Eur. J. Biochem. 267, 3323±3329 (2000) q FEBS 2000

Cloning of MMP-26A novel matrilysin-like proteinase

Ame lie Benoit de Coignac1, Greg Elson1, Yves Delneste1, Giovanni Magistrelli1, Pascale Jeannin1,Jean-Pierre Aubry1, Odile Berthier2, Daniel Schmitt2, Jean-Yves Bonnefoy1 and Jean-FrancËois Gauchat1

1Centre d'immunologie Pierre Fabre, St Julien-en-Genevois, France; 2INSERM U 346, HoÃpital Edouard Herriot, Lyon, France

A cDNA encoding a novel human matrix metalloproteinase (MMP), named MMP-26, was cloned from fetal

cDNA. The deduced 261-amino-acid sequence is homologous to macrophage metalloelastase (51.8% identity). It

includes only the minimal characteristic features of the MMP family: a signal peptide, a prodomain and a

catalytic domain. As with MMP-7, this new MMP does not comprise the hemopexin domain, which is believed to

be involved in substrate recognition. A study of MMP-26 mRNA steady states levels reveals, among the tissue

examined, a specific expression in placenta. MMP-26 mRNA could also be detected in several human cell lines

such as HEK 293 kidney cells and HFB1 lymphoma cells. Recombinant MMP-26 was produced in mammalian

cells and used to demonstrate a proteolytic activity of the enzyme on gelatin and b-casein.

Keywords: matrix metalloproteinase; cDNA; matrixin; cancer; metastasis.

The matrix metalloproteinases (MMPs) are zinc dependentendopeptidases that can digest various components of theextracellular matrix. They play a major role in tissueremodeling during morphogenesis, wound healing, angio-genesis and apoptosis [1] and have been implicated in variouspathologies involving degradation processes such as rheuma-toid arthritis, tumor invasion, tumor metastasis, and cardiacrupture following infarction [2±4]. Whereas they exhibitdifferent substrate specificity, members of this family sharecommon properties such as the requirement of zinc and calciumions for catalytic activity. They are related by their amino-acidsequence and secondary structure and are subdivided into fourdifferent families: collagenases, gelatinases, stromelysins andmembrane-type MMPs (MT-MMPs) although some memberssuch as macrophage metalloelastase (MMP-12) [5], matrilysin(MMP-7) [6±8], MMP-19 [9] and enamalysin (MMP-20) [10]cannot be classified within these subgroups. They are translatedas inactive zymogen forms requiring the cleavage of aprodomain for activation [11]. The prodomain, locatedimmediately after the signal peptide, comprises a free cysteinewhich can interact with the Zn1 ion of the catalytic domainto maintain the proteinase in an inactive state. Among theMMPs which are activated intracellularly, the prodomain isseparated from the catalytic domain by a furin-cleavage site[12]. The catalytic domain contains a highly conservedHEXGHXXGXXHS/T zinc binding sequence as well asseveral calcium binding sites. This catalytic domain isseparated from the C-terminal hemopexin-like domain by a

proline rich linker region commonly referred to as the hingeregion. The hemopexin-like domain contains four repeatshomologous to hemopexin and vitronectin. Although notessential for enzyme activity, this domain does seem to befundamental for substrate specificity (reviewed in [13]). Inaddition to the domains described above, some MMPs displayadditional features. The gelatinase subclass MMPs (MMP-2and -9) contain three fibronectin type II domain repeats insertedin the catalytic domain which increase the affinity for gelatinsubstrates. In the membrane-type MMP subclass, the hemo-pexin domain is followed by a membrane spanning hydro-phobic region [14,15]. In contrast to the other MMPs, MMP-7(matrilysin), the smallest MMP identified to date, lacks thehemopexin-like domain [16] as does the recently clonedMMP-23 [17].

We describe here the cloning of a novel MMP lacking thehemopexin domain. This MMP has tentatively been calledMMP-26. Sequence analysis indicates that the encoded proteincontains the same domains as MMP-7 and is closely related toMMP-12 (macrophage metalloelastase, 51.8% amino acididentity). The expression pattern of MMP-26 mRNA wasanalyzed in normal human tissues and in various human celllines. A recombinant form of the proteinase was also expressedin mammalian cells and used for a preliminary enzymaticcharacterization.

