Collagenase 1

472 CLAN MA(M) - M10A 125. CoUagenase 1

125. CoUagenase 1 Databanks

Activity and Specificity

MMP-1 cleaves collagen types I, II, III, VII and X (Gadher etal., 1989; Seltzer etal, 1989). Welgus et al. (1980) calculated that only 22 molecules of collagen are degraded per molecule of coUagenase per hour and this turnover rate is one of the slowest observed for an enzyme-catalyzed reaction. Highly cross-linked collagen is slowly attacked by MMP-1 but the rate increases if other proteinases, such as stromelysin (Chapter 131), are present. Gelatin and the core protein of proteoglycan can be cleaved (both to a limited extent). The activity against gelatin is low compared with that of coUagenase 3/MMP-13 (Chapter 127), which has a broad proteolytic activity (Knäuper et al., 1996). MMPName and History 1 cleaves itself within the polypeptide linker between the N- and C-terminal domains (Clark & Cawston, 1989) at The name coUagenase was first used to describe a bactePro269-(-Ile270-Gly in human MMP-1. The corresponding rial enzyme that degraded collagen; then Gross & Lapiere Pro-Ser-Gly sequence in pig is more resistant to cleavage (1962) identified an enzyme produced by resorbing tadpole but Ala238-^Ile239 is slowly cleaved at high concentration tail in culture that specifically degraded triple-helical colin pig MMP-1 (Clark et al., 1995). A human enzyme lagen at neutral pH. This discovery provoked an important with these residues mutated to the pig linker sequence is shift in understanding concerning the susceptibility of collarelatively stable (O'Hare et al., 1995). This autoprocessing gen to attack by endogenous proteinases. The term interstitial may be physiologically relevant but could represent an coUagenase was used to describe this enzyme as it cleaved artifact of isolation. the triple helix of interstitial collagens (types I, II and III) When the C-terminal domain is lost then the N-terminal across all three a chains at a single point three-quarters of domain containing the catalytic zinc retains the ability to act the way from the N-terminal end of the molecule. The human as a proteinase (and it can also act as an activator of the a 1(1) chain is cleaved between Gly-Pro-Gln-Gly775-^Ile-Alaproenzyme) but it is unable to cleave triple-helical collagen. Gly-Gln. The enzyme belongs to the matrixin family and This implicates the C-terminal domain in collagen binding has been designated matrix metalloproteinase 1 (MMP-1) although little is known of the properties of the C-terminal (Nagase et ah, 1992). It has also been called vertebrate coldomain once cleaved. It may block the action of coUagenase lagenase, mammalian coUagenase orfibroblast coUagenase. or enhance activity if the C-terminal domain is involved in More recently it has been called coUagenase 1 (Coll) to dis- loosening the triple helix. tinguish it from enzymes subsequently described from the a2-Macroglobulin is cleaved in a susceptible sequence matrixin family that cleave interstitial collagens in the same Gly-Pro-Glu-Gly679-fLeu680-Arg-Val-Gly adjacent to the way, namely neutrophil coUagenase (MMP-8; coUagenase 2) bait region (Mortensen et al., 1981) and catalysis proceeds (Chapter 126), coUagenase 3 (MMP-13) (Chapter 127) and much more rapidly than for any other protein substrate so far coUagenase 4 (MMP-18) (Chapter 128). However, the name studied for this enzyme. MMP-1 also cleaves oil-proteinase coUagenase 1 could still be confused with some of the bacinhibitor at two sites (Met-Phe-|-Leu-Glu and Ile-Pro-|-Metterial collagenases. Thus, in order to avoid any ambiguity, Ser), áé-antichymotrypsin at one site (Ser-Ala-fLeu-Val) the best name to use is MMP-1. Gelatinase A (MMP-2) (Desrochers et al., 1991) and serum amyloid A (SAA) at (Chapter 129) also cleaves interstitial collagen at the threesome point in the sequence Gly-Gly-Val-Trp-Ala-Ala-Gluquarters/one-quarter site (Aimes & Quigley, 1995) as does Val. The cleavage of a2-macroglobulin allows MMP-1 to MT1-MMP (MMP-14) (Chapter 137) (Ohuchi et al., 1997). be rapidly inhibited, serpin cleavage is likely to have a Until recently no rodent MMP-1 could be detected but physiological function and as SAA is known to induce two enzymes named Mcol-A and Mcol-B are found within coUagenase secretion, cleavage of this protein may act to the cluster of MMP genes located at the A1-A2 position limit tissue destruction (Mitchell et al., 1993). on murine chromosome 9. These enzymes are most similar Netzel-Arnett et al. (1991b) measured the rate of hydrolto human MMP-1, sharing 74% identity (nucleotides) and ysis of 60 oligopeptides covering the P4 to the P5' subsites 58% identity (amino acids). One of these enzymes, Mcol-A, of the substrate. They modeled these peptides on the known cleaves collagen at the specific cleavage site and occupies cleavage sites in collagens as well as on other protein suba position sytenic to the human MMP-1 locus at llq22. strates such as a2-macroglobulin. The amino acids in subsites Both enzymes are expressed during mouse embryogenesis, P4 to P4' all influence the rate of hydrolysis. Some substituparticularly in mouse trophoblast giant cells although neither tions not found in collagens led to a higher rate of hydrolysis, enzyme is as widely distributed as MMP-1 in other species particularly at subsites PI, PI' and P2'. Ala is preferred in (BalbinetaL, 2001). subsite PI and Tip or Phe in subsite P2'. MMP-1 does not MEROPS name: CoUagenase 1 MEROPS classification: clan MA(M), family M10A, peptidase M10.001 IUBMB: EC 3.4.24.7 Species distribution: Chordata Sequence known from: Bos taurus, Equus caballus, Homo sapiens, Oryctolagus cuniculus, Ovis aries, Rana catesbeiana, Sus scrofa Tertiary structure: Available

