Plasma Membrane-dependent Activation of the 72-kDa Type IV ...

VOl. 268, No. 19,Issue of July 5,PP, 14033-14039,1993 Printed in U.S. A .

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Plasma Membrane-dependent Activation of the 72-kDa Type IV Collagenase Is Prevented by Complex Formation with TIMP-2* (Received for publication, December 30, 1992)

Alex Y. Strongin, BarryL. Marmer, GregoryA. Grant$, and GregoryI. Goldberg From the Division of Dermatology and the $Department of Molecular Biology and Pharmacology, Washington UniversitySchool of Medicine, St. Louis, Missouri 63110-1093

Human 72-kDa type IV collagenase (72T4C1) is secreted as a proenzyme that canform a specific stoichiometric complex with the tissue inhibitor of metalloproteases TIMP-2 via interaction with the carboxylterminal domain of the enzyme. Both complexed and free enzymes can be activated by treatment with organomercurials. The mechanism of the 72T4C1 activation under physiological conditions is not known. Here we describe a “plasma membrane-dependent” activation of inhibitor-free 72T4C1 and identify thefirst conversion intermediate as a 64-kDa species resulting from cleavage of the AsnS7-Leu peptide bond in the presence of plasma membranes from 12-0-tetradecanoylphorbol-13-acetate-inducedHTlOSO cells. This reaction isspecific for 72T4C1in thata closely related proenzyme (92-kDa typeIV collagenase) is resistant to activation under thesame conditions. Formation of the 72T4Cl.TIMP-2 complex inhibitsactivation at the level of the initial Asns7-Leu cleavage. Addition of TIMP-1 has no effect on this reaction, but blocks the autocatalytic conversion of the LeuSBintermediate into a 62-kDa activeenzyme with an amino-terminal Tyr”. Membrane-dependent activation of 72T4C1 is competitively inhibited in the presence of a 26-kDa peptide derived from the carboxyl-terminaldomain of the enzyme. The results suggest that interaction of the carboxyl-terminal domain of the enzyme with a membrane-associated component(s) causes initiation of enzyme activation through an autoproteolytic mechanism.

initial cleavage between Arg5 andA d 6 , generating a 46-kDa conversion intermediate leading to the formationof the 42kDa stable activecollagenase. Treatment with organomercurial compounds results first in activation of the proenzyme without loss in molecular mass, followed by conversion t o a 44-kDa intermediate and finally to the 42-kDa stable active enzyme form. Bothtrypsinandorganomercurialsactivate procollagenase byinitiating an intramolecular autoproteolytic reaction resulting in the removal of 81 amino acids from the amino-terminal portionof the molecule (4). Prostromelysin canbe activated in an analogous fashion by treatment with either trypsin or organomercurials, with concomitant loss of 84 (13) or 82 (14, 15) amino acids from the amino terminus, resulting in a 45-kDa stable active enzyme species.A physiologically relevantpathway of collagenase activation involves the urokinase-dependent proteolytic cascade (5) in which urokinase plasminogen activator converts plasminogen into plasmin. Plasmin is capable of activating purified procollagenase and prostromelysin. Plasmin-dependentactivation of procollagenase generates enzymespecies identical to those generated by limited proteolysis with trypsin ortreatmentwithorganomercurial compounds. Catalytic amounts of activated stromelysin can, in turn, convert plasmin- or trypsin-activated collagenase into a fully active enzyme, resulting in an additional 5-8-fold increase in specific activity (5). Activation of procollagenase with stromelysin in the presence of plasmin is 24,000-fold faster than with stromelysin alone (16). Unlikeinterstitial collagenase andstromelysin, less is known about the activation of the type IV procollagenases. Both type IV procollagenases share a common feature with interstitial procollagenase andprostromelysin inhavinga Secretedextracellularmatrixmetalloproteasesinitiate turnover of extracellular matrixmacromolecules, thus playing conserved amino acid sequence in the amino-terminalregion an essential role in the control of tissue remodeling (1-3). that is essential in the maintenance of the proenzyme state The extracellular activity of these enzymes is controlled a t (17, 18). We have previously demonstrated that 92- and 72the level of gene expression, proenzyme activation, and inter- kDa type IV procollagenases, in contrast to interstitialcollaactionwith specific tissueinhibitors of metalloproteases genase, form specific complexes with TIMP-1 and the related (TIMP-1andTIMP-2).Althoughmechanisms of enzyme inhibitor TIMP-2, respectively(17, 19). Recently, we have activation in solution have been a focus of intensive investi- shown (8) that 92T4C1’ can form a covalent homodimer and a novel complex with interstitial collagenase. The formation gation (4-11), mechanisms involved in the activation and of a 92T4C1 proenzyme complex with TIMP-1 prevents dicompartmentalization of theenzymesintheextracellular space are poorly understood. Purified interstitial collagenase merization, formation of the complex with interstitial collacan be activated by partial proteolysis with trypsin or by genase, and activation of the 92T4C1 proenzyme by strometreatment with organomercurial compounds(4, 12). Trypsin- lysin. In the presence of TIMP-1, interstitial collagenase is induced activation of procollagenase occurs as a result of the displaced from the 92T4C1-interstitial collagenase complex, resulting in a 92T4Cl.TIMP-1 complex and free procollagen* This work was supported by National Institutes of Health Grants ase interstitial. Formationof the covalent92T4C1 homodimer R01 AR39472 and R01 AR40618 and Training Grant T32 AI307284 and by a Monsanto Co./Washington University biomedical research The abbreviations used are: 92T4C1 and 72T4C1,92- and 72-kDa agreement. The costs of publication of this article were defrayed in type IV collagenases, respectively; PAGE, polyacrylamidegel electropart by the payment of page charges. This article must therefore be phoresis; HPLC, high pressure liquid chromatography; APMA, p hereby marked “advertisement” in accordance with 18U.S.C. Section aminophenylmercuric acetate; TPA, 12-0-tetradecanoylphorbol-131734 solely to indicate this fact.

