Macrophage Metalloelastase Degrades Matrix and Myelin Proteins and Processes a Tumour Necrosis Factor-α Fusion Protein

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 228, 421–429 (1996)ARTICLE NO. 1677

Macrophage Metalloelastase Degrades Matrix and Myelin Proteins andProcesses a Tumour Necrosis Factor-a Fusion Protein

Stephen Chandler,1 Judy Cossins, Jon Lury, and Graham Wells

British Biotech Pharmaceuticals Ltd., Watlington Road, Cowley, Oxford, OX4 5LY, UK

Received October 7, 1996

The matrix metalloproteinases (MMPs) are a group of enzymes which have the ability to degradeextracellular matrix. They also cleave non-matrix proteins such as myelin basic protein and a1-antitrypsinand they are able to process tumour necrosis factor-a (TNF) to its mature form. We have cloned, expressedand purified human macrophage metalloelastase (EC 3.4.24.65), an MMP recognised for its ability todegrade elastin, but whose substrate specificity has not yet been defined. With the exception of type Icollagen this enzyme degraded all matrix proteins tested, namely: type IV collagen, type I gelatin, fibronectin,laminin, vitronectin and proteoglycan. It also degraded myelin basic protein, cleaved a1-antitrypsin andreleased TNF from a pro-TNF fusion protein. Thus, in common with several other MMPs, macrophagemetalloelastase has a broad substrate range which extends beyond that of elastin alone. q 1996 Academic

Press, Inc.

The matrix metalloproteinases (MMPs) are a family of Zn2/-dependent neutral endoprotei-nases which includes both soluble and membrane-bound types (1, 2). Collectively these en-zymes can degrade all components of extracellular matrix (1, 3, 4) and they are involved inboth normal tissue turnover (5) and pathological matrix remodelling (6-8). During inflammationMMPs are responsible for the matrix degradation required for leukocyte extravasation (9) andthey have been shown to process the inflammatory cytokine, tumour necrosis factor-a (TNF),to its mature form (10). There is also evidence that MMPs are involved in the demyelinatingneuroinflammatory disease multiple sclerosis (11, 12) and all the MMPs tested so far candegrade myelin basic protein (MBP), the major extrinsic membrane protein of central nervoussystem (CNS) myelin (13, 14).

Ten soluble MMPs are known (1, 3): interstitial collagenase, neutrophil collagenase andcollagenase-3 which cleave fibrillar collagens; 72kDa and 92kDa gelatinase which degradetype IV collagen and gelatin; stromelysins-1,-2 and matrilysin which have a broad substratespecificity; and stromelysin-3 and macrophage metalloelastase (MME) whose specificities arenot yet fully defined. MME (EC 3.4.24.65) was only recently identified as an MMP withelastolytic activity (15, 16). The mouse enzyme also cleaves a1-antitrypsin (a1-AT) (17) andhas been stated to degrade other proteins (15, 18), but no data are available; only elastin hasbeen identified as a substrate for human MME. Here we describe the purification of recombinanthuman MME and show that it has a broad substrate specificity against matrix proteins, MBPand a pro-TNF fusion protein. A preliminary report of this work has appeared elsewhere (19).

1 Corresponding author. Fax: /44 1865 781115. E-mail: [email protected]: MMP, matrix metalloproteinase; TNF, tumour necrosis factor-a; MBP, myelin basic protein; CNS,

central nervous system; MME, macrophage metalloelastase; a1-AT, a1-antitrypsin; EHS, Engelbreth-Holm-Swarmtumour; PG, proteoglycan; McaPLGLDpaAR, (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4dinitrophe-nyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH2; McaPL, (7-methoxycoumarin-4-yl)acetyl-Pro-Leu; CHO, Chinese ham-ster ovary; APMA, 4-aminophenylmercuric acetate; GST-TNF, glutathione S-transferase pro-TNF fusion protein.