CLAN MA(M) - M10A 125. CoUagenase 1 473

tolerate aromatic residues in subsite PI'. Of peptides tested the best sequence appears to be Ala-Leu-Ala+Leu-Arg-ValThr. Tyr and Met could be easily substituted at P2; His, Phe Pro, Gin, Val and Met in PI; Val, Met and Phe in PI'; Glu and Leu in PI'; Ser, Met and Ala in P3' and Pro in P4'. Only Val in subsite PI and Phe in subsite PI' are predicted to be bad substitutions. It is not clear why only one site in each collagen a chain is cleaved while many potentially cleavable sites are unaffected. Fields (1991) suggests that the cleavage site in the interstitial collagens is characterized by a region of tightly coiled triple helix preceding the cleavage site, with a loosely coiled region immediately after. Arg is the only charged residue, always found at P5 or P8', in a 25 amino acid sequence that is found around the collagen cleavage site. There must not be an imino acid adjacent to the Gly-)-Ile/Leu bond cleaved. Careful examination of 31 identified sites with the required sequence, but that remain uncleaved, reveal that none fulfilled all of the above properties. There may be other groups and side chains along the collagen fibril that are not present at the cleavage site and so allow access for cleavage. Triple-helical peptides can compete with collagen and the Cterminal domain could contain a triple-helix recognition site (Netzel-Arnett et al., 1994) and such peptides have been used to study the mechanism of action of MMP-1 (Lauer-Fields et al., 2000). Collagen cleavage is unusual as three peptide bonds must be cleaved and the active-site cleft is too small and rigid to accommodate all three a chains of the collagen molecule at one time, being only 5 Ä wide whilst the diameter of triplehelical collagen is about 15 Ä. Chung et al., (2000) estimated that the closest peptide bond of collagen is about 7 Ä away from the catalytic zinc atom so changes in conformation of either enzyme, substrate or both is required for catalysis. It is likely that MMP-1 binds to collagen, cleaves one peptide bond, then the second and third prior to disengaging (Welgus et al., 1985). The binding of the C-terminal domain to collagen may perturb the triple helix, perhaps where it is loosely coiled, to extend the first chain into the activesite cleft. Subsequent cleavages could then presumably occur with increasing ease. One proposal is that the C-terminal domain interacts with collagen on the opposite side of the helix to the active site, sandwiching and trapping the substrate at the active-site cleft (Gomis-Rüth et al., 1996). Two molecules of MMP-1 could be involved with the Cterminal domain of one molecule, allowing the other catalytic domain to cleave. Alternatively the C-terminal domain may bind to a point along the helix distant from the cleavage point and the length of the linker may then position the active site at the cleavage point whilst destabilizing the triple helix. Nine residues, 183RWTNNFREY191, which are located between the fifth ß strand and the second a helix in the catalytic domain of MMP-1 are important for activity against collagen although additional structural elements in the catalytic domain and linker are also required. Tyr 191 is conserved in all MMPs that cleave collagen and is important for maximal activity (Chung et al., 2000). A collagenasecharacteristic cw-peptide bond, Glul88-Tyrl89 in MMP-1 and Asnl89-Tyrl90 in MMP-8 is proposed as being a critical feature in this region that determines the recognition of the triple helix of collagen (Brandstetter et al., 2001).Knauper

et al. (2001) also showed that the region in MMP-1 coded for by exon 5 is involved in substrate specificity. Insertion of this region from MMP-3 into MMP-1 reduced activity but the chimeric enzyme could still cleave collagen. Different possible mechanisms have been summarized by Overall (2001) and it is clear that recognition sites on both N- and C- terminal domains are important, followed by intercalation of the proline-rich linker with the collagen triple helix to separate the three chains. Overall (2001) proposed that the most likely sites for collagen binding are on the rim of the second hemopexin blade that is closest to the catalytic cleft and the area around the S3' subsite on the N-terminal domain. Assay of activity uses radiolabeled collagen at pH 7.6 (the pH optimum of the enzyme), and calcium (1-lOmM) is also included to confer stability at 37 °C. Collagen is allowed to form fibrils and at the end of the assay period undigested collagen is removed by centrifugation (Cawston & Barrett, 1979). This assay has now been adapted to a 96well plate format (Koshy et al., 1999). Other assay systems have been employed using 96-well plate formats with collagen bound to each well. Care needs to be taken that collagen is not denatured to ensure that assays measure coUagenase activity. Trypsin resistance is usually taken as a reliable measure of collagen integrity. To definitively show that collagenase cleavage at the three-quarters/one-quarter position occurs involves cleavage followed by separation by SDSPAGE to identify the three-quarters and one-quarter products. Different assay systems and the labeling methods for collagen are described by Dioszegi et al. (1995). Activity assays do not distinguish among various enzymes that cleave collagen and for reliable assays that just measure MMP-1, ELISAs are used (Cooksley et al., 1990; Clark et al., 1992; Zhang et al., 1993). Some immunoassays can recognize neoepitopes revealed after specific cleavage of collagen by MMP-1 and other collagenases and these assays can be used to demonstrate coUagenase activity in vivo and in vitro (Hollander et al., 1994). Collagen zymography has also been proposed as a sensitive and specific method for measuring interstitial coUagenase and has the advantage that total coUagenase can be measured in that any TIMP-MMP complexes are dissociated (Gogly et al, 1998). Heparin-enhanced zymography can also detect MMP-1 (Yu & Woessner, 2001). Quenched fluorescent substrates (Stack & Gray, 1989; Knight, 1995; Beekman et al., 1996) can be made that are preferentially cleaved by MMP-1 although these are never totally specific; other MMPs usually cleave at a lower rate. The substrate Dnp-Pro-Leu-Ala-fLeu-Trp-Ala-Arg was developed as a specific substrate for MMP-1 (NetzelArnett et al., 1991a) and Knight et al., (1992) have reported a more sensitive assay for MMP-1 using Mca-Pro-LeuGly+Leu-Dpa-Ala-Arg-NH2 (Dpa: N-3-(2,4-dinitrophenyl)L-2,3-diaminopropionyl). MMP-1 is inhibited stoichiometrically by TIMP-1 (tissue inhibitor of metalloproteinases 1) (Cawston et al., 1983) and TIMP-2 (DeClerck et al., 1991). It is rapidly inhibited by a2-macroglobulin (Cawston & Mercer, 1986) in preference to TIMP-1 and inhibited by chelators such as 1,10-phenanthroline (2mM) and EDTA (lOmM). Specific inhibitors with hydroxamate or chelating groups coupled to peptides that mimic the cleavage sequence are effective in the nanomolar range (Henderson et al., 1990; Cawston,