acetate.

14033

14034

Membrane Activation of 72-kDA Type IV Collagenase

precludes the formation of a proenzyme-TIMP-1 complex, inhibitors (10 mM N-ethylmaleimide, 10 pg/ml aprotinin, 1 pg/ml thus allowing the enzyme to enter into the proteolytic cascade pepstatin A, 1 pg/ml leupeptin, and 1 mM phenylmethylsulfonyl of activation. All TIMP-1-free forms of the proenzyme can fluoride) and homogenized in a Dounce homogenizer. The homogenate was centrifuged at 3000 X g for 10 min in a refrigerated centribe activated under physiological conditions by stromelysin. fuge, and the resulting supernatant was centrifuged at 100,000 X g The interstitial collagenase component of the 92T4C1-inter- for 2 h. The pellet was resuspended in 25 mM Tris and 50 mM NaCl stitial collagenase complex can be activated in a way similar (pH 7.4) containing inhibitors (as described above) and separated to that of free interstitial collagenase, yielding a complex furtherona discontinuous sucrose gradient (20,30,50, and 60% active against both gelatin and fibrillar type I collagen. A sucrose in water) centrifuged a t 100,000 X g for 2 h at 4 "C. The novel role for TIMP-1 as an inhibitor of physiological acti- plasma membrane-enriched fraction appearing as a visible band at vation of 92T4Cl suggests that ananalogous complex between the 30/50% sucrose interface was collected, pelleted at 100,000 X g for 2 h, and stored at -80 "C. 72T4C1 and TIMP-2 may serve a similar role in the metaboExtraction of Plasma Membranes with Nonionic Detergents-The lism of this enzyme. plasma membrane-enriched fractions were resuspended in 50-500 pl While this work was in progress, Okada et al. (20) reported of 25 mM HEPES/KOH (pH 7.5) containing 0.1 mM CaC12and 0.25that nine tested endopeptidases, including trypsin, plasmin, 1%nonionic detergent (n-octyl glucoside, Triton X-100, Triton Xand stromelysin, lack the ability to activate 72T4C1. This 114, Nonidet P-40, or Lubrol PX) toreach a final protein concentracontrasts with the proteolytic activation of 92T4C1, procolla- tion of 1-2 mg/ml. After extraction for 1 h at 4 "C, the remaining was separated by centrifugation a t 100,000 X g for 1.5 h at 4 "C genase, and prostromelysin. In addition, TPA- or transform- pellet and discarded. The resulting extract was kept frozen at -80 "C until ing growth factor /31-induced HT1080 cells (21) and fibro- use. blasts stimulated with concanavalin A (11,22) were shown to Activation of 72T4Cl Proenzyme-Enzyme activation with organproduce a high ratio of activated 72T4C1. Activation of the omercurials, stromelysin, plasmin, and plasminogen activator was as enzyme by membranes from the concanavalin A-stimulated described previously (4, 5, 8, 17-19). Between 15 and 50 ng of the fibroblasts was shown to be sensitive to TIMP-2(11, 23) and 72T4C1 proenzyme was used for activation with plasma membranes (1-4 pg of plasma membrane protein) or plasma membrane extract required the carboxyl-terminal domain since the truncated (1-2 pgof the extracted proteins) in a 10-pl final volume of 25 mM enzyme was not susceptible to activation in this system (23). HEPES/KOH (pH 7.5) containing 0.1 mM CaClZ.The reaction was Here we demonstrate that 72T4C1 activation requires a incubated at 37 "C for 120 min (unless indicated otherwise), termimembrane-associated component, that complex formation nated by addition of sample buffer, and subjected to gelatin zymogram analysis. Samples containing a nonionic detergent were precipitated with TIMP-2 blocks this activation pathway, and that the carboxyl- terminal domain peptide competitively inhibits the with 10 volumes of cold acetone for 1 h a t -70 "C and centrifuged at 15,000 X g for 15 min at 4 "C. The pellets were solubilized in 12.5 mM membrane-dependent activation reaction. MATERIALS ANDMETHODS