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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MATERIALS AND METHODSMaterials. Porcine type I skin collagen was obtained from Pentapharm Ltd. (Basel, Switzerland) and mouse

Engelbreth-Holm-Swarm tumour (EHS) type IV collagen from Collaborative Biomedical Products (Bedford, MA,USA). Bovine CNS MBP was from Upstate Biotechnology Inc. (Lake Placid, NY, USA), bovine plasma vitronectinfrom Life Technologies Ltd. (Paisley, Scotland) and proteoglycan (PG) monomer and polysaccharide lyases fromICN Biomedicals Ltd. (Thame, Oxon., UK). Elastin-fluorescein (400 mesh) and k-elastin (ETNA-elastin) were fromElastin Products Co., Inc. (Owensville, MO, USA) and human neutrophil elastase, leupeptin, pepstatin, a1-AT, elastasepeptide substrates, (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH2 (McaPLGLDpaAR) and (7-methoxycoumarin-4-yl)acetyl-Pro-Leu (McaPL) were from Calbiochem-Novabiochem (UK) Ltd. (Nottingham, UK). Except where stated chromatography resins were from Pharmacia BiotechLtd. (St. Albans, Herts., UK) and other reagents from Sigma (Poole, Dorset, UK).

Enzymes. Human recombinant interstitial collagenase (EC 3.4.24.7), 72kDa gelatinase (EC 3.4.24.24), matrilysin(EC 3.4.24.23) and 92kDa gelatinase (EC 3.4.24.35) were purified as described by Chandler et al (14). The humanMME sequence was amplified by PCR using as target a cDNA from phorbol 12-myristate 13-acetate-stimulated U937cells and cloned into pGEM-T (Promega Ltd., Southampton, UK). The sequence contained 4 silent base changes: 12T(ACT r ACC), 34E (GAG r GAA), 234A (GCT r GCC) and 434Y (TAT r TAC) (16). It was sub-cloned into pGW1-H and a xanthine-guanine phosphoribosyltransferase gene inserted on a Bam HI fragment (20). Expression in Chinesehamster ovary (CHO) cells was as previously described (14). To purify MME, the medium was adjusted to pH7.7with NaOH solution, passed through soyabean trypsin inhibitor agarose (Sigma) and gelatin Sepharose and ontoheparin Sepharose; all three columns were equilibrated with 50mM Tris-HCl pH7.4 / 5mM CaCl2 / 0.05% Brij 35 /0.02% NaN3 (buffer A). The heparin column was washed with buffer A and MME eluted with a linear NaCl gradient(0-1M) in buffer A. Enzyme-containing fractions were pooled, diluted to 0.15M salt and adjusted to pH8.0 withNaOH solution. The pool was loaded onto chelating Sepharose which had been charged with 0.3M ZnCl2 andequilibrated with 50mM Tris-HCl pH8.0 / 0.15M NaCl / 5mM CaCl2 / 0.05% Brij 35 / 0.02% NaN3 (buffer B). Thecolumn was washed with buffer B, buffer A pH6.5 and buffer A pH6.5 / 0.8M NaCl. MME was eluted with 50mMacetate buffer (Na) pH4.7 / 0.8M NaCl / 5mM CaCl2 / 0.05% Brij 35 / 0.02% NaN3 and desalted into buffer A onSephadex G-25. The desalted eluate was loaded onto buffer A-equilibrated Q and SP Sepharose columns mounted intandem and the enzyme eluted from the SP column as from heparin Sepharose (above). Enzyme-containing fractionswere pooled and desalted into buffer A on Sephadex G-25. MME concentration was estimated spectrophotometrically(e280 Å 75,140M01cm01; Genetics Computer Group, Madison, WI, USA).

Enzyme activation. Interstitial collagenase, 72kDa gelatinase and matrilysin were activated by incubation with 1mM4-aminophenylmercuric acetate (APMA) for 1h at 377C; 92kDa gelatinase was activated likewise but for 48h. MMEwas not activated (see below).