474 CLAN MA(M) - M10A 125. Collagenase 1 1996; Skiles et al, 2001). These inhibitors can be modeled on cleavage sites in substrates, propeptide cleavage sites, propeptide sequences that interact with zinc, autolytic cleavage sites or sequences cleaved in other inhibitors such as serpins or oi2-macroglobulin. Some specificity is seen with these inhibitors for individual enzymes and the structural information available on MMPs has led to MMP-1-specific inhibitors or inhibitors that spare MMP-1 (Toba et al., 1999; Szardenings et al., 1999). Tetracyclines are also reported to inhibit MMP-1 both in vivo and in vitro (Greenwald, 1994; Greenwald et al., 1987). Inhibition of MMPs as therapeutic targets in cancer or the arthritides has had only limited success. New roles for MMPs and approaches to therapy in cancer have been reviewed (Egeblad & Werb, 2002).

Structural Chemistry MMP-1 is synthesized as a preproenzyme of 469 amino acids with a prepropeptide (19 amino acids), a propeptide domain (81 amino acids), a catalytic domain (162 amino acids), a linking peptide (16 amino acids) and the C-terminal domain (192 amino acids) as shown in Figure 125.1. Its sequence is closely related to those of neutrophil collagenase (Chapter 129) and collagenase 3 (Chapter 127) as well as other matrix metalloproteinases. The proenzyme of Mr 51929 is activated to an Mr of 42570. This activation is accomplished by organomercurials, proteolysis by stromelysin (MMP-3; Chapter 131), trypsin, plasmin and other serine proteinases and by chaotropic or chemical agents (Springman et al., 1990). Activation depends on proteolytic cleavages that disrupt the interaction between the catalytic zinc and the conserved cysteine in the propeptide sequence Pro-Arg-Cys92-(Val/Asn)-Pro-Asp-(Val/Leu)(Ala/Gly) with release of the propeptide (Windsor et al., 1991). The final step to give maximal activation is a cleavage at Gln99+Phel00 (Suzuki et al, 1990). A number of X-ray structures are published for the Nterminal domain of MMP-1 (Borkakoti et al., 1994; Lowry etal, 1992; Spurlino et al, 1994; Lovejoy et al, 1994). This domain consists of three a helices with five ß sheets; two zinc atoms are found, one in the catalytic site and one in a structural role. Some biochemical studies have suggested that native MMP-1 contains only one zinc ion (Willenbrock et al, 1995) and it is possible that the inclusion of zinc in refolding mixtures for recombinant proteins may introduce a second ion into the molecule. The structural studies suggest that the second zinc ion is tightly held and performs a structural role although other studies suggest that the second zinc may not be an absolute requirement or could prepro peptide

propeptide

be replaced by other similar ions (Springman et al, 1995). Two to three calcium ions are also present in this domain. The full-length structure of the pig MMP-1 (Figure 125.2) shows the N-terminal domain linked by an unstructured proline-rich linker peptide, which is highly exposed (thus explaining the susceptibility to autocatalytic cleavage). This links to the C-terminal domain which has a unique fourbladed ß-propeller structure stabilized by a calcium ion and a disulfide bond that links the first unit of ß sheet to the fourth unit at the C-terminus of the protein (Li et al, 1995). In Figure 125.2, a peptide-based inhibitor, co-crystallized with the protein, is shown binding to the active-site zinc atom. The other zinc and calcium ions are also shown. The position of the two domains, relative to each other, will change in solution as there is little interaction between the domains and little structure within the linking peptide (Bode et al, 1999). Knowledge of the structure of the catalytic site (Figure 125.3) allows the design of new specific inhibitors that prevent the action of MMP-1 and act as therapeutic molecules preventing collagen turnover. The SI' specificity pocket is the most prominent pocket within the MMP catalytic domain and largely determines specificity. This pocket is relatively small in MMP-1; its

Figure 125.2 The structure of MMP-1 showing the N-terminal domain which contains two zinc ions and three calcium ions. A highly exposed linker peptide joins to the C-terminal domain which has a four-bladed ß-propeller structure. The first blade of the propeller is joined to the fourth by a disulfide bond and a calcium ion is found in the central channel where the four blades meet.

catalytic domain

hinge

C-terminal domain

192

QPRC92GVP

Figure 125.1 Domain structure of MMP-1.