Cell Culture"HT1080 fibrosarcoma cells were grownin monolayer culture inRPMI 1640 mediumsupplemented with 4% fetal calf serum and 2 mM glutamine in the presence of 5% COz and treated with TPA (50 ng/ml for 24-48 h). The p6Rhyg expression plasmid was constructed as described earlier (8).The complete 72T4C1 cDNA(18, 24) was subcloned into the Hind111 site of the p6Rhyg vector. The resulting expression plasmid (p6R72hyg) was transfected into E1Aexpressing p2AHT2a cells (24) using the calcium phosphate method (25) as described for 92T4C1 (8). The stably transfected cell line p2AHT7211A expressing 72T4C1was selected in the presence of hygromycin (200 pg/ml). Enzyme Purification"72T4Cl procollagenase was purified from conditioned medium of transfected p2AHT2a cells as described (8). Fractions from reactive red-agarose (Sigma) containing gelatinolytic activity were applied to a gelatin-Sepharose (Sigma) affinity column, and the TIMP-2-free enzyme was eluted using a 0-10% dimethyl sulfoxide gradient. Gelatin-Sepharose-purifiedenzyme was dialyzed into 0.005 M Tris-HC1 (pH 7.5) containing 0.0001 M CaClzand 0.005% Brij 35 and stored a t -80 "C. TIMP-2 was purified from the 72T4C1 proenzyme-inhibitor complex using an Aquapore RP-300 reverse-phase column (7 pm) developed with a 5-70% gradient of acetonitrile in 0.1% trifluoroacetic acid as described earlier (18).The TIMP-2-containing fractions were repeatedly lyophilized to remove acetonitrile and trifluoroacetic acid and stored in water at -80 "C. Gel-filtration chromatography was performed on an AcA-44 column (0.5 X 88.5 cm) equilibrated in 20 mM Tris-HC1 (pH 7.5) containing 200 mM NaCl and 0.01% Brij 35 and developed with the same buffer at a rate of 1 ml/h. 200-4 fractions were collected, and proteins were analyzed by SDS-PAGE. Zymogram, Western blot, gel electrophoresis, and protein sequence analyses of activated enzyme species were performed as described (18).Protein concentration inthe samples was determined according to Lowry et al. (34) using bovine albumin as astandard. Mass spectrometry was performed using a Vestec Model 201 single quadrapole electrospray mass spectrometer equipped with a Teknivent Vector-two data system. Fractionation of HT1080 Cells"HT1080 cells were treated with TPA, washed with phosphate-buffered saline, scraped, and centrifuged a t 1000 x g for 5 min. Cells were resuspended in 25 mM TrisHC1 (pH 7.4) containing 8.5% sucrose, 50 mM NaCl, and protease