M A T E R I A L S A N D M E T H O D S

Source of cells and culture conditions

The fibroblastic cells lines HEK 293, COS-1, the B-cell linesRPMI 8226 and RPMI 8866, HT29, Raji, THP-I, the epi-dermoid larynx carcinoma cell line HEP-2, the breastcarcinoma cell line MCF-7 and the primary colonic tumorcell line Caco-2 were obtained from the American Type CultureCollection (Manassas, VA, USA) and cultured according totheir specifications. The mast cell line HMC-1 was a kind giftfrom J. Butterfield (Mayo Clinic, Rochester, MN, USA) andwas maintained in Dulbecco's modified Eagle's medium and

Correspondence to J.-F. Gauchat, Centre d'immunologie Pierre Fabre,

5 Avenue Napoleon III, BP 97, 74164 Saint Julien-en-Genevois, France.

Fax: 1 33 450 35 35 90, Tel.: 1 33 450 35 35 83,

E-mail: jean.francois.gauchat@pierre-fabre.com

Abbreviations: AP, anchored primer; MMPs, matrix metalloproteases;

MT-MMP, membrane-type MMP; EST, expressed sequence tag; RT-PCR,

reverse transcription PCR.

Note: the nucleotide sequence reported in this paper has been submitted to

the GenBankTM/EMBL Data Bank with accession number AF230354.

(Received 7 February 2000, revised 31 March 2000, accepted 3 April 2000)

Ham's F12 nutrient mix supplemented with 10% fetal bovineserum. The rhabdomyosarcoma cell line KYM-1 was a kind giftfrom A. Meager (National Institute for Biological StandardsControl, South Mimms, UK) and was maintained in RPMI 1640supplemented with 10% fetal bovine serum. The source andculture conditions of the myeloma cell line U266, Jijoye,the B-cell lymphoma HFB1 and the human T-cell clone JF7have been previously reported [18,19]. The melonoma celllines TIC3 and IC8 (invasive and noninvasive, respectively),were cultured as monolayers in McCoy 5A medium (LifeTechnologies, Paisley, UK) supplemented with 10% fetalbovine serum. Human peripheral blood monocytes wereisolated from buffy coats by centrifugation over Ficoll-Hypaque (Amersham-Pharmacia biotech, Les Ulis, France).

EST database screening

The EST database (http://www.ncbi.nlm.nih.gov/) was screenedwith TBLASTN using as query a conserved MMP motif(DINTFRLSADDIRGIQSLYG) derived from the catalyticdomain of MMP-12. Retrieved ESTs were compared withthe MMP sequences deposited in the Swissprot database(http://www.ncbi.nlm.nih.gov/). An EST (accession No.AI743415) of 593 bp encoding a new putative MMP wasidentified and the corresponding cloned cDNA namedMMP-26.

cDNA cloning

Two PCR primers designed using EST AI743415 were usedto amplify the 593 bp MMP-26 fragment using Fetal RaceReady cDNA as template (Clontech, Palo Alto, CA, USA).The MMP-26 cDNA fragment was cloned into the TA cloningvector pCR2.1 (Invitrogen, Groningen, the Netherlands) andsequenced. The nucleotide sequence of MMP-26 was used todesign primers for the cloning of the remaining MMP-26coding sequence by anchored PCR using Fetal Race ReadycDNA as template. To clone the 5 0-end of the cDNA, an initialPCR amplification was carried out with the primer MMPZ2(5 0-TGGATATCATCGGCACTGAG-3 0), derived from theAI743415 EST sequence along with the supplied anchoredprimer (AP1). A nested PCR amplification was performedwith the second supplied anchored primer (AP2) and theMMP-26 specific primer MMPZ6 (5 0-ATGGCCTAAGATAC-CACCTG-3 0). The same approach was used to clone the 3 0 endof MMP-26. The first PCR reaction was performed withprimers MMPZ1 (5 0-AAGATGGTTGGCCCTTTGATGG-3 0)along with AP1 and the nested PCR amplification, withprimers MMPZ3 (5 0-CTTAGGCCATGCCTTTTTAC-3 0) andAP2. Amplified fragments were cloned in pCR2.1 andsequenced. The full length ORF of MMP-26 cDNA wasthen amplified by PCR using the high fidelity cDNA PCRkit (Roche Diagnostics, Meylan, France) with the primersMMPZ11 (5 0-TTGGCATGCAGCTCGTCATC-30) and MMPZ16(5 0-CGGTTCAGCTAACCTGACTA-3 0) following manu-facturers guidelines. Amplified cDNA products with theexpected size (930 bp) were directly cloned in pCR2.1 andfully sequenced. To further confirm the cDNA sequence, PCRwas performed on lymph node cDNA (Clontech) with primersMMPZ11 and MMPZ16. The amplified cDNA fragment wascloned in pCR2.1 and fully sequenced. The MMP-26 cDNAwere recloned in the pcDNA3.1-MYC-HIS vector (Invitrogen)for transient transfection in COS cells.