Enzyme assays. Fluorometric assays were done in a Fluostar microplate fluorometer (SLT Labinstruments GmbH,Grodig, Austria). Continuous assays with McaPLGLDpaAR (6mM) were done at 377C in 0.1M Tris-HCl pH7.5 /0.1M NaCl / 10mM CaCl2 / 0.05% Brij 35 / 0.02% NaN3 (assay buffer) using 320nm excitation / 405nm emission(21). The instrument was calibrated using McaPL and the conversion of substrate never exceeded 10%. Elastaseassays (22) were done for 18h at 377C in assay buffer (0.2ml) containing 125mg insoluble elastin-fluorescein. Reactionswere stopped with 0.8ml 0.1M Tris-HCl pH7.5 / 25mM EDTA, centrifuged and the fluorescence measured using485nm excitation / 538nm emission.

Preparation of PG monomer. Lyophilised PG monomer from rat chondrosarcoma was dissolved in 4M guanidine-HCl / 0.1M Tris-HCl pH7.5 / 0.25mM PMSF / 10mM E-64 (Boehringer Mannheim UK Ltd., Lewes, UK) / 20mMleupeptin / 1mM pepstatin and then dialysed into assay buffer also containing inhibitors. These were necessary to stopendogenous proteolytic activity in the PG preparation.

Substrate degradation and SDS-PAGE. Protein substrates were incubated with enzyme in 50ml of assay buffer (seeabove). The MMP inhibitor BB-2116 (10) was prepared at 10mM in dimethyl sulphoxide and used at 10mM; dimethylsulphoxide alone (0.1%) had no effect on enzyme activity. Reactions were stopped with 51 loading buffer to give1% SDS / 0.5% 2-mercaptoethanol and heated in boiling water for 3min. SDS-PAGE (23) was done using pre-cast4-20 or 16% gels (Novex, San Diego, CA, USA) followed by staining with Colloidal Coomassie G-250 (Novex).

Zymography. k-elastin zymography (24) was done using a 10% resolving gel containing 0.1% k-elastin and a 4%stacking gel; both contained 0.1% SDS. Sample was prepared in loading buffer containing 1% SDS without mercapto-ethanol or heating. After electrophoresis at ambient temperature the gel was washed in 2.5% Triton X-100 to removeSDS and incubated overnight at 377C in assay buffer (see above) to allow proteolysis. The gel was stained withCoomassie Brilliant Blue G-250.

Protein sequencing. N-terminal sequence analysis was done by M-Scan Ltd. (Ascot, Berks., UK).

RESULTS AND DISCUSSION

Purification and characterization of MME. Here we describe the first purification of recombi-nant human MME expressed by mammalian cells. The enzyme was purified by sequential

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FIG. 1. (A) SDS-PAGE analysis of human MME. MME (0.6mg) was electrophoresed under reducing conditionsand stained as described in Materials and Methods. 1, molecular mass standards; 2, MME. (B) Zymogram analysisof MME. MME (0.4mg) was analysed by k-elastin zymography under non-reducing conditions (Materials and Methods).The positions of the molecular mass standards are shown. (C) Cleavage of a1-AT by human MME and matrilysin.Enzymes were incubated (75min/377C) with a1-AT (0.2mg/ml) and the samples were analysed as described in Materialsand Methods. 1, molecular mass standards; 2, MME (5mg/ml) plus BB-2116; 3, MME (5mg/ml); 4, MME (0.5mg/ml); 5, no enzyme; 6, matrilysin (0.5mg/ml).

