrophages
As stromelysin-l but much lower activity
2
Gelatinase A
72
Most cell types
Denatured collagens; collagens IV, V, VII, X, and XI; elastin
9
Gelatinase B
95
Monocytes, connective tissue, tumor cells
As gelatinase A
7
Pump (matrilysin)
28
Immature monocytes, mesangial, tumor cells (not fully defined)
As stromelysins, elastin
11
"Stromelysin-3"
51
Stromal cells of tumors
Unknown
12
Metallo-elastase
57
Macrophages (mouse)
Elastin, fibronectin
* Molecular masses are for the human enzymes as estimated by SDS-PAGE except for MMPll (eDNA-predicted mass) and MMP12 (mouse). Minor glycosylated species of higher molecular mass are observed for MMPI and MMP3.
(Figure 1), including a propeptide of 77 to 87 amino acids lost during activation, a catalytic Zn2+- and Ca't-binding domain of 162 to 173 residues, and domains with sequence similarities to a number of extracellular matrix structural proteins (18). Of these, the C-terminal vitronectin-like domain of 202 to 213 amino acids is found in all MMPs except pump. The gelatinases also have a fibronectin-like gelatinbinding sequence inserted into the catalytic domain (Figure 1). The 95-kD gelatinase B has a further collagen-like insertion C-terminal to the catalytic domain. These extra gelatinase domains are contained within a discrete set of exons. The precise mechanism by which the propeptide domain maintains latency is not yet clear. Sequential processing events occur as part of the activation process involving exogenous and endogenous cleavages (11); the physiologic rele-
been named "stromelysin-3" (14). No specific catalytic activity has yet been defined for this protein; however, it is clear that it is structurally somewhat distant from the stromelysins (15). The most recently identified MMP cDNA is that for mouse macrophage metallo-elastase (16), but no human macrophage counterpart has yet been reported. This enzyme could potentially be important in matrix turnover since it degrades many matrix macromolecules, including elastin (17). The cDNA-predicted amino acid sequences of all the MMPs can be aligned, demonstrating a high degree of conservation between each type of enzyme across several mammalian species (about 80% similarity between collagenases and stromelysins). The similarity between types is lower but still a highly significant 50 %. Domains within these sequences can be delineated with apparent specific functions
Figure 1. Domain structure of the matrix metalloproteinases. All members of the matrix metalloproteinase family contain a definable propeptide that is lost during activation and contains the conserved motif PRCGV/NPD (see text), as well as a catalytic domain containing two putative Zn2 + -binding ligands (HEFI L/IGH). Stromelysin-3 has a unique extension of 10 amino acids between the propeptide and catalytic domains. Collagenases, stromelysins, and gelatinases all have a third C-terminal domain with similarities to members of the hemopexin family, including vitronectin. It contains a conserved disulfide bond linking the N- and C-terminal portions of the domain and is linked to the catalytic domain by a proline-rich sequence of five to ten amino acids. Both gelatinases have an extra domain related to the collagen-binding region of fibronectin that is inserted into the catalytic domain. Gelatinase B has a fifth domain, with similarities to several fibrillar collagens, inserted between the catalytic and C-terminal domains.
DOMAIN STRUCTURE OF MATRIX METALLOPROTEINASES Propeptide
Catalytic
C-terminal (vitronectin-like)
gelatinase A ( fibronectin-Iike)
C / / ",
""
"
PRCGXPD
pump C-terminus
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vance of this is discussed below. One aspect of the latent nature of pro MMPs may be explained by the fact that the propeptide contains a conserved sequence PR~GV/NPD in which the free cysteine may interact with the Zn 2+ of the adjoining catalytic domain, displacing the H 20 molecule required for catalytic activity (19). However, other features of the propeptide apparently stabilize the cysteine-Zn" interaction, since cleavage at sites upstream from the cysteinecontaining motif in the propeptide of MMPs such as stromelysin and pump will initiate further autocatalytic cleavages that lead to complete removal of the propeptide (11, 20). The catalytic domain of the MMPs has been defined by both biochemical and protein engineering approaches. In the case of pump, this is the only domain other than the propeptide. Both stromelysin and collagenase catalytic domains display proteinase activity against substrates such as casein, the former being identical to that of the full-length enzyme. However, the collagenase catalytic domain loses its unique ability to cleave triple helical collagen in the absence of the C-terminal vitronectin-like domain (21-24). Gelatinase A, expressed as a truncated form consisting of the catalytic and gelatin-binding domains, also shows identical substrate specificity to the full-length enzyme (25, 26). The catalytic domain of all the MMPs contains a short sequence of identity with thermolysin. In thermolysin, the sequence, HELTH is known to contain two of the three histidine residues (-142 and -146) that coordinate the zinc, as well as the catalytic glutamic acid. In the absence of structural information, the precise Zn2+ ligands of the MMPs are not known, but it seems likely that the histidine residues of the conserved HEL/I/FGH motif are involved, although the third