CLAN MA(M) - M10A 125. Collagenase 1 475

Figure 125.3 A high-power view of the active-site zinc with a hydroxamate peptide inhibitor bound to the zinc and filling the active sites. depth is defined by Arg214, at the bottom of the pocket in a position equivalent to Leu218 in MMP-13. MMP-13, -3, -8 have large open pockets and this difference can be used to make inhibitors specific for MMP-1. However in some compound series it is surprising that those with large ÑÃ groups are potent MMP-1 inhibitors. Such compounds require Arg214 to change position so that the ST pocket can open (Lovejoy et al., 1999). The structure of the N-terminus of neutrophil collagenase (MMP-8) (Chapter 126) is well-ordered if Phe is present as the N-terminal amino acid but disordered if other residues are at the N-terminus of the protein (Reinemer et al., 1994). The authors suggest that this is likely to also be the case for MMP-1. An ammonium group from the N-terminal PhelOO forms a salt-link with the side-chain carboxylate group of the strictly conserved Asp252. Thus stabilization of the catalytic site could be conferred by strong hydrogen bonds made by the strictly conserved Asp252 with the characteristic conserved Met236, which lies underneath the active-site residues. These structural observations explain the highspecific-activity forms of collagenase that can be purified if Phe is present at the N-terminus after activation in the presence of stromelysin (Cawston & Tyler, 1979).

Preparation MMP-1 is widely distributed; it is produced by fibroblasts, chondrocytes, macrophages, endothelial cells and keratinocytes and is upregulated by a variety of stimuli (Goldring, 1993; Vincenti, 2001). It has been purified from conditioned culture medium of different tissues or cells from a variety of species (early references listed in Barrett & McDonald, 1980; Woessner, 1992). Standard Chromatographie procedures can be used to successfully purify this enzyme from these sources (Cawston & Murphy, 1981). Commonly used Chromatographie steps include zinc-chelate affinity chromatography, heparin-Sepharose or CM-Sepharose and gel filtration. Zinc-chelate affinity chromatography is a useful first step as it binds the enzyme but

allows TIMP-1 (often found in cell-conditioned media) to pass through the column without binding. Antibody affinity chromatography has also been used. Other methods involve the use of specific inhibitors coupled to Sepharose (Moore & Spilburg, 1986). Coupling of these inhibitors to activated Sepharose should be allowed to proceed for 24-48 h to ensure good coupling efficiency if proline is the amino acid at the N-terminus of the peptide-hydroxamate. These peptide-hydroxamate matrices only purify active forms of the enzyme and are rarely totally specific, so that MMP-1 preparations may be contaminated with other MMPs unless different matrices are used prior to this step. MMP-1 is relatively stable to exposure to either high (9.5) or low (4.5) pH providing this is kept to short time periods. The inclusion of calcium ions and a detergent such as Brij-35 is essential for maximum yields. Preparations of wild-type porcine MMP-1 are remarkably stable at neutral pH and 1M salt at 4°C. MMP-1 has been purified 135-fold from human skin fibroblasts (Stricklin et al., 1977), 235-fold from pig synovial tissue conditioned media (Cawston & Tyler, 1979) and 8500-fold from cattle nasal cartilage extract (Boukla, 1990). Large yields have been obtained from gingival fibroblasts in culture stimulated with interleukin Iß yielding 5 mgliter -1 of conditioned culture medium (Lark et al., 1990). MMP-ls from rabbit, pig, bovine, bull frog and human have been cloned. MMP-1 has been expressed in COS cells (Murphy et al., 1987), E. coli as inclusion bodies (Windsor et al., 1991), E. coli as a fusion protein (O'Hare et al, 1992) and in baculovirus-based expression systems (Vallon & Angel, 2001). Recombinant proteins can be purified as described above or fusion proteins can be expressed and specific purification strategies used for the fusion partner followed by subsequent cleavage of MMP-1. Refolding of MMP-1 is best performed by slowly removing denaturant and many protocols include low levels of zinc ions in addition to calcium ions (see Zhang & Gray, 1996). When selecting a vector for expression of active enzyme, care needs to be taken to ensure that the correct N-terminal Phe is expressed, providing enzyme of the highest specific activity.