Northern blot analysis

For the analysis of MMP-26 transcript expression, a 930-bpMMP-26 cDNA fragment was labelled with [a32P]dCTP (220TBq´mmol21; Amersham-Pharmacia Biotech) by randompriming [20]. This radiolabeled cDNA fragment was used toprobe the human 12-lane Multiple Tissue Northern blot as wellas the human Immune System Blot II (Clontech) membranes.Hybridizations, performed in ExpressHyb solution (Clontech),and washes were carried out according to manufacturer'sguidelines. For the analysis of MMP-26 mRNA expression incell lines by Northern blot assay, polyA1 RNA was isolatedusing the guanidium thiocyanate-CsTFA procedure followed bychromatography on oligo-dT sepharose [21]. Aliquots of RNA(2 mg) were subjected to electrophoresis in 1% agarose, 6%formaldehyde gels, electrotransferred to Nylon1 membranesand fixed with UV [22]. Hybridization were performed asdescribe above. For control hybridizations with a house keepinggene, the MMP-26 signal was stripped by incubating themembranes in boiling 0.5% SDS. A human GAPDH cDNAprobe (Clontech) was radiolabeled and used for Northern blotassays as described above for MMP-26 mRNA detection.

Transient transfection

COS cells were transiently transfected with plasmidsMMP26-pcDNA3.1-MYC-HIS using the transfection reagentFuGENETM 6 (Roche Diagnostics) following manufacturer'sguidelines.

Western blot analysis

COS cells and culture supernatants were harvested 72 h aftertransfection. Supernatants were cleared from cellular debris byhigh speed centrifugation and then concentrated using 10 kDacut-off concentrators (AMICON, Millipore, Bedford, MA, USA).Cells were washed twice in NaCl/Pi and resuspended in lysisbuffer (RIPA: 50 mm Tris pH 8; 150 mm NaCl; 1% Nonidet P40;0,5% sodium deoxycholate; 0,1% SDS). Cell lysates andsupernatant were first precleared using protein G-Sepharose(Amersham-Pharmacia biotech), and subsequently incubatedwith an anti-(c-Myc) antibody (10 mg´mL21) for 1 h at 4 8C.Then, protein G-Sepharose was added for an overnightincubation. Protein-G-sepharose immunocomplexes werewashed twice with incubation buffer (RIPA or RPMI,respectively) and resuspended in Tris/glycine/SDS samplebuffer containing 750 mm 2-mercapthoethanol. Eluted proteinswere denatured, subjected to SDS/PAGE on 10% SDS-polyacrylamide gels and electrotransferred to nitrocellulosemembranes [23]. Membrane were blocked overnight at 4 8C in1 � Tris/NaCl/Pi/Tween (20 mm Tris, 154 mm NaCl, 0,1%Tween 20) containing 5% dried milk, followed by a subsequent2 h incubation at room temperature in the same buffercontaining a peroxidase-coupled mAb anti-(c-Myc) tag, diluted1/1000 (Roche Diagnostics). Bound antibody was detectedusing ECL (Amersham-Pharmacia biotech) following themanufacturer's guidelines.

Gelatin and casein zymography

Aliquots of immunoprecipitated protein (300 ng) were incu-bated for 20 min at 37 8C in SDS sample buffer (Bio-Rad,Hercules, CA, USA) in the absence of reducing agent andsubjected to electrophoresis on 10% gelatin or 12% caseinpolyacrylamide gels (Bio-Rad). Electrophoresis gels were

3324 A. Benoit de Coignac et al. (Eur. J. Biochem. 267) q FEBS 2000

subsequently washed in 2.5% Triton X100 for 1 h at roomtemperature, incubated overnight in development buffer(50 mm Tris/HCl pH 7.5, 200 mm NaCl, 2 mm CaCl2, 0.02%Brij 35), and stained with 0.5% Coomassie blue. Thegelatinolytic or caseinolytic activities were detected as clearbands on a uniform background staining.