Tris-HC1 (pH 7.5) containing 5% SDS and 40% glycerol prior to SDS-PAGE. To activate 72T4C1 in amounts sufficient for protein sequence analysis, 30 pg of proenzyme was mixed with 400 pl of the 1%Lubrol extract and incubated for 3 h at 37 "C. o-Phenanthroline was added to the reaction mixture to a final concentration of 10 mM, and the sample was diluted 10-fold with 5 mM Tris-HC1 (pH 7.5) containing 0.005% Brij 35, 400 mM NaCl, and 0.5 mM EDTA and applied to a gelatin-agarose column. The affinity column was eluted with 5 mM Tris-HC1 (pH 7.5) containing 0.001% Brij 35, 400 mM NaCl, 0.5 mM EDTA, and 10% dimethyl sulfoxide; dialyzed against 5 mM Tris-HC1 (pH 7.5) containing 0.001% Brij 35 and 0.5 mM EDTA; and subjected to SDS-PAGE, electroblotting, and amino acid sequence analysis as described earlier (18). Zsolution of 72T4Cl-deriuedPeptides-1 mg of 72T4C1 was activated with 1 mM APMA for 1 h a t 37 "C in 25 mM Tris-HC1 (pH 7.5) containing 0.1 mM CaC12and 0.05% Brij 35; adjusted to 0.1% trifluoroacetic acid; and dialyzed against 20 mM Tris-HC1 (pH 7.5) containing 5 mM CaC12,150 mM NaCl, and 0.005% Brij 35. The dialyzed sample was applied to a gelatin-Sepharose affinity column, and the gelatin-binding peptides were eluted with the same buffer containing 10% dimethyl sulfoxide. The 26-kDa carboxyl-terminal peptide that does not bind gelatin was further purified by chromatography on a reactive red-agarose column, eluted with 20 mM Tris-HC1 (pH 7.5) containing5 mM CaClZ and 2.5 M NaC1, and dialyzed overnight against 20 mM HEPES/KOH (pH 7.5) containing 0.005% Brij 35. The isolated 26-kDa carboxyl-terminal fragment was reduced and alkylated or cleaved with CNBr as indicated. Reduction of the peptide was achieved using 10 mM ditbiothreitol for 2 h at 37 "C under nitrogen. The resulting fragment was alkylated with 200 mM iodoacetamide for 4 h at ambient temperature and further purified using an Aquapore RP-300 reverse-phase column (7 rm). Peptide cleavage with CNBr (130, w/w) was in 6 M guanidine, 0.1 N HCl for 18 h at room temperature under nitrogen in the dark. The resulting peptides were separated using a reverse-phase HPLC ~ 0 1 umn as described above. Peptide Synthesis-Peptide synthesis was performed using an Applied Biosystems Model431 peptide synthesizer and standardtbutyloxycarbonyl chemistry protocols. The peptides were cleavedwith anhydrous HF, purified by reverse-phase HPLC, and subjected to amino acid analysis and mass spectrometry. The disulfide bridge in peptide 1666Bwas formed by air oxidation of peptide 1666A. Disulfide bridge formation was verified by mass spectrometric analysis.