Biological Aspects The structure of the MMP-1 gene contains ten exons and nine introns in 8-12kbp of DNA and is located on the long arm of chromosome 11 in the human at 1 lq22 (Huhtala et al., 1991). The sequence of the cDNA clone is published (Goldberg et al., 1986). MMP-1 is induced by the proinflammatory cytokines IL-1 and tumor necrosis factor a (TNFa), various growth factors such as EGF, PDGF, basic FGF and oncostatin M. The promoter region contains TATA, API and PEA3 elements. Studies with rabbit MMP-1 have shown that interleukin 1 (IL-1) does not strongly induce API-binding activity but does increase MMP-1 gene transcription through sequences in the distal promoter. It is clear that the regulation of collagenase gene expression by proinflammatory cytokines such as IL-1 requires both transcriptional and posttranscriptional mechanisms (Vincenti et al., 1994). Phorbol esters activate regulatory elements in the proximal promoter including an API site, a PEA3-like element and an API-like element (TTAATCA) located 186bp from the transcriptional start site (Vincenti et al., 1996). These authors have shown

476 CLAN MA(M) - M10A 125. Collagenase 1 that activation of rabbit collagenase transcription by proinflammatory cytokines involves the activation of an src-related tyrosine kinase (Vincenti et al, 1996; Vincenti, 2001). Maximal activation of collagenase transcription can occur when the cytokines oncostatin M and IL-1 are added together (Korzus et al, 1997). This study also reported that both AP1and STAT-binding sites in the MMP-1 promoter are involved in astrocytes. Later studies in chondrocytes found no evidence for this oncostatin M response element and synergistic cooperation required STAT binding to the c-fos promoter to accomplish maximum rates of transcription. In cartilage cultures oncostatin M and IL-1 synergize to promote collagen loss through upregulation of MMP-1, demonstrating that this mechanism could be important in disease (Catterall et al, 2001). Other agents can also regulate MMP-1 and these include serotonin, leukoregulin, calcium ionophore, lipopolysaccharide, substance P, UV irradiation, connective tissue fragments (e.g. fibronectin) and mechanical loading. Heparin and also calmodulin have been shown to affect the production of collagenase in some systems. The response to individual agents can be specific to individual cell types (Goldring, 1993). It can be modulated by several hormones such as progesterone and 1,25-dihydroxyvitamin D3 and estradiol (Delaisse et al, 1988). Induction of expression can occur with physical rather than chemical stimuli, such as cell shape change, phagocytosis of particles such as crystals, heat shock, treatment with cytochalasin B, altered cell-matrix interactions or direct contact with inflammatory cells. Cell-cell contact between activated T cell clones (or cell membranes prepared from T cells) and either monocytes or dermal fibroblasts upregulates MMP-1 production (Miltenburg et ai, 1995). MMP-1 is downregulated by a variety of agents and these include IL-4, transforming growth factor ß (Urii et al., 1998), interferon ã, retinoic acid and glucocorticoids. MMP-1 is implicated in a wide variety of physiological and pathological processes where collagen degradation occurs. These include rheumatoid arthritis, osteoarthritis, periodontal disease, respiratory disease, tumor invasion, metastasis, emphysema, angiogenesis, bone growth, corneal ulceration, tissue remodeling, wound healing, inflammatory bowel disease, atherosclerosis, aneurysm, restenosis and fibrotic diseases (for review see Birkedal-Hansen et al., 1993). The role of MMP-1 in bone resorption is not completely clear. It is produced by osteoblasts and is responsible for removing the layer of osteoid to allow osteoclasts to then bind to the exposed bone surface (Birkedal-Hansen et al., 1993). Studies also suggest that MMP-1 can be found in osteoclasts and may be left on the bone surface as these cells move over the bone. Once the cell has moved on and as the pH rises then collagen cleavage occurs (Okada et al., 1995). A single nucleotide polymorphism is located in the promoter region of the human MMP-1 gene that partially regulates gene expression (Rutter et al., 1998). The 2G/2G genotype enhances transcriptional activity and is associated with an increased risk of lung cancer, colorectal cancer invasiveness but not of erosive changes within the joint although raised levels of MMP-1 within synovial tissue are associated with erosive disease (Cunanne et al., 2001). There is ample evidence that in many biological situations the activation of proMMP-1 is the critical step that

determines if connective tissue turnover occurs but this is a neglected control point and it is still not clear exactly how this occurs in vivo. Plasminogen activators are produced by many cells that also produce MMP-1 and in the presence of plasminogen, plasmin (Chapter 515) can be generated and MMP-1 activated. Stromelysin (Chapter 131) activates MMP-1 along with other MMPs. In cartilage thiol proteinases may also sometimes be involved (Buttle et al., 1993) and recent evidence also implicates serine proteinases (Milner et al., 2001). In many biological systems MMP-1 can be upregulated and secreted from the cell but no collagen breakdown occurs as the enzyme is not activated. Thus activation may be the limiting control point in these systems (van der Zee et al., 1996). Some studies show that collagenolytic activity is associated with the plasma membrane of certain cells (O'Grady et al, 1982). Li et al. (2000) showed that plasma membrane-associated MMP-1 is responsible for the motility of smooth muscle cells on collagen specifically cleaving collagen beneath the leading edge and retracting cell tail after stimulation by FGF-2. It is not yet clear if this activity is a membrane-associated form of one of the recognized collagenases or if this is one of the membrane-associated metalloproteinases of the matrixin family known as MT-MMPs. It is known that MT1-MMP (Chapter 137) can degrade interstitial collagens at the three-quarters/one-quarter cleavage site (Ohuchi et al., 1997). MMP-1 also interacts with the 012 subunit of the oi2ßi-integrin via both hemopexin domain and the linker region and this can also localize MMP-1 on the cell surface as a mechanism to regulate its biological activity (Strieker et al, 2001).