ligand can only be speculated upon (15,27). Attempts at mutagenesis of a number of conserved histidine and aspartic acid residues in stromelysin that are potential candidates have all yielded inactive enzyme forms. The C-terminal domain, which is present in all the MMPs except pump, shows some similarity to the hemopexin family of proteins, which includes vitronectin. The biologic relevance of this domain is only just being elucidated with the preparation of C-terminally deleted enzyme forms by both biochemical and protein engineering approaches. This domain mediates the interaction of both collagenase and stromelysin to collagen fibers (24, 28), a phenomenon that can be observed by immunohistochemical studies on rapidly resorbing tissues. In the case of collagenase, binding only occurs when the intact enzyme is in the active form, presumably due to the unmasking of some essential components of the binding domain. The isolated collagenase or stromelysin C-terminal domain will bind to collagen, but apparently to different sites. The presence of the C-terminal domain is essential for the specific collagenolytic activity of collagenase. However, this domain cannot confer collagenolytic activity to the catalytic domain of stromelysin when a hybrid enzyme is engineered and expressed. Similarly, the stromelysin C-terminal domain can mediate binding of the collagenase catalytic domain to collagen but not its specific ability to cleave collagen (24). The C-terminal domain of progelatinase A has an even more intriguing role, apparently being required for the action of a gelatinase activator that has been localized to the cell (26, 29). The C-terminal domain also interacts weakly with the inhibitor TIMP-2 when the enzyme
is in its proform, such that progelatinase A is frequently isolated as a complex with TIMP-2 (25, 30). Consequently, TIMP-2 is an effective and specific inhibitor of the action of the membrane activator (see below; 27). In the case of all the MMPs studied thus far, it appears that the C-terminal domain may also have a role in the binding of active enzyme forms to both TIMP-l and TIMP-2 (24-26). The precise role of the extra domains expressed in the gelatinases has not yet been analyzed. Both enzymes have a domain consisting of three repeats similar to those found in the type II domains of the gelatin-binding region of fibronectin. Expression of the isolated domain from 72-kD gelatinase has been carried out and binding to gelatin Sepharose demonstrated (31). The 95-kD gelatinase has a small extra domain with a sequence similar to that found in many collagens (8). It is not yet known if the catalytic domain of the gelatinases is functional in the absence of these domains. Tissue Inhibitors of Metalloproteinases The major physiologic inhibitors of the MMPs are a2macroglobulin, which is restricted in its sites of activity due to its large size (780 kD), and a family of inhibitors that are specific for the MMPs and are produced by many cell types, including those of connective tissues. Four inhibitors have been reported, and two inhibitors of this family have been fully characterized and cloned. These TIMPs have closely related structures and inhibitory properties. TIMP-l is a 30-kD glycoprotein (32), and TIMP-2 a 23-kD unglycosylated protein (33). Their cellular distribution appears to be very similar, although TIMP-l is generally found in larger amounts. The occurrence of TIMP-2 is, however, not yet fully documented. The TIMPs act specifically against the active forms ofMMPs, TIMPs-l and -2 having very similar activities (30) and forming essentially irreversible complexes with equimolar stoichiometry. Initial estimates suggest that TIMP-l has a K, of 10-9 to 10-11 M for most MMPs (15). It has been shown that TIMP-l may be recovered by acid dissociation of complexes with stromelysin and is fully active and apparently unmodified (34). The TIMPs have 12 identically conserved cysteine residues forming six disulfide bonds, which confer marked stability to pH and temperature (35). A two-domain structure of three loops each can be delineated (Figure 2). The precise folding of these inhibitors appears to be essential since reduction and alkylation leads to a loss of activity. It has been shown that the N-terminal loops of TIMP-l encoded by amino acids 1 through 126 can fold independently of the C-terminal domain and act as an efficient inhibitor of most MMPs (36). A similarly truncated form of TIMP-2 behaves in the same manner. In each case, the unique binding to the proform of 95-kD and 72-kD gelatinases B and A, respectively, is abolished (36; G. Murphy, unpublished observations). We are still a long way from an understanding of the mechanism of TIMP inhibition, which will be achieved by a combination of specific mutagenesis studies and X-ray crystallography.
Extracellular Regulatory Mechanisms Activation Given the plethora of substrates that active MMPs can degrade, MMP regulation is necessarily stringent and in most
Update
123
Human TIMP-l
Figure 2. The loop structure of TIMP-l. The eDNA-predicted sequence of TIMP-l can be depicted as a six-loop structure on the basis of the distribution of the six disulfide bonds (36). The first two loops contain carbohydratebinding sites (CHO); TIMP-l is a complex glycoprotein and is variably substituted with sialic acid giving it a heterogeneous charge. The first three loops, when expressed alone, retain metalloproteinase-inhibitory properties.