Membrane Activation of 72-kDA Type

IV Collagenase

14035

RESULTS

HT1080 Fibrosarcoma Cells Contain Specific Membraneassociated Activator of 72-kDa Type ZV Collagenase Proenzyme-The 72T4C1 proenzyme can be activated by treatment with the organomercurial APMA and converted into a 62kDa protein with amino-terminal sequence starting atTyr*l (6). This activation is unaffected by the formation of the proenzyme complex with the inhibitor TIMP-2 (19, 26-28). To investigate a physiologically relevant mechanism of 72T4C1activation and therole of TIMP-2 in thisprocess, we have used the “metalloprotease-negative’’ cell line p2AHT2a (24) to overexpress 72T4C1 relative to TIMP-2 and topurify inhibitor-free enzyme. The 72T4C1 cDNA from the p72T4ClBS plasmid (18, 24) was subcloned into the p6Rhyg expression vector (8) and transfected into p2AHT2a cells using the calcium phosphatemethod (25). Synthesis of 72T4C1 in stably transfectedp2AHT7211A cells resulted from the expression of the cDNA plasmid since the transcription of the chromosomal 72T4C1 gene in the p2AHT2a host is repressed by the adenovirus E1A protein (24). 72T4C1was purified from the conditioned medium of p2AHT7211A cells by affinity chromatography on reactive red-agarose and gelatin-Sepharose gels. Although host cells produce TIMP-2, the expressed enzyme is sufficiently overproduced so that the inhibitor-free enzyme can be purified from the complex onagelatin-Sepharoseaffinity column developed with a 0-10% gradient of dimethyl sulfoxide (Fig. lA, lane 3). The purified recombinant TIMP-2-free enzyme is resistant to activation with urokinase and plasmin; and in contrast to theclosely related enzyme 92T4C1 (8), 72T4C1 is also resistant toactivation by stromelysin (lanes 1-6). To investigate further a possible mechanism of72T4C1 activation, we have used the observation that theconditioned medium from TPA-stimulated HT1080 cells consistently contains a substantial amountof activated 72T4C1 in proportion to the proenzyme (8, 18). To determine whether a soluble 72T4C1 activator is present in this system, medium conditioned by HT1080 cells was concentrated and chromatographed on gelatin affinity and AcA-44 size fractionation columns to separate secreted enzymes and their inhibitors. The resulting fractionslacked any detectable activitycapable of activating exogenously added TIMP-2-free 72T4C1 (data not shown), suggesting that the putativeactivator is associated with cells. To verify this hypothesis, we fractionated TPA-stimulated HT1080 cells using a discontinuous sucrose gradient (see “Materials and Methods”). The plasma membrane-enriched fraction was capable of activating the TIMP2-free 72T4C1 proenzyme (Fig. lA, lanes 7 and 8). The activation was observed by a semiquantitative zymogram assay in which the appearance of a lower molecular mass activated enzyme species can be readily observed in the presence of a background of membrane proteins. The activation reaction was specific since this membrane preparation was unable to activate the closely related TIMP-1-free 92T4Cl enzyme under the same conditions (lanes 9 and 10).The results presented in Fig. 1B demonstrate that activation of the exogenously added enzyme increaseswithincubationtime and amount of the added membrane protein. Moreover, the “activator” can be extracted from plasma membranes with the nonionic detergent Lubrol (lanes 11-14),but is resistant to extraction with octyl glucoside and salt (data notshown). The results demonstratethat a“membrane-dependent” activation of the 72T4C1 proenzyme results inthe appearance of 64-, 62-, and 42-kDa enzyme forms. The apparentmolecular masses of these forms are in good agreement with that of the APMA-activated 72-kDa enzyme species generated by auto-

+

+ + +

+

+

94 -

67 43 1 2 3 4 5 6 7 8 9 1 0

120 min

1 2 3 4 5 6 7214CI (ng) 15 I5 15 15 15 15 Membrane (pg) Extract (pg)

- 2 2 2 2 2 - - - - - -

7 8 9 IO 11 12 13 14 50 50 50 50 50 50 50 50 4 1,34415 - - 1.2 .A 13.04

-

”

“

FIG. 1. Activation of 72T4C1 by plasma membranes from HT1080 cells. A, 1 pg (lanes 1-6) or 50 ng (lanes 7-10) of purified inhibitor-free 92T4Cl and 72T4Cl proenzymes was activated with 50 ng of stromelysin (Str) (lanes 2 and 4 ) . 500 ng of plasmin (lane 51, 500 ng of urokinase-type plasminogen activator (uPA) (lane 61, or 8 pg of plasma membranes (Membr) (lanes8 and 10) for 2 h at 37 “C and subjected to SDS-PAGE on 10% acrylamide or zymogram gels. Plasma membrane activation was in 25 mM HEPES/KOH (pH 7.5) containing 0.1 mM CaC12 ina 10-pl reactionmixture. B, purified inhibitor-free 72T4C1 (lane I ) was activated with plasma membranes as described for A using the indicated amounts of enzyme, plasma membranes, and Lubrol extract and the indicated incubation times. Lubrol extract (lanes 11-14) was prepared as described under “Materials and Methods.”