Distinguishing Features As substrate, MMP-1 prefers type III collagen, neutrophil collagenase (MMP-8) (Chapter 126) prefers type I, and collagenase 3 (MMP-13) (Chapter 127) prefers type II collagen. Care must be taken when comparing different collagenases to ensure that the high-specific-activity forms of each enzyme are being compared. Collagenase 3 (MMP13) is able to cleave type I collagen at an additional amino telopeptide locus (Krane et al, 1996). The sequence of amino acids cleaved in peptide substrates is very similar for MMP1 and neutrophil collagenase (MMP-8). MMP-1 is smaller in size than neutrophil collagenase (MMP-8) and differs only slightly in size (approximately 3kDa) from collagenase 3 (MMP-13). Although both MMP-1 and collagenase 3 (MMP-13) bind to many of the same Chromatographie matrices during purification, it is reported that Sephadex QAE does not bind MMP-1 but binds collagenase 3 (MMP-13) (Van der Stappen et al, 1992). MMP-1 can be shown to be distinct from MMP-8 using specific antibodies and western blotting. The collagenases are often differentially regulated; for example MMP-1 is downregulated by retinoic acid whilst collagenase 3 (MMP-13) is often upregulated by this agent. MMP-8 is usually produced constitutively and stored within neutrophil but recent studies have shown this enzyme can be upregulated by proinflammatory signals in some cell types. Collagenase 3 (MMP-13) has a broad proteolytic activity. Gelatinase A (MMP-2) (Chapter 129), which can also cleave collagen, can be separated from MMP-1 by gelatin-Sepharose. Rabbit and sheep polyclonal antibodies

CLAN MA(M) - M10A

have been produced for a wide variety of species and monoclonal antibodies to the human enzyme and are available from a range of suppliers that include Cambio and Oncogene Research Products. A commercial ELISA kit for MMP-1 is available from Amersham (see Appendix 2 for full names and addresses of suppliers). A polyclonal antibody to pig MMP-1 is available from Chemicon International.

Further Reading Extensive reviews may be found in Birkedal-Hansen et al. (1993), Nagase & Woessner (1999), Dioszegi et al (1995) and Brinckerhoff & Matrisian (2002). References Aimes, R.T. & Quigley, J.P. (1995) Matrix metalloproteinase-2 is an interstitial collagenase. J. Biol Chem. 270, 5872-5876. Balbin, M., Fueyo, A., Knäuper, V., Lopez, J.M., Alvarez, J., Sanchez, L.M., Quesada, V., Bordallo, J., Murphy, G. & LopezOtin, C. (2001) Identification and enzymic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation. J. Biol. Chem. 276, 10253-10262. Barrett, AJ. & McDonald, J.K. (1980) Vertebrate collagenase. In: Mammalian Proteases: A Glossary and Bibliography, vol. 1. London: Academic Press, pp. 359-378. Beekman, B., Drijfhout, J.W., Bloemhoff, W., Ronday, K.H., Tak, P.P. & te Koppele, J.M. (1996) Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media. FEBS Lett. 390, 221-225. Birkedal-Hansen, H., Moore, W.G.I., Bodden, M.K., Windsor, L.J., Birkedal-Hansen, B., DeCarlo, A. & Engler, J.A. (1993) Matrix metalloproteinases: a review. Crit. Rev. Oral Biol. Med. 4, 197-250. Bode, W., Fernandez-Catalan, C , Tschesche, H., Grams, F., Nagase, H., Maskos, K. (1999) Structural properties of matrix metalloproteinases Cell. Mol. Life Sei. 55, 639-652. Borkakoti, N., Winkler, F.K., Williams, D.H., D'Arcy, A., Broadhurst, M.J., Brown, P.A., Johnson, W.H. & Murray, EJ. (1994) Structure of the catalytic domain of human fibroblast collagenase complexed with an inhibitor. Struct. Biol. 1, 106-110. Boukla, A. (1990) Purification and properties of bovine nasal hyaline cartilage collagenase. Int. J. Biochem. 11, 1273-1282. Brandstetter, H., Grams, F., Glitz, D.,Lang, A.,Huber, R.,Bode, W., Krell, H.,Engh, R.A. (2001)The 1.8 Ä crystal structure of a matrix metalloproteinase 8-barbiturate inhibitor complex reveals a previously unobserved mechanism for collagenase substrate recognition. J. Biol. Chem. 276, 17405-17412. Brinckerhoff, C.E. & Matrisian, L.M. (2002) Matrix metalloproteinases: a tail of a frog that became a prince. Nature Rev. Mol. Cell. Biol. 3, 207-214. Buttle, DJ., Handley, C.J., Ilic, M.Z., Saklatvala, J., Murata, M. & Barrett, AJ. (1993) Inhibition of cartilage proteoglycan release by a specific inactivator of cathepsin B and an inhibitor of matrix metalloproteinases: evidence for two converging pathways of chondrocyte-mediated proteoglycan degradation. Arthritis Rheum. 36, 1709-1717. Catterall, J.B., Carrere, S., Koshy, P.J.T., Degnan, BA., Shingleton, W.D., Brinckerhoff, C.E., Rutter, J., Cawston, T.E. & Rowan, A.D. (2001) Synergistic induction of matrix metalloproteinase 1 by interleukin-Àá and oncostatin M in human chondrocytes involves signal transducer and activator of transcription