R E S U LT S A N D D I S C U S S I O N

Identification and cloning of a cDNA coding for a new humanMMP

Sequences of human origin in the EST database were searchedusing TBLASTN with a conserved amino-acid motif(DINTFRLSADDIRGIQSLYG) derived from the catalyticdomain of MMP-12 as query. ESTs in the resulting BLASToutput were further selected for the presence of long openreading frames (ORFs). The predicted amino-acid sequencesfrom these ESTs were compared with the protein sequencesdeposited in the Swissprot database using BLASTP. ESTs withORFs having amino-acid sequences homologous but notidentical to known MMPs were retained. The amino-acidsequence of the ORF from human EST AI743415 shared 56%identity with human macrophage metalloelastase (MMP-12). Apanel of different human cDNAs (brain, bone marrow, lymphnodes and fetus) were screened by RT-PCR using specificoligonucleotides, for the expression of mRNA encoding thenovel MMP, represented by EST AI743415. A cDNA fragmentwith a sequence identical to EST AI743415 was amplified fromfetal cDNA.

To clone the full length ORF, we employed an anchored PCRapproach with fetal cDNA as a template. A region of 400 bp ofthe cDNA located 5 0 from the area covered by the EST wasobtained. The fragment included a putative methionine startcodon preceded by a Kozac consensus sequence. The EST

AI743415 includes a stop codon and a poly A tail. To identifyputative splice variants of the novel MMP mRNA, a 3 0-RACEPCR was carried out, but all the cDNA isolated had a sequenceidentical to EST AI74415. The MMP cDNA coding region wascloned by RT-PCR using oligonucleotides primers designed toamplify the full length ORF and the 3 0-untranslated region. ThecDNAs amplified in two independent PCR reactions hadcompletely identical nucleotide sequences. The same sequencewas obtained when a third cDNA, isolated by RT-PCR fromhuman lymph node, was analyzed. The 930 bp cDNA obtainedencodes a 261 amino-acid proteinase comprising the samefeatures as MMP-7 (matrilysin). The prepro-proteinase has apredicted molecular mass of 29.6 kDa (Fig. 1B). The initiationmethionine is followed by a putative signal peptide, containing24 hydrophobic residues, suggesting that this novel MMP issecreted (Fig. 1A). The signal peptide is followed by a putativeprodomain containing a unique cysteine (at position 82), whosepresence in other metalloproteinases is known to inactivate theproenzyme by binding to the zinc of the catalytic domain(`cysteine switch') [11]. Interestingly the highly conservedPRCGVPD sequence is replaced in MMP-26 by PHCGVPD, achange which is however, unlikely to affect on the function ofthe prodomain as both amino acids are basic and positivelycharged. No furin cleavage site was detected between the pro-and the catalytic domain suggesting that MMP-26 is notactivated intracellularly by this enzyme. The alignment of theMMP-26 protein sequence with MMP-3, -7, -12 and -13 revealsthat in the region of the putative pro-peptide cleaving-site,MMP-26 is remarkably unconserved. The predicted 172 amino-acid catalytic domain comprises the highly conserved zincbinding motif (HEIGHSLGLQHS) as well as a methionineresidue, located seven residues downstream of this motif, whichis conserved in all MMPs. This residue has been proposed toplay an essential role in the structure of the active site of theseenzymes [24]. Like MMP-7, MMP-26 does not have a

Fig. 1. Predicted amino-acid sequence (A)

and domain structure of human MMP-26 (B).

(A) The putative signal peptide (amino acids

M1±A24) in bold is followed by the prodomain

in italic (amino acids D25±D89) and the catalytic

domain in regular characters (amino acids

T90±I260). The cysteine switch region (amino

acids P80±D86) and the catalytic domain

Zn-binding site (amino acids H208±S219) are

boxed. The putative glycosylation sites are

underlined. (B) MMP-26 and MMP-7

(matrilysin) zymogens have similar organization.

q FEBS 2000 Human MMP-26 (Eur. J. Biochem. 267) 3325

hemopexin domain and therefore represents the second shorterMMP describe to date (Fig. 1A,B). Taken together, thisstructural comparison suggests that the open reading frameencodes a putative new member of the MMP family that wehave named MMP-26, as MMP-25 (leucolysin) is the mostrecent member of this family to be identified and cloned [25].