cases occurs predominantly at the level of gene expression. This aspect of their control will not be discussed here and has been excellently reviewed by Matrisian (37). A second level of regulation occurs extracellularly and is thought to be of some importance, ultimately controlling the level of enzyme activity in terms of matrix destruction, namely the activation of secreted proenzyme forms and their subsequent inhibition by TIMPs (1, 18). As was discussed above, the stepwise process of MMP activation by loss of the propeptide may be initiated by exogenous proteolytic cleavage in the case of the collagenases, stromelysins-l and -2, pump, and gelatinase B. Nagase and associates (11) have systematically analyzed the action of potential physiologic enzyme activators, which include plasmin, plasma kallikrein, cathepsin B, cathepsin G, and neutrophil elastase. Plasmin has long been thought to be an important activator of MMPs, and this concept has been supported by a number of studies using cell model systems with both connective tissue and tumor cells (38). The study of plasmin generation from plasminogen by the action of plasminogen activators is a particularly active and exciting field (39). It has shown that plasmin generation and activity is highly focused, largely occurring at the cell surface where both plasminogen and the activators are specifically bound at cell adhesion plaques. It is likely that in localized pericellular regions, the action of a2 anti-plasmin is limited because of the location of plasmin on the cell membrane or local excess of free enzyme. Stromelysin-l, which can be sequestered on the collagenous matrix, is particularly susceptible to plasmin activation and once activated can potentiate collagenase activity and act as a gelatinase B activator (Figure 3). TIMPs may regulate the activation process to
some extent, since it has been shown that they can slow down or prevent the autocatalytic cleavages that occur after initiation of activation by the exogenous proteinases described above (26). Gelatinase A is unlike the other MMPs in that it is regulated differently at the transcriptional level. Furthermore, it differs in its mechanism of extracellular activation. The propeptide of this enzyme has no apparent cleavage site susceptible to plasmin and other proteinases, although it can undergo self-cleavage reactions to lose the propeptide and become active (11, 30). However, this enzyme can be activated by a fibroblast- or tumor cell membrane-mediated process that is sensitive to metalloproteinase inhibitors. The membrane activator which is specific for gelatinase A and does not activate the other pro MMPs (29) can be induced by a number of effectors including concanavalin A, phorbol esters, and transforming growth factor-S (29, 37). It is not yet known if the activator is itself a proteinase or merely binds gelatinase A (as discussed above) and initiates autocatalytic cleavages. This activation process is efficiently prevented by TIMP-2 when bound to the proenzyme but not by TIMP-l, which only binds to active gelatinase A (25, 26, 30). Gelatinase A is a particularly widespread proteinase and appears to be constitutively expressed by many cells. It may therefore be postulated that the membrane activation mechanism plays an important regulatory role, by controlling the precise locations at which this enzyme may be activated. Inhibition The importance of TIMPs as regulators of MMP activity can only be demonstrated equivocally. In many cell model systems, the ability of TIMPs to prevent matrix degradation has
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scuPA-tcuPA
8 -t--t
Prostromelysin
1
Plasminogen - - - - - - - -... PLASMIN
I-/----:~------+~ _-
been demonstrated (40). In some disease situations, e.g., osteoarthritic cartilage, it has been shown that TIMP levels rise but do not compensate for the much larger increase in degradative activity of MMPs (41). Immunolocalization studies of TIMP-1 expression in in vivo models of resorption have demonstrated the precise spatial and temporal expression of this inhibitor relative to MMPs. In the developing growth plate of the young rabbit, TIMP-1 is specifically localized to certain regions of the tissue where collagenase and gelatinase expression are high (2). In a rabbit colon wound-healing model studied at specific intervals after anastomoses, it is evident that collagenase is expressed within the first 12 h but TIMP-1 expression does not occur until 24 h (42). TIMP-1 expression in the developing embryo has been studied by in situ hybridization and shown to be particularly localized in osteogenic tissues and the ovary (43), but no concomitant MMP studies have yet been made. TIMPs have been shown to be sequestered in tissues such as cartilage but no evidence of direct matrix binding has been obtained. It can be postulated that, like the plasminogen activator inhibitors, they may be limited in their action by the focal nature of enzyme activation at certain sites on the cell surface.
Conclusions and Future Directions Knowledge of structure-function relationships in the MMPs and TIMPs is only at an early stage following the rapid cloning of many examples from a number of species. Early studies are defining the importance of domain structures in enzyme-inhibitor and matrix interactions. Further work covering the function of specific residues by site-directed
Figure 3. The central role of plasmin and stromelysin in the pericellular activation cascade of matrix metalloproteinases. Urokinase-type plasminogenactivator (uPA) bound to cell receptors (R) is functional in both the singlechain (sc) and two-chain (tc) forms, activating cell- or matrixbound plasminogen, and is regulated by plasminogen activator inhibitors (PAIs). The plasmin that is generated can activate prostromelysin and gelatinase B directly, the process being regulated by Q!2-antiplasmin (Q! 2AP) and also by TIMPs, since the final stages of activation are autocatalytic. Plasmin and stromelysin can activate collagenase by sequential cleavages. These events are also regulated by Q!2AP and TIMPs. The concept of localized and limited activation at cellmatrix adhesion sites is supported by the abundance of prometalloproteinases, TIMPs, and PAIs observed in the culture medium of connective tissue cells.
mutagenesis and X-ray crystallographic analyses is now required. The importance of extracellular regulatory mechanisms in determining the activity of the MMPs has become apparent, and studies are now under way to determine in vivo events surrounding the activation and inhibition processes. The focal nature of activators, including receptor-bound plasmin or the specific gelatinase A membrane activator, has been defined. Reagents to analyze the location of activators and active versus latent forms of MMPs, in relation to the cell membrane and cell-matrix adhesion sites, are now being developed. Acknowledgments: Dr. Murphy is supported by the Arthritis and Rheumatism Council, UK. We acknowledge the contributions of many whose work has been mentioned here but not cited in the restricted reference list.