catalytic processing (6,10,18). Theamino-terminal sequences of the 62-kDa (6) and 42-kDa (10) species obtained upon treatment of the enzyme with organomercurial were determined previously. The structure of the 64-kDa conversion intermediate is of particular interest since it is likely to result from the membrane-induced initial cleavage in the aminoterminal domain of the enzyme, while lower molecular mass forms result from further autocatalytic processing (6, 19). This interpretation of the results is supported by the observation (Fig. lB, lanes 1-6) that a membrane-dependent activation reaction leads to theappearance of the 64-kDa enzyme prior to accumulation of the lower molecular mass species. To verify the hypothesis that the plasma membrane-dependent activation of 72T4C1 results in the processing of the aminoterminal domain, we have determined the amino acid sequence of the 64-kDa conversion intermediate. To accumulate a sufficient amount of the 64-kDa form for sequence analysis, a Lubrol extract of the membrane fractionwas usedto activate 30 pgof 72T4C1. The activation reaction was terminated after 3 h by addition of o-phenanthroline to prevent further autocatalytic conversion. The reaction products were separated from the membraneproteins on a gelatin-agarose affinity column, resolved onSDS-PAGE, and subjected to aminoterminal amino acid sequence analysis. The amino-terminal sequence LFVLKDXLK (Fig. 2) of the 64-kDa enzyme suggests that theinitial cleavage of the 72T4C1 proenzyme catalyzed in the presence of the plasma membrane occurs at Asn”Leu, 43 amino acid residues upstream from the amino-termi-

LFVLK

Membrane Activation of 72-kDA Type IV Collagenase

14036 Peptide Peptide Peptide Peptide

1666B 166614 1666 1667

n CPKESCNLFVLK \

"N

APSPIIKFPGDVAPKTDKELAVQYLNTFYGCPKESCNtFVLKDTLKKMQK 5 0 FFGLPQTGDLDQNTIETMRKEGNPDVANYNFFPRKPKWDKNQITYRII 100

72T4CI +-

4

FIG. 2. Structure of plasma membrane-activated 72T4C1. The 72T4C1 proenzyme was activated as described under "Materials and Methods" and purified from plasma membrane proteins on a gelatin-agarose affinity column, and activated species were separated by SDS-PAGE on 7.0% gels. The protein species were electroblotted on an Immobilon membrane and subjected to amino acid sequence analysis on an Applied Biosystems Model 470A Gas-phase Sequencer as described earlier (17). The amino-terminal sequences of the 72kDa proenzyme and 64- and 62-kDa activation products start from A'PSPII (6,17), L3'FVLKDT (upper arrow), and Y'INFFPRK (lower arrow). The conserved activation sequence PRC is underlined. The amino acid sequences of peptides 1666, 1667, 1666A, and 1666B are also shown. Half-cysteine residues in peptide 1666B are linked by the disulfide bridge as indicated (see "Materials and Methods").

nal Tyr" of the 62-kDa enzyme form (6). Activation of 72T4C1 by Plasma Membranes Is Prevented by Complex Formation with TIMP-2"We have previously demonstrated that 92- and 72-kDa type IV procollagenases, in contrast to interstitial collagenase, form specific complexes with TIMP-1 and therelated inhibitor TIMP-2,respectively (17,19). However, the physiological significance of the proenzyme-inhibitor complex and the mechanism of activation of type IV collagenases has remained unclear. Recently, we have demonstrated (8) that the formation of a 92T4C1 proenzyme complex with TIMP-1 prevents dimerization, formation of the complex with interstitial collagenase, and activation of the 92T4C1 proenzyme by stromelysin (a related metalloprotease). Based on these observations, we hypothesized that formation of the 72T4Cl.TIMP-2 complex serves an analogous function in the metabolism of this enzyme. To verify this hypothesis, we investigated the effect of various inhibitors, including TIMP-1 and TIMP-2, onplasma membranedependent activationof 72T4C1.Both plasma membrane(Fig. 3 B ) and detergent-solubilized (data not shown) forms of the 72T4C1 processing activity were sensitive to the metalloprotease inhibitors EDTA and o-phenanthroline, whereas phenylmethylsulfonyl fluoride, N-ethylmaleimide, bestatin, leupeptin, pepstatin, aprotinin, and chymostatin had no effect. Addition of TIMP-1 to the reactionresulted in the accumulation of the 64-kDa intermediate and inhibition of autocatalytic conversion into the 62-kDa form. Addition of purified TIMP-2 caused complete inhibition of the 72T4C1 conversion (lane 6). The same result was achieved when an in vitro reconstituted 72T4Cl.TIMP-2 complex was used as a substrate for the membrane-dependent activation reaction. The complex was reconstituted by incubation of recombinant pro-72T4Cl with purified TIMP-2 (Fig. 3A) and was separated from excess inhibitor onan AcA-44 gel-filtration column. The appearance of a small amount of the 64-kDa form (Fig. 3B, lane 8) is due to partial dissociation of the complex during the isolation procedure and can be preventedby addition of a small amount of TIMP-2, but not TIMP-1 (lanes 9 and 10). These results suggest that membrane-dependent activation of 72T4C1 can be explained by either of two mechanisms. (i) 72T4C1 can interact with a component of the membrane that altersits conformation so that the enzyme is capable of autoproteolytic processing similar to that induced by treatment with organomercurials; (ii) alternatively, a membraneassociated protease is capable of specific cleavage of the