Pairwise protein sequence comparisons between MMP-26and the other MMPs was performed. Even though MMP-26 byits structure is more closely related to matrilysin (MMP-7),having the minimal number of domains required for secretionand activity, sequence comparisons indicate that MMP-26shares highest homology with human macrophage metallo-elastase (MMP-12). When the sequence comparison wasrestricted to the predicted catalytic domains, MMP-26 wasfound to be 54% identical to human macrophage metallo-elastase (MMP-12) and 51% identical to human stromelysin 1(MMP-3). As with matrilysin, MMP-26 can not be classifiedinto one of the four groups of the MMP family, as it doesn'tshare the characteristic motifs of these groups. The three aminoacids around the zinc binding site (Tyr214, Asp235, Gly237 incollagenase 3 numbering) that have been described asfundamental for collagenase specificity [26] are absent in

MMP-26 rendering it unlikely to be a collagenase. Thecharacteristic hydrophobic region comprising nine aminoacids found at the C terminus of the catalytic domain ofstromelysins is also absent in MMP-26. Similarly, the absenceof fibronectin-like type II domain repeats in MMP-26 makes itunlikely that it belongs to the gelatinase subgroup.

MMP catalytic domains are known to bind two zincmolecules. In agreement with Soler et al. [27], Browner andcolleagues [28] determined that there are four metal ions boundto the matrilysin molecule, a catalytic zinc ion, a structuralzinc ion, and two calcium ions. The catalytic zinc ion wasfound to be complexed to the three His residues of theHEXGHXXGXXH region, and the structural zinc is bound in asimilar fashion. Becker et al. [29] demonstrated that for MMP-3,the structural zinc interacts with the side chains of Asp153,His151, His166 and His179. A similar motif is found in MMP-26at positions His158, Asp160, His173, and His186 suggesting thatMMP-26 may also bind two zinc molecules. MMP-26 has alsothree putative N-glycosylation sites located at positions 64±67(NGTD), 133±136 (NVTP) and 221±224 (NQSS).

Finally, among the MMP families characterize so far,MMP-26, like matrilysin, is unique in that its gene encode only

Fig. 2. Analysis of MMP-26 mRNA expression in human tissues (A) and cell lines (B). Aliquots of polyA1 RNA isolated from indicated human tissue

and cell lines were subjected to Northern blot assay with a specific radio-labeled probe (Upper panels). Positions of the molecular mass marker are indicated

on the left. Filters were subsequently hybridized with a human house keeping gene as a control for RNA integrity and equal loading (GAPDH; Lower panels).

Hybridization signal was detected by autoradiography.

3326 A. Benoit de Coignac et al. (Eur. J. Biochem. 267) q FEBS 2000

the minimal domains required for activity. Although thehemopexin domain present in other MMPs can be removedin vitro by proteolysis, it is not clear when or where thisprocessing occurs in vivo, suggesting that the structure ofmatrilysin and now of MMP-26, is most likely functionallydistinctive to other MMPs.

Distribution of MMP-26 mRNA in human tissues and cell lines

The expression of MMP-26 mRNA was analyzed by Northernblot assay on polyA1 RNA isolated from various human tissues(Fig. 2A). The 1.0-kb MMP-26 transcript was detected only inplacenta. The MMP-26 mRNA could not be detected in heart,brain, kidney, spleen, small intestine, liver, thymus, colon orskeletal muscle.

The placenta is an active remodeling/involuting tissuecomprising endothelial cells, stromal fibroblast-likemesenchymal cells and Hofbauer cells of macrophageorigin. Interestingly, MMP-12, to which MMP-26 is homo-logous, was also initially detected only in placenta, in which itwas further localized to macrophages and stromal cells [5]. Avery restricted expression pattern was also observed for MMP-7(to which MMP-26 is related by its minimal MMP domainstructure) with mRNA transcripts detected only in theendometrium and prostate (reviewed in [30]).

Human pregnancy is associated with extensive growth andremodeling of the uterus and placenta, and restructuring ofthese tissues during specific stages of gestation is likely toinvolve the degradative activity of various MMPs such asMT-MMPs, MMP-1, -2, -3, -7 and -9. MMP-7 is known to beinvolved in the activation of latent form of MMPs [31,32]. Theexpression of MMP-26 in the placenta and its structural analogyto MMP-7 suggest that this MMP could also participate in thepregnancy remodeling associated process, either directly bedegrading the extracellular matrix or indirectly by activatingother MMPs.

Analysis of MMP-26 mRNA expression was then extendedto human cell lines (Fig. 2B). MMP-26 transcript was detectedin the adenovirus-transformed kidney cell line HEK 293 and the

human lymphoma B cell line HFB1. Production of MMP-7 bykidney cells has been reported [8,33]. It remains to bedetermined if the observed expression in 293 cells reflects aproduction by normal kidney cells or was the result ofadenovirus transformation. In the HFB-1 cell line two splicevariants of 0.24 and 2.0 Kb were observed. A weaker signalcould also be detected in the melanoma cell lines TIC3 and IC8,in the monocytic cell line THP-1 and in the rhabdomyosarcomacell line KYM-1.