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metalloproteinases-2. J. Bioi. Chem. 266: 13064-13069. 26. Murphy, G., F. Willenbrock, R. V. Ward, M. I. Cockett, D. Eaton, and A. J. P. Docherty. 1992. The C-terminal domain of 72 kDa gelatinase is not required for catalysis but it is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases. Biochem. J. 283:637-641. 27. Vallee, B. L., and D. S. Auld. 1990. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29:56475659. 28. Allan, J. A., R. M. Hembry, S. Angal, J. J. Reynolds, and G. Murphy. Binding of latent and high M r active forms of stromelysin to collagen is mediated by the C-terminal domain. J. Cell Sci. 99:789-795. 29. Ward, R. V., S. J. Atkinson, P. M. Slocombe, A. J. P. Docherty, J. J. Reynolds, and G. Murphy. 1991. Tissue inhibitorofmetalloproteinases-2 inhibits the activation of 72 kDa progelatinase by fibroblast membranes. Biochim. Biophys. Acta 1079:242-246. 30. Ward, R. V., R. M. Hembry, J. J. Reynolds, and G. Murphy. 1991. The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-l , Biochem. J. 278: 179-187. 31. Banyai, L., and L. Patthy. 1991. Evidence for the involvement of type II domains in collagen binding by 72 kDa type IV procollagenase. FEBS Lett. 282:23-25. 32. Docherty, A. J. P., A. Lyons, B. J. Smith et al. 1985. Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroidpotentiating activity. Nature 318:66-69. 33. Boone, T. c., M. J. Johnson, Y. A. De Clerck, and K. E. Langley. 1990. cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases. Proc. Natl. Acad. Sci. USA 87: 2800-2804. 34. Murphy, G., P. Koklitis, and A. F. Came. 1989. Dissociation of tissue inhibitor of metalloproteinases (TIMP) from enzyme complexes yields fully 2.etive inhibitor. Biochem. J. 261: 1031-1034. 35. Williamson, R. A., F. A. O. Marston, S. Angal et al. 1990. Disulphide bond assignment in human tissue inhibitor ofmetalloproteinases (TIMP). Biochem. J. 268:267-274. 36. Murphy, G., A. Houbrechts, M. I. Cockett, R. A. Williamson, M. O'Shea, and A. J. P. Docherty. 1991. The N-terminal domain of tissue inhibitor of metalloproteinases retains metalloproteinase inhibitory activity. Biochemistry 30:8097-8102. 37. Matrisian, L. M. 1990. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet. 6:121-125. 38. Murphy, G., S. Atkinson, R. Ward, J. Gavrilovic, and J. J. Reynolds. 1992. The role of plasminogen activators in the regulation of connective tissue metalloproteinases. Ann. NY Acad. Sci. In press. 39. Vassalli, J.-D., A.-P. Sappino, and D. Belin. 1991. The plasminogen activator/plasmin system. J. Clin. Invest. 88:1067-1072. 40. Moscatelli, D., andD. B. Rifkin. 1988. Membrane and matrix localization of proteinases: a common theme in tumor cell invasion and angiogenesis. Biochim. Biophys. Acta 948:67-85. 41. Dean, D. D., J. Martel-Pelletier, J.-P. Pelletier, D. S. Howell, and J. F. Woessner. 1989. Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage. J. Clin. Invest. 84:678-685. 42. Chowcat, N. L., F. J. Savage, R. M. Hembry, andP. B. Boulos. 1988. Role of collagenase in colonic anastomoses: a reappraisal. Br. J. Surg. 75: 330-334. 43. Nomura, S., B. L. M. Hogan, A. J. Wills, J. K. Heath, andD. R. Edwards. 1989. Developmental expression of tissue inhibitor of metalloproteinase (TIMP) RNA. Development 105:575-583.
A number of metalloproteinases that degrade the extracellular matrix of connective tissues and two specific tissue inhibitors of metalloproteinases (TIMPs) have now been isolated, characterized, and cloned. Comparison of the enzyme sequences has allowed the delineation of domain structures, and initial studies have been carried out to assess the contribution of these domains to their biochemical and biologic properties, including activation, inhibition by TIMPs, and matrix binding. Such events represent the major levels of extracellular regulation of metalloproteinase activity, which is thought to be an important aspect of their control. Activation is probably a cell surface phenomenon, involving the plasminogen activator cascade or other membrane-associated mechanisms. The inhibitory action of TIMPs is postulated to be as important in activation as in the subsequent regulation of enzyme degradation of the matrix.