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protein 1 transcription factors via a novel mechanism. Arthritis Rheum. 44, 2296-2310. Cawston, T.E. (1996) Metalloproteinase inhibitors and the prevention of connective tissue breakdown. Pharmacol. Ther. 70, 163-182. Cawston, T.E. & Barrett, AJ. (1979) A rapid and reproducible assay for collagenase using [l-14C]acetylated collagen. Anal. Biochem. 99, 340-345. Cawston, T.E. & Mercer, E. (1986) Preferential binding of collagenase to a2-macroglobulin in the presence of the tissue inhibitor of metalloproteinases. FEBS Lett. 209, 9-12. Cawston, T.E. & Murphy, G. (1981) Mammalian collagenases. Methods Enzymol. 80, 711-722. Cawston, T.E. & Tyler, JA. (1979) Purification of pig synovial collagenase to high specific activity. Biochem. J. 183, 647-656. Cawston, T.E., Murphy, G., Mercer, E., Galloway, W.A., Hazleman, B.L. & Reynolds, JJ. (1983) The interaction of purified rabbit bone collagenase with purified rabbit bone metalloproteinase inhibitor. Biochem. J. 211, 313-318. Chung, L., Shimokawa, K., Dinakarpandian, D., Grams, F., Fields, G.B., Nagase, H.(2000) Identification of 183RWTNNFREY191 region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity. J. Biol. Chem. 275, 29610-29617. Clark, I.M. & Cawston, T.E. (1989) Fragments of human fibroblast collagenase: purification and characterization. Biochem. J. 263, 201-206. Clark, I.M., Wright, J.K., Cawston, T.E. & Hazleman, B.L. (1992) Polyclonal antibodies against human fibroblast collagenase and the design of an enzyme-linked immunosorbent assay to measure TIMP-collagenase complex. Matrix 12, 108-115. Clark, I.M., Mitchell, R.E., Powell, L.K. & Bigg, H.F. (1995) Recombinant porcine collagenase: purification and autolysis. Arch. Biochem. Biophys. 316, 123-127. Cooksley, S., Hipkiss, J.B., Tickle, S.P., Holmes-Leavers, E., Docherty, A.J.P., Murphy, G. & Lawson, A.D.G. (1990) Immunoassays for the detection of human collagenase, stromelysin, tissue inhibitor of metalloproteinases (¹ÌÑ) and enzyme-inhibitor complexes. Matrix 10, 285-291. Cunnane, G., FitzGerald, O., Hummel, K.M., Youssef, P.P., Gay, R.E. Gay, S. & Bresnihan, B. (2001) Synovial tissue protease gene expression and joint erosions in early rheumatoid arthritis. Arthritis Rheum. 44, 1744-1753. DeClerck, Y.A., Yean, T.-D., Lu, H.S., Ting, J. & Langley, K.E. (1991) Inhibition of autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MI/TIMP-2. J. Biol. Chem. 266, 3893-3899. Delaisse, J.M., Eeckhout, Y. & Vaes, G. (1988) Bone resorbing agents affect the production and distribution of procollagenase as well as the activity of collagenase in bone tissue. Endocrinology 123, 264-276. Desrochers, P.E., Jeffrey, JJ. & Weiss, SJ. (1991) MMP-1 (matrix metalloproteinase-1) expresses serpinase activity. J. Clin. Invest. 87, 2258-2265. Dioszegi, M., Cannon, P. & Van Wart, H.E. (1995) Vertebrate collagenases. Methods Enzymol. 248, 413-431. Egeblad, M. & Werb, Z. (2002) New functions for the matrix metalloproteinases in cancer progression. Nature Rev. Cancer 2, 161-174. Fields, G.B. (1991) A model for interstitial collagen catabolism by mammalian collagenases. J. Theor. Biol. 153, 585-602.

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Krane, S.M., Byrne, M.H., Lemaitre, V., Henriet, P., Jeffrey, J.J., Witter, J.P., Liu, X., Wu, H., Jaenisch, R. & Eeckhout, Y. (1996) Different collagenase gene products have different roles in degradation of type 1 collagen. /. Biol. Chem. 45, 28509-28515. Lark, M.W., Walakovits, L.A., Shah, T.K., Vanmiddlesworth, J., Cameron, P.M. & Lin, T.-Y. (1990) Production and purification of prostromelysin and procollagenase from IL-1 beta-stimulated human gingival fibroblasts. Connect. 7w.s. Res. 25, 49-65. Lauer-Fields, J.L., Tuzinski, K.A., Shimokawa, K., Nagase, H. & Fields, G.B. (2000) Hydrolysis of triple-helical collagen peptide models by matrix metalloproteinases. /. Biol. Chem. 275, 13282-13290. Li, J., Brick, P., O'Hare, M.C., Skarzynski, T., Lloyd, L.F., Curry, V.A., Clark, I.M., Bigg, H.F., Hazleman, B.L., Cawston, T.E. & Blow, D.M. (1995) Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calciumlinked, four-bladed ß-propeller. Structure 3, 541-549. Li, S.H., Chow, L.H. & Pickering, J.G. (2000) Cell surface-bound collagenase-1 and focal substrate degradation stimulate the rear release of motile vascular smooth muscle cells. J. Biol. Chem. 275, 35384-35392. Lovejoy, B., Cleasby, A., Hassell, A.M., Longley, K., Luther, M.A., Weigle, D., McGeehan, G., McElroy, A.B., Drewry, D., Lambert, M.H. & Jordan, S.R. (1994) Structure of the catalytic domain of fibroblast collagenase complexed with an inhibitor. Science 263, 375-377. Lovejoy, B., Welch, A.R., Carr, S., Luong, C , Broka, C , Hendriks, R.T., Campbell, J.A., Walker, K.A.M., Martin, R., Van Wart, H. & Browner, M.F. (1999) Crystal structures of MMP1 and -13 reveal the structural basis for selectivity of collagenase inhibitors. Nature Struct. Biol. 6, 217-221. Lowry, C.L., McGeehan, G. & LeVine, H. (1992) Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase. Proteins: Struct. Funct. Genet. 12, 42-48. Milner, J.M., Elliott, S.-F., Cawston, T.E. (2001) Activation of procollagenases is a key control point in cartilage collagen degradation. Arthritis Rheum. 44, 2084-2096. Miltenburg, A.M.M., Lacraz, S., Welgus, H.G. & Dayer, J.-M. (1995) Immobilized anti-CD3 antibody activates T cell clones to induce the production of MMP-1, but not tissue inhibitor of metalloproteinases, in monocytic THP-1 cells and dermal fibroblasts. J. Immunol. 154, 2655-2667. Mitchell, T.I., Jeffrey, J.J., Palmiter, R.D. & Brinckerhoff, C.E. (1993) The acute phase reactant serum amyloid A (SAA3) is a novel substrate for degradation by the metalloproteinases collagenase and stromelysin. Biochim. Biophys. Acta 1156, 245-254. Moore, W.M. & Spilburg, C.A. (1986) Purification of fibroblast collagenase with a peptide-hydroxamic acid affinity column. Biochemistry 25, 5189-5195. Mortensen, S.B., Sottrup-Jensen, L., Hansen, H.F., Petersen, T.E. & Magnusson, S. (1981) Primary and secondary cleavage sites in the bait region of a2-macroglobulin. FEBS Lett. 135, 295-300. Murphy, G., Cockett, M.I., Stephens, P.E., Smith, B.J. & Docherty, A.J.P. (1987) Stromelysin is an activator of procollagenase: a study with natural and recombinant enzymes. Biochem. J. 248, 265-268. Nagase, H. & Woessner, J.F., Jr. (1999) Matrix metalloproteinases. J. Biol Chem. 274, 21491-21494.