heparin, chelating and ion-exchange chromatography as described in Materials and Methods.Contaminating CHO cell 92kDa gelatinase in the starting material was first depleted on gelatinSepharose and finally removed on Q Sepharose in the final stage of the purification. Theproduct contained 3 species with molecular masses of 45, 28 and 20kDa, of which the 45 and20kDa forms were active on k-elastin (Figure 1A, B). N-terminal sequence analysis of the28kDa species gave QRLPN, residues 269-272 of the human MME sequence at the junctionof the catalytic and haemopexin domains (16). The mixture was therefore concluded to containthe 45kDa activated enzyme (lacking propeptide) and two fragments generated by a furtherprocessing step between the catalytic and haemopexin domains. The tendency of MME toundergo autolytic processing to a low molecular weight catalytic fragment has been observedwith both the human and mouse enzymes expressed in E. coli (15, 16) and probably reflectsevents which occur in vivo (18). It is not known if cleavage at N268-Q269 is the primary eventor the result of subsequent processing.

We tested the activity of human MME on insoluble elastin, a range of chromogenic elastasesubstrates and the fluorogenic MMP substrate, McaPLGLDpaAR (21). Activity on elastin waspoor when compared with human neutrophil elastase (Table 1) although it was comparablewith the activity of the mouse MME purified by Banda and Werb (25) which gave a value,in our units, of 34 rising to 104 for a homogenous preparation of the 22kDa species alone.The ability of MME to degrade elastin is not unique among the MMPs; the gelatinases,stromelysins 01 and 02 and matrilysin all degrade this substrate (26, 27). No release ofnitroaniline was detectable with any of the following p-nitroanilide (pNA) substrates, all ofwhich were cleaved by neutrophil elastase: MeO-Suc-A2PV-pNA, Boc-A2PA-pNA, Suc-A2P-

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TABLE 1Specific Activity of MME with Insoluble Fluorescein-Elastin

and the Fluorogenic Peptide McaPLGLDpaAR

Insoluble elastin McaPLGLDpaAREnzyme (mg/18 h/mg enzyme) (mmol/min/mg enzyme)

MME (as purified) 25 0.80MME (APMA/1 h/377C) — 0.43Human neutrophil elastase 630 —

Neutrophil elastase was dissolved in 50 mM acetate (Na) pH 5.5/0.2 M NaCl for use.Assays were done at 377C as described in Materials and Methods.

2-aminobutyryl-pNA, pyroglutamyl-PV-pNA, Suc-A3-pNA or Suc-A2V-pNA. However, MMEshowed good activity with the peptide McaPLGLDpaAR (Table 1), a substrate known to becleaved by other MMPs (21, 28). APMA-treatment decreased the activity, consistent with thefact that the enzyme was already activated (see above). We also tested human MME with a1-AT, a protein known to be cleaved by the mouse enzyme (17). As shown in Figure 1(c) ourenzyme cleaved this substrate as effectively as matrilysin, the most active MMP so far describedon a1-AT (29).

Degradation of collagen type I, collagen type IV and gelatin. Activity on type I collagenwas tested at 267C to avoid non-specific collagenolysis (30). Human interstitial collagenasecleaved collagen to characteristic 1

4- and 34-length fragments whereas MME was inactive (Figure

2A). With the exception of the collagenases (1, 3) only 72kDa gelatinase degrades this substrate

FIG. 2. (A) Cleavage of type I collagen by human interstitial collagenase and MME. Enzymes were incubated(20h/267C) with type I collagen (0.2mg/ml) and the samples analysed by SDS-PAGE (Materials and Methods). 1,molecular mass standards; 2, no enzyme; 3, interstitial collagenase (5mg/ml); 4, MME (5mg/ml). (B) Degradation oftype I gelatin by human MME, 72kDa and 92kDa gelatinase. Enzymes were incubated (20h/377C) with heat-denatured(10min/567C) type I collagen (0.2mg/ml) and the samples were analysed by SDS-PAGE. 5, MME (5mg/ml) plus BB-2116; 6, MME (0.5mg/ml); 7, MME (50ng/ml); 8, 72kDa gelatinase (5ng/ml); 9, 92kDa gelatinase (5ng/ml); 10,molecular mass standards.