The matrix metalloproteinases (MMPs) are a family of zincand calcium-dependent endopeptidases that have the combined ability to degrade the various components of connective tissue matrices (1). They are synthesized and secreted by connective tissue and some hematopoietic cells and are known to be important in both normal remodeling processes (2) and in the accelerated destruction occurring in many diseases (3, 4). Regulation of the MMPs is stringent, occurring not only at the level of gene expression but extracellularly, after secretion, by the action of activators of the proenzyme forms and of specific inhibitors. The major natural inhibitor, tissue inhibitor ofMMPs (TIMPs), which is produced by the same cells, has been identified in two forms to date. This report will review our newly developing knowledge of the structure of MMPs and TIMPs and the potential contribution of their definable domain structures to their biochemical behavior. One of the major questions confronting biologists is the importance of MMP activation as a regulatory mechanism in vivo. In relation to this, our current understanding of possible activation pathways will be discussed.
Matrix Metalloproteinase Structure Nine MMPs have been identified by cDNA cloning and sequencing. Comparison of these sequences, in conjunction (Received in original form February 24, 1992 and in revised form March 17, 1992) Address correspondence to: Dr. Gillian Murphy, Cell and Molecular Biology Department, Strangeways Research Laboratory, Worts' Causeway, Cambridge CBl 4RN, United Kingdom. Abbreviations: matrix metalloproteinase, MMP; tissue inhibitor of metalloproteinases, TIMP. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 120-125, 1992
with biochemical and immunologic data, have indicated that two forms of collagenase, two gelatinases (type IV collagenases), and two stromelysins occur (Table 1). Three other enzymes, pump (punctuated metalloproteinase, matrilysin), "stromelysin-S," and metallo-elastase have somewhat different sequences and properties (Table 1). The interstitial collagenases are the most specific of the MMPs, cleaving the native helix of the fibrillar collagens (types I, II, and III) at a single locus. One form, MMP1, is produced by most connective tissue cells whereas MMP8 is confined to neutrophil granules (5, 6). The, gelatinases degrade types IV, V, VII, and X collagens and may act synergistically with interstitial collagenases in the degradation of collagens, since they efficiently degrade their denatured gelatin forms. They also degrade elastin. Two separate but similar cDNAs encoding a 72-kD gelatinase A (MMP2) and a 95-kD gelatinase B (MMP9) have been identified to date (Table 1), and the corresponding proteins have been shown to be associated with monocyte/macrophages, as well as many stimulated connective tissue cells (7, 8). The stromelysins 1 (MMP3) and 2 (MMP10) (5, 9) are potentially very important members of the MMP family, having broad pH optima and substrate specificities. They are able to degrade many extracellular matrix proteins, including proteoglycans and laminin, and are also potentiators of collagenase and 95-kD gelatinase activity (10, 11). A more recently discovered MMP, punctuated metalloproteinase (pump, MMP7, matrilysin), appears to be distantly related to the stromelysins but lacks the C-terminal domain (see below and Figure 2). Pump has broad proteolytic activity that includes elastin and proteoglycans (12) and has been shown to be associated with immature monocytes (13). A further putative MMP identified in the stroma of tumors, the trophoblast, and fetal lung fibroblasts by both cloning and immunologic studies has
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Update
TABLE 1
The matrix metalloproteinase family* MMP No.
8
Source
Substrates
Trivial Name
kD
Interstitial collagenase
55
Connective tissue cells
Fibrillar collagens I, II, and III; collagen X; gelatin; proteoglycans
Neutrophil collagenase
75
Neutrophils
Fibrillar collagens, gelatin, proteoglycans
Connective tissue cells, macrophages
Proteoglycan; cross-link: regions of collagens II, IV, and IX; collagens X and XI; procollagens; fibronectin; laminin; gelatin; collagenase; gelatinase B
3
Stromelysin-l
57
10
Stromelysin-2
57
Macrophages
As stromelysin-l but much lower activity
2
Gelatinase A
72
Most cell types
Denatured collagens; collagens IV, V, VII, X, and XI; elastin
9
Gelatinase B
95
Monocytes, connective tissue, tumor cells
As gelatinase A
7
Pump (matrilysin)
28
Immature monocytes, mesangial, tumor cells (not fully defined)
As stromelysins, elastin
11
"Stromelysin-3"
51
Stromal cells of tumors
Unknown
12
Metallo-elastase
57
Macrophages (mouse)
Elastin, fibronectin
* Molecular masses are for the human enzymes as estimated by SDS-PAGE except for MMPll (eDNA-predicted mass) and MMP12 (mouse). Minor glycosylated species of higher molecular mass are observed for MMPI and MMP3.