--

- 94

BI

72T4Cl/ TIMP-2

72T4CI 1 N

N

-67 43

+ + + +

+ +

- 30

TIMP-2-

166

-

- 64 '62

- 20

1 2 3

-++

1 2 3 4 5 6 7 8 9 1 0

- + + + + + - + + + FIG. 3. Complex formation of 72T4C1 proenzyme with TIMP-2 prevents its activation by plasma membranes. A, the purified inhibitor-free 72T4C1 proenzyme (lane 1 )was incubatedwith a 3-fold molar excess of purified TIMP-2, and the enzyme-inhibitor complex (lane 2) was separated from free TIMP-2 (lane 3) by gelfiltration chromatography on a 5 X 900-mm AcA-44 column equilibrated with 20 mM Tris-HC1 (pH 7.5) containing 200 mM NaCI, 5 mM CaCI2, and 0.01% Brij 35 at a rate of 1 ml/h. The eluted peaks were subjected to SDS-PAGE on a 13%gel. B, 15 ng of 72T4C1 (lanes 1-6) or the 72T4Cl.TIMP-2 complex (lanes 7-10;also see A , lane 2 ) was incubated without (lanes 2 and 8)or with the protease inhibitors EDTA (5 mM; lane 3),o-phenanthroline (OP;10 mM; lane 4 ) , TIMP1 (100 ng; lanes 5 and 9 ) , or TIMP-2 (100 ng; lanes 6 and 10) for 15 min at 4 "C and then activated with 2 pg of plasma membranes as described for Fig. L4 for 120 min. ACA-44

Membrane

proenzyme a t A ~ n ~ ~ - Lthus e u , initiating enzyme activation. The fact that membrane-dependent activation is completely inhibited by the metalloprotease inhibitors EDTA and 0phenanthroline suggests that if the initial cleavage is effected by a membrane-bound protease, it is a metalloenzyme. However, since APMA-induced autocatalytic conversion is also sensitive to the same inhibitors, this observation does not permit discrimination between the two possible activation mechanisms. In addition, formation of the specific complex of 72T4C1 with TIMP-2 blocks the initial step of activation, whereas TIMP-1, unable to form the complex with the proenzyme, blocks the reaction at thenext level of autoproteolytic conversion to the62-kDa species. Membrane-dependent Activation of 72T4C1Is Competitively Inhibited by 72T4Cl Carboxyl-terminal Domain-derived Peptide-To further understand the mechanism of membranedependent cleavage of the amino-terminal domain of 72T4C1 at A~n~~-L wee usearched , for a specific competitive inhibitor of this reaction. Initially, we synthesized a series of peptides (Fig. 2) corresponding to the sequence of72T4C1 spanning the initial cleavage site ( A ~ n ~ ~ - L e These u). peptides were used to determine whether the membrane fraction contains a protease capable of specific cleavage a t A ~ n ~ ~ - and, L e u if so, whether it is sensitive to addition of the metalloprotease inhibitors EDTA and o-phenanthroline. Both peptide 1666A and its isomer (1666