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FIG. 3. Degradation of type IV collagen by human MME. MME was incubated (20h) with EHS type IV collagen(0.2mg/ml) at 267C (lanes 2 & 3), 307C (lanes 4 & 5) or 377C (lanes 6 - 9) and the samples were analysed by SDS-PAGE (Materials and Methods). 1, molecular mass standards; 2, 4 and 6, no MME; 3 and 5, MME (5mg/ml); 7,MME (0.5mg/ml); 8, MME (50ng/ml); 9, MME (5mg/ml) plus BB-2116.

(31). At 377C, heat-denatured type I collagen (gelatin) was degraded by MME although theactivity was at least 100-fold lower than that observed with recombinant human gelatinases(Figure 2B). In contrast to type I, activity against type IV collagen was detectable at 267C,albeit at a low level (Figure 3). The activity increased at 307C and further still at 377C, possiblyas a result of thermal denaturation of the substrate (32). At 377C, all activity was abolishedby the MMP inhibitor BB-2116. Conflicting results have been obtained for the degradation ofEHS type IV collagen by MMPs. Murphy et al (26) described its cleavage by the two gelatinasesat 297C whereas Mackay et al (32) were unable to obtain degradation with either enzyme at25 or 307C. It is possible that these discrepancies reflect differences between batches of typeIV collagen. We were unable to obtain degradation at 267C with either of the recombinanthuman gelatinases at 5mg/ml (results not shown). This result suggests that MME has type IVcollagenase activity which exceeds that of the gelatinases.

Degradation of structural glycoproteins. We tested MME against 3 members of this classof matrix proteins. The results in Figure 4 show that MME was able to degrade fibronectin,laminin and vitronectin, vitronectin being the most susceptible substrate. All activities weresensitive to BB-2116. The combined ability of MME to degrade the basement membraneproteins laminin and type IV collagen (above) is consistent with the recent finding that perito-neal macrophages from MME-deficient mice have an impaired ability to penetrate such mem-branes (18).

Degradation of proteoglycan. Figure 5 shows the results of an experiment in which MMEwas used to digest PG monomer. Samples were analysed both before and after polysaccharidelyase treatment. In untreated samples PG monomer did not enter the gel (lanes 7 and 10,Figure 5). After digestion with MME a fragment with a molecular mass of approximately55kDa was generated (lanes 8 and 9) and at 5mg/ml enzyme an additional product of molecularmass approximately 40kDa also appeared (lane 8). Removal of the glycosaminoglycans withpolysaccharide lyases allowed the undigested PG to enter the gel and decreased slightly themolecular mass of both product species (lanes 3 and 4), although the 40kDa species wasobscured by the polysaccharide lyases. This indicated that neither product carried significantamounts of glycosaminoglycan. At least 5 MMP cleavage sites have been identified betweenthe G1 and G2 domains of the PG core protein aggrecan, depending on the enzyme anddigestion conditions (33). Cleavage at the major MMP site by interstitial collagenase, 72kDa

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FIG. 4. Degradation of glycoproteins by human MME. Fibronectin (lanes 1-3), laminin (lanes 4-6) and vitronectin(lanes 7-9) at 0.2mg/ml were incubated (20h/377C) with MME and the samples were analysed by SDS-PAGE (Materialsand Methods). 1, 4 and 7, MME (5mg/ml) plus BB-2116; 2, 5 and 8, MME (5mg/ml); 3,6 and 9, no MME; 10,molecular mass standards.

gelatinase, stromelysin-1 or -2, matrilysin, neutrophil collagenase, 92kDa gelatinase or colla-genase-3 releases the G1 domain as a 56kDa fragment which carries little glycosaminoglycan.Such cleavage by MME would account for the 55kDa species but not the 40kDa species. Thismay have arisen because of the denaturing conditions under which our PG was prepared sincethis has been found to increase the number of products generated in such digests (34). Therewas no degradation in the presence of BB-2116 indicating that proteolysis could not be ascribedto proteases in the PG sample (see Materials and Methods).