Expression of MMP-26 in transiently transfected COS cells

To verify that the cloned cDNA was capable of coding for asecreted metalloproteinase zymogen, the MMP-26 codingsequence was cloned into a mammalian expression vector.The construct comprises a c-Myc antibody epitope recognitiontag coding sequence (22 amino acids) in frame with 3 0-end ofthe cDNA ORF, allowing the detection of the recombinantprotein with an antitag mAb. The cDNA was transfected intoCOS cells. The recombinant MMP-26 was then isolated fromcell lysates and cell culture medium by immunoprecipitationwith the antitag mAb and analyzed by Western blot assay. Twomajor products with apparent molecular masses close to the onepredicted for the tagged proenzyme (29.4 kDa) were detectedin both the cell lysate and supernatant of transfected cells(Fig. 3A). These two products could reflect differences in thepost-translational modifications of the protein as putative N-glycosylation sites were predicted from the MMP-26 amino-acid sequence. Two contaminating bands around 22 kDa wereobserved in all lanes. The tagged form of MMP-26 could alsobe immunoprecipitated from transfected cell culture super-natants (Fig. 3A, lanes 3 and 4) indicating that, as predicted bythe presence of a putative signal peptide, the MMP-26 zymogenis secreted.

Protease Activity of recombinant MMP-26

To test the metalloproteinase activity of the recombinantprotein, MMP-26 was immunoprecipitated from cell lysatesand culture medium and analyzed for proteinase activity by

Fig. 3. Analysis of MMP-26 expression in

transfected COS cells by Western blotting (A)

and zymography (B,C,D). Cells were

transfected with a cDNA construct encoding the

c-Myc tagged MMP-26. Aliquots of proteins

purified by immunoaffinity chromatography were

analyzed by SDS/PAGE and Western blot with an

anti-c-Myc tag mAb coupled to HRP (A) or

zymography (B,C and D). A: Lanes 1 and 2;

transfected cell lysates. Lanes 3 and 4;

concentrated supernatants. Lanes 1, 3; mock

transfected cells. Lanes 2, 4; MMP-26

transfected cells. B and C: Analysis of the

protease activity by casein (B) and gelatin (C)

zymography. Lane 1; mock transfected COS

cells. Lane 2; MMP-26 transfected cells. D:

Inhibition of caseinolytic activity of MMP-26

(lane 1) by 50 mm EDTA (lane 2)

and 2 mm 1,10 phenantroline (lane 3).

q FEBS 2000 Human MMP-26 (Eur. J. Biochem. 267) 3327

gelatin and casein zymography. Several bands of proteinaseactivity could clearly be observed around 29 kDa in theproteins immunoprecipitated from the MMP-26 transfected celllysates (Fig. 3B,C). No activity was detected in the fractionsisolated from mock transfected cells (Fig. 3B,C, lane 1)indicating that they reflect the gelatinolytic and caseinolyticactivity of MMP-26. The fact that several bands are observedcould reflect variations in the glycosylation of MMP-26. Aweaker band was observed around 21 kDa (Fig. 3B) whichcould represent the tagged enzyme activated by cleavage of theprodomain during the elution of the imunoprecipitationcomplex by detergents (predicted size of the unglycosylatedpolypeptide fragment: 22 kDa). The same bands were observedwhen proteins were immunoprecipitated from the cell culturemedium and analyzed by zymography, the signal beinghowever, less intense (data not shown).

The proteolytic signal was reproducibly stronger on caseinthan on gelatin substrate (Fig. 3B,C, and data not shown).Therefore the effects on the protease activitity of the ionchelator EDTA or of the broad metalloproteinase inhibitorphenantroline were tested on this substrate. A partial inhibitionby EDTA was observed whereas the protease activity wascompletely abolished by phenantroline (Fig. 3D). These resultsfurther support that the activity detected is due to the presenceof recombinant MMP-26.

The availability of the MMP-26 cDNA and recombinantprotein will allow further biochemical characterization of thisproteinase and the study of its biological functions.

A C K N O W L E D G E M E N T S

We thank Pr. A. Meager for providing the rabdomyosarcoma cell line

KYM-1 and Dr J. Holzwarth for bioinformatics studies. We are grateful to

Pr. F. Woessner for kind help in attributing the MMP-26 number.

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