(Figure 1), including a propeptide of 77 to 87 amino acids lost during activation, a catalytic Zn2+- and Ca't-binding domain of 162 to 173 residues, and domains with sequence similarities to a number of extracellular matrix structural proteins (18). Of these, the C-terminal vitronectin-like domain of 202 to 213 amino acids is found in all MMPs except pump. The gelatinases also have a fibronectin-like gelatinbinding sequence inserted into the catalytic domain (Figure 1). The 95-kD gelatinase B has a further collagen-like insertion C-terminal to the catalytic domain. These extra gelatinase domains are contained within a discrete set of exons. The precise mechanism by which the propeptide domain maintains latency is not yet clear. Sequential processing events occur as part of the activation process involving exogenous and endogenous cleavages (11); the physiologic rele-
been named "stromelysin-3" (14). No specific catalytic activity has yet been defined for this protein; however, it is clear that it is structurally somewhat distant from the stromelysins (15). The most recently identified MMP cDNA is that for mouse macrophage metallo-elastase (16), but no human macrophage counterpart has yet been reported. This enzyme could potentially be important in matrix turnover since it degrades many matrix macromolecules, including elastin (17). The cDNA-predicted amino acid sequences of all the MMPs can be aligned, demonstrating a high degree of conservation between each type of enzyme across several mammalian species (about 80% similarity between collagenases and stromelysins). The similarity between types is lower but still a highly significant 50 %. Domains within these sequences can be delineated with apparent specific functions
Figure 1. Domain structure of the matrix metalloproteinases. All members of the matrix metalloproteinase family contain a definable propeptide that is lost during activation and contains the conserved motif PRCGV/NPD (see text), as well as a catalytic domain containing two putative Zn2 + -binding ligands (HEFI L/IGH). Stromelysin-3 has a unique extension of 10 amino acids between the propeptide and catalytic domains. Collagenases, stromelysins, and gelatinases all have a third C-terminal domain with similarities to members of the hemopexin family, including vitronectin. It contains a conserved disulfide bond linking the N- and C-terminal portions of the domain and is linked to the catalytic domain by a proline-rich sequence of five to ten amino acids. Both gelatinases have an extra domain related to the collagen-binding region of fibronectin that is inserted into the catalytic domain. Gelatinase B has a fifth domain, with similarities to several fibrillar collagens, inserted between the catalytic and C-terminal domains.
DOMAIN STRUCTURE OF MATRIX METALLOPROTEINASES Propeptide
Catalytic
C-terminal (vitronectin-like)
gelatinase A ( fibronectin-Iike)
C / / ",
""
"
PRCGXPD
pump C-terminus
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vance of this is discussed below. One aspect of the latent nature of pro MMPs may be explained by the fact that the propeptide contains a conserved sequence PR~GV/NPD in which the free cysteine may interact with the Zn 2+ of the adjoining catalytic domain, displacing the H 20 molecule required for catalytic activity (19). However, other features of the propeptide apparently stabilize the cysteine-Zn" interaction, since cleavage at sites upstream from the cysteinecontaining motif in the propeptide of MMPs such as stromelysin and pump will initiate further autocatalytic cleavages that lead to complete removal of the propeptide (11, 20). The catalytic domain of the MMPs has been defined by both biochemical and protein engineering approaches. In the case of pump, this is the only domain other than the propeptide. Both stromelysin and collagenase catalytic domains display proteinase activity against substrates such as casein, the former being identical to that of the full-length enzyme. However, the collagenase catalytic domain loses its unique ability to cleave triple helical collagen in the absence of the C-terminal vitronectin-like domain (21-24). Gelatinase A, expressed as a truncated form consisting of the catalytic and gelatin-binding domains, also shows identical substrate specificity to the full-length enzyme (25, 26). The catalytic domain of all the MMPs contains a short sequence of identity with thermolysin. In thermolysin, the sequence, HELTH is known to contain two of the three histidine residues (-142 and -146) that coordinate the zinc, as well as the catalytic glutamic acid. In the absence of structural information, the precise Zn2+ ligands of the MMPs are not known, but it seems likely that the histidine residues of the conserved HEL/I/FGH motif are involved, although the third ligand can only be speculated upon (15,27). Attempts at mutagenesis of a number of conserved histidine and aspartic acid residues in stromelysin that are potential candidates have all yielded inactive enzyme forms. The C-terminal domain, which is present in all the MMPs except pump, shows some similarity to the hemopexin family of proteins, which includes vitronectin. The biologic relevance of this domain is only just being elucidated with the preparation of C-terminally deleted enzyme forms by both biochemical and protein engineering approaches. This domain mediates the interaction of both collagenase and stromelysin to collagen fibers (24, 28), a phenomenon that can be observed by immunohistochemical studies on rapidly resorbing tissues. In the case of collagenase, binding only occurs when the intact enzyme is in the active form, presumably due to the unmasking of some essential components of the binding domain. The isolated collagenase or stromelysin C-terminal domain will bind to collagen, but apparently to different sites. The presence of the C-terminal domain is essential for the specific collagenolytic activity of collagenase. However, this domain cannot confer collagenolytic activity to the catalytic domain of stromelysin when a hybrid enzyme is engineered and expressed. Similarly, the stromelysin C-terminal domain can mediate binding of the collagenase catalytic domain to collagen but not its specific ability to cleave collagen (24). The C-terminal domain of progelatinase A has an even more intriguing role, apparently being required for the action of a gelatinase activator that has been localized to the cell (26, 29). The C-terminal domain also interacts weakly with the inhibitor TIMP-2 when the enzyme