Degradation of MBP. We tested MME for its ability to degrade MBP and compared theactivity with that of 72kDa gelatinase, the most active MMP so far tested on this substrate(14). As shown in Figure 6, MME had the same activity, on a weight for weight basis, as72kDa gelatinase. Both enzymes were inhibited by BB-2116 as expected. Nothing is knownabout the expression of MME in the CNS but this tissue is now known to express a numberof other MMPs (35). In the demyelinating neuroinflammatory disease multiple sclerosis, whichis characterised by macrophage involvement (12), it is possible that MME contributes to myelindestruction.

Cleavage of pro-TNF. The ability to process pro-TNF is a feature common to several MMPs(10). MME was able to cleave a glutathione S-transferase pro-TNF fusion protein (GST-TNF)to mature TNF (Figure 7). N-terminal sequence analysis of the 16kDa species showed thatapproximately two thirds of the material had an N-terminus of VRSSS, characteristic of solubleTNF (36), whereas the remainder had an N-terminus two residues upstream of this (QAVR).Although the enzyme responsible for the release of TNF from the cell surface is not an MMP(37) this result indicates that MME has the potential to contribute to TNF-driven inflammationunder appropriate conditions.

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FIG. 5. Cleavage of PG monomer from rat chondrosarcoma by human MME. MME was incubated (20h/377C)with PG (2.4mg/ml) in the presence of protease inhibitors. Samples were either stopped (lanes 7-10) or treated with0.1U chondroitinase ABC and 0.2U keratanase for a further 5h/377C (lanes 1-5). All were then analysed by SDS-PAGE (Materials and Methods). 1, polysaccharide lyases alone; 2 and 7, MME (5mg/ml) plus BB-2116; 3 and 8,MME (5mg/ml); 4 and 9, MME (0.5mg/ml); 5 and 10, no MME; 6, molecular mass standards. The two product speciesare arrowed (see text).

FIG. 6. Degradation of MBP by human MME and 72kDa gelatinase. Enzymes were incubated (20h/377C) withMBP (0.2mg/ml) and the samples were analysed by SDS-PAGE (Materials and Methods). 1 and 10, no enzyme; 2,MME (5mg/ml) plus 10mM BB-2116; 3, MME (5mg/ml); 4, MME (0.5mg/ml); 5, MME (50ng/ml); 6, molecular massstandards; 7, 72kDa gelatinase (0.5mg/ml) plus BB-2116; 8, 72kDa gelatinase (0.5mg/ml); 9, 72kDa gelatinase (50ng/ml).

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FIG. 7. Cleavage of GST-TNF by human MME. MME was incubated (2h/377C) with GST-TNF (0.2mg/ml) (10)and the samples were analysed by SDS-PAGE (Materials and Methods). 1, MME (5mg/ml) plus BB-2116; 2, MME(5mg/ml); 3, MME (0.5mg/ml); 4, no MME; 5, molecular mass standards.

We have shown that MME degrades a range of matrix and non-matrix substrates. Of themacromolecular substrates tested, only type I collagen was not cleaved. Although named forits ability to degrade elastin, the enzyme exhibits characteristics common to other MMPs.Thus it cleaves a1-AT, PG core protein, GST-TNF and the peptide McaPLGLDpaAR. It doesnot act on a range of arylamide elastase substrates and its activity on insoluble elastin issignificantly lower than that of neutrophil elastase. MME should therefore be considered asan MMP with a broad substrate specificity, which, like a number of other MMPs, has theability to degrade elastin.

ACKNOWLEDGMENTSWe wish to thank Mr. G. Catlin for cloning and sequencing the MME cDNA, Mr. P. Nayee for providing the GST-

TNF and the British Biotech MMP programme for their contribution to this work.

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Macrophage Metalloelastase Degrades Matrix and Myelin Proteins and Processes a Tumour Necrosis Factor-α Fusion Protein