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480 CLAN MA(M) - M10A 126. Neutrophil collagenase Welgus, H.G., Jeffrey, J.J., Stricklin, G.P., Roswit, W.T. & Eisen, A.Z. (1980) Characteristics of the action of human skin fibroblast collagenase on fibrillar collagen. J. BioL Chem. 255, 68066813. Welgus, H.G., Jeffrey, J.J., Eisen, A.Z., Roswit, W.T. & Stricklin, G.P. (1985) Human skinfibroblastcollagenase: interaction with substrate and inhibitor. Collagen Relat. Res. 5, 167-179. Willenbrock, F., Murphy, G., Phillips, I.R. & Brocklehurst, K. (1995) The second zinc atom in the matrix metalloproteinase catalytic domain is absent in the full-length enzymes: a possible role for the C-terminal domain. FEBS Lett. 358, 189192. Windsor, L.J., Birkedal-Hansen, H., Birkedal-Hansen, B. & Engler, J.A. (1991) An internal cysteine plays a role in the maintenance

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Tim E. Cawston Rheumatology, School of Clinical Medical Sciences, University of Newcastle, The Medical School, Newcastle Upon Tyne NE2 4HH, UK Email: T.E. Cawston @ newcastle. ac. uk Handbook of Proteolytic Enzymes 2nd Edn Copyright © 2004 Elsevier Ltd ISBN 0-12-079610-4 All rights of reproduction in any form reserved

126. Neutrophil collagenase Databanks MEROPS name: Collagenase 2 MEROPS classification: clan MA(M), family M10A, peptidase M10.002 IUBMB: EC 3.4.24.34 CAS registry: 9001-12-1 Species distribution: Mammalia Sequence known from: Cavia porcellus, Homo sapiens, Mesocricetus auratus, Mus musculus, Rattus norvegicus Tertiary structure: Available

Name and History The name collagenase denotes an enzyme capable of cleaving triple-helical collagen. A collagenase isolated from human polymorphonuclear leukocytes (PMNLs) was first described by Lazarus et al. (1968). Later it was shown to be localized to the specific granules (Murphy et al., 1977). Because of its origin the enzyme was given the name PMNL-collagenase or neutrophil collagenase. As a member of the matrixin family the neutrophil collagenase is also referred to as matrix metalloproteinase 8 (MMP-8) (Nagase et al., 1992). In 1990 the cDNA encoding human neutrophil collagenase was cloned

and sequenced using a gtll cDNA library constructed from mRNA extracted from the peripheral leukocytes of a patient with chronic granulocytic leukemia (Hasty et al, 1990). Human neutrophil collagenase was thought to be expressed exclusively by neutrophil leukocytes, but the enzyme and its mRNA were recently found also in normal human articular chondrocytes (Cole et al., 1996), in mononuclear fibroblastlike cells and human endothelial cells (Hanemaaijer et al, 1997), in odontoblasts (Palosaari et al., 2000), in a T cell line (Kim et al., 2001), and also in plasma cells (Wahlgren et al., 2001), and bronchial epithelial cells (Prikk et al, 2001). Following the discovery of a third form of collagenase in humans (MMP-13; Chapter 127), the neutrophil collagenase is frequently referred to as collagenase 2.

Activity and Specificity Neutrophil collagenase is stored intracellularly as a latent proenzyme in the specific granules of polymorphonuclear leukocytes (Murphy et al, 1977). Activation of the procollagenase occurs in the extracellular space after secretion. The active enzyme is capable of cleaving types I, II and III triple-helical collagen into the characteristic one-quarter and three-quarter fragments by hydrolyzing the Gly7754-Leu776