is in its proform, such that progelatinase A is frequently isolated as a complex with TIMP-2 (25, 30). Consequently, TIMP-2 is an effective and specific inhibitor of the action of the membrane activator (see below; 27). In the case of all the MMPs studied thus far, it appears that the C-terminal domain may also have a role in the binding of active enzyme forms to both TIMP-l and TIMP-2 (24-26). The precise role of the extra domains expressed in the gelatinases has not yet been analyzed. Both enzymes have a domain consisting of three repeats similar to those found in the type II domains of the gelatin-binding region of fibronectin. Expression of the isolated domain from 72-kD gelatinase has been carried out and binding to gelatin Sepharose demonstrated (31). The 95-kD gelatinase has a small extra domain with a sequence similar to that found in many collagens (8). It is not yet known if the catalytic domain of the gelatinases is functional in the absence of these domains. Tissue Inhibitors of Metalloproteinases The major physiologic inhibitors of the MMPs are a2macroglobulin, which is restricted in its sites of activity due to its large size (780 kD), and a family of inhibitors that are specific for the MMPs and are produced by many cell types, including those of connective tissues. Four inhibitors have been reported, and two inhibitors of this family have been fully characterized and cloned. These TIMPs have closely related structures and inhibitory properties. TIMP-l is a 30-kD glycoprotein (32), and TIMP-2 a 23-kD unglycosylated protein (33). Their cellular distribution appears to be very similar, although TIMP-l is generally found in larger amounts. The occurrence of TIMP-2 is, however, not yet fully documented. The TIMPs act specifically against the active forms ofMMPs, TIMPs-l and -2 having very similar activities (30) and forming essentially irreversible complexes with equimolar stoichiometry. Initial estimates suggest that TIMP-l has a K, of 10-9 to 10-11 M for most MMPs (15). It has been shown that TIMP-l may be recovered by acid dissociation of complexes with stromelysin and is fully active and apparently unmodified (34). The TIMPs have 12 identically conserved cysteine residues forming six disulfide bonds, which confer marked stability to pH and temperature (35). A two-domain structure of three loops each can be delineated (Figure 2). The precise folding of these inhibitors appears to be essential since reduction and alkylation leads to a loss of activity. It has been shown that the N-terminal loops of TIMP-l encoded by amino acids 1 through 126 can fold independently of the C-terminal domain and act as an efficient inhibitor of most MMPs (36). A similarly truncated form of TIMP-2 behaves in the same manner. In each case, the unique binding to the proform of 95-kD and 72-kD gelatinases B and A, respectively, is abolished (36; G. Murphy, unpublished observations). We are still a long way from an understanding of the mechanism of TIMP inhibition, which will be achieved by a combination of specific mutagenesis studies and X-ray crystallography.
Extracellular Regulatory Mechanisms Activation Given the plethora of substrates that active MMPs can degrade, MMP regulation is necessarily stringent and in most
Update
123
Human TIMP-l
Figure 2. The loop structure of TIMP-l. The eDNA-predicted sequence of TIMP-l can be depicted as a six-loop structure on the basis of the distribution of the six disulfide bonds (36). The first two loops contain carbohydratebinding sites (CHO); TIMP-l is a complex glycoprotein and is variably substituted with sialic acid giving it a heterogeneous charge. The first three loops, when expressed alone, retain metalloproteinase-inhibitory properties.
cases occurs predominantly at the level of gene expression. This aspect of their control will not be discussed here and has been excellently reviewed by Matrisian (37). A second level of regulation occurs extracellularly and is thought to be of some importance, ultimately controlling the level of enzyme activity in terms of matrix destruction, namely the activation of secreted proenzyme forms and their subsequent inhibition by TIMPs (1, 18). As was discussed above, the stepwise process of MMP activation by loss of the propeptide may be initiated by exogenous proteolytic cleavage in the case of the collagenases, stromelysins-l and -2, pump, and gelatinase B. Nagase and associates (11) have systematically analyzed the action of potential physiologic enzyme activators, which include plasmin, plasma kallikrein, cathepsin B, cathepsin G, and neutrophil elastase. Plasmin has long been thought to be an important activator of MMPs, and this concept has been supported by a number of studies using cell model systems with both connective tissue and tumor cells (38). The study of plasmin generation from plasminogen by the action of plasminogen activators is a particularly active and exciting field (39). It has shown that plasmin generation and activity is highly focused, largely occurring at the cell surface where both plasminogen and the activators are specifically bound at cell adhesion plaques. It is likely that in localized pericellular regions, the action of a2 anti-plasmin is limited because of the location of plasmin on the cell membrane or local excess of free enzyme. Stromelysin-l, which can be sequestered on the collagenous matrix, is particularly susceptible to plasmin activation and once activated can potentiate collagenase activity and act as a gelatinase B activator (Figure 3). TIMPs may regulate the activation process to
some extent, since it has been shown that they can slow down or prevent the autocatalytic cleavages that occur after initiation of activation by the exogenous proteinases described above (26). Gelatinase A is unlike the other MMPs in that it is regulated differently at the transcriptional level. Furthermore, it differs in its mechanism of extracellular activation. The propeptide of this enzyme has no apparent cleavage site susceptible to plasmin and other proteinases, although it can undergo self-cleavage reactions to lose the propeptide and become active (11, 30). However, this enzyme can be activated by a fibroblast- or tumor cell membrane-mediated process that is sensitive to metalloproteinase inhibitors. The membrane activator which is specific for gelatinase A and does not activate the other pro MMPs (29) can be induced by a number of effectors including concanavalin A, phorbol esters, and transforming growth factor-S (29, 37). It is not yet known if the activator is itself a proteinase or merely binds gelatinase A (as discussed above) and initiates autocatalytic cleavages. This activation process is efficiently prevented by TIMP-2 when bound to the proenzyme but not by TIMP-l, which only binds to active gelatinase A (25, 26, 30). Gelatinase A is a particularly widespread proteinase and appears to be constitutively expressed by many cells. It may therefore be postulated that the membrane activation mechanism plays an important regulatory role, by controlling the precise locations at which this enzyme may be activated. Inhibition The importance of TIMPs as regulators of MMP activity can only be demonstrated equivocally. In many cell model systems, the ability of TIMPs to prevent matrix degradation has
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
scuPA-tcuPA
8 -t--t
Prostromelysin
1
Plasminogen - - - - - - - -... PLASMIN
I-/----:~------+~ _-
been demonstrated (40). In some disease situations, e.g., osteoarthritic cartilage, it has been shown that TIMP levels rise but do not compensate for the much larger increase in degradative activity of MMPs (41). Immunolocalization studies of TIMP-1 expression in in vivo models of resorption have demonstrated the precise spatial and temporal expression of this inhibitor relative to MMPs. In the developing growth plate of the young rabbit, TIMP-1 is specifically localized to certain regions of the tissue where collagenase and gelatinase expression are high (2). In a rabbit colon wound-healing model studied at specific intervals after anastomoses, it is evident that collagenase is expressed within the first 12 h but TIMP-1 expression does not occur until 24 h (42). TIMP-1 expression in the developing embryo has been studied by in situ hybridization and shown to be particularly localized in osteogenic tissues and the ovary (43), but no concomitant MMP studies have yet been made. TIMPs have been shown to be sequestered in tissues such as cartilage but no evidence of direct matrix binding has been obtained. It can be postulated that, like the plasminogen activator inhibitors, they may be limited in their action by the focal nature of enzyme activation at certain sites on the cell surface.
Conclusions and Future Directions Knowledge of structure-function relationships in the MMPs and TIMPs is only at an early stage following the rapid cloning of many examples from a number of species. Early studies are defining the importance of domain structures in enzyme-inhibitor and matrix interactions. Further work covering the function of specific residues by site-directed
Figure 3. The central role of plasmin and stromelysin in the pericellular activation cascade of matrix metalloproteinases. Urokinase-type plasminogenactivator (uPA) bound to cell receptors (R) is functional in both the singlechain (sc) and two-chain (tc) forms, activating cell- or matrixbound plasminogen, and is regulated by plasminogen activator inhibitors (PAIs). The plasmin that is generated can activate prostromelysin and gelatinase B directly, the process being regulated by Q!2-antiplasmin (Q! 2AP) and also by TIMPs, since the final stages of activation are autocatalytic. Plasmin and stromelysin can activate collagenase by sequential cleavages. These events are also regulated by Q!2AP and TIMPs. The concept of localized and limited activation at cellmatrix adhesion sites is supported by the abundance of prometalloproteinases, TIMPs, and PAIs observed in the culture medium of connective tissue cells.
mutagenesis and X-ray crystallographic analyses is now required. The importance of extracellular regulatory mechanisms in determining the activity of the MMPs has become apparent, and studies are now under way to determine in vivo events surrounding the activation and inhibition processes. The focal nature of activators, including receptor-bound plasmin or the specific gelatinase A membrane activator, has been defined. Reagents to analyze the location of activators and active versus latent forms of MMPs, in relation to the cell membrane and cell-matrix adhesion sites, are now being developed. Acknowledgments: Dr. Murphy is supported by the Arthritis and Rheumatism Council, UK. We acknowledge the contributions of many whose work has been mentioned here but not cited in the restricted reference list.
References 1. Docherty, A. J. P., and G. Murphy. 1990. The tissue metalloproteinase family and the inhibitor TIMP: a study using cDNAs and recombinant proteins. Ann. Rheum. Dis. 49:469-479. 2. Brown, C. C., R. M. Hembry, andJ. J. Reynolds. 1989. Immunolocalization of metalloproteinases and their inhibitor in the rabbit growth plate. J. Bone Joint Surg. [Am.] 71-A:580-593. 3. Gravallese, E. M., J. M. Darling, A. L. Ladd, J. N. Katz, and L. H. Glimcher. 1991. In situ hybridisation studies of stromelysin and collagenase messenger RNA expression in rheumatoid synovium. Arthritis Rheum. 34:1076-1084. 4. Henney, A. M., P. R. Wakeley, M. J. Davies et al. 1991. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hy. bridization. Proc. Natl. Acad. Sci. USA 88:8154-8158. 5. Whitham, S. E., G. Murphy, P. Angel et al. 1986. Comparison of human stromelysin and collagenase by cloning and sequence analysis. Biochem. J. 240:913-916. 6. Hasty, K. A., T. F. Pourmotabbed, G. I. Goldberg et al. 1990. Human neutrophil collagenase. A distinct gene product with homology to other matrix metalloproteinases. J. Bioi. Chem. 265: 11421-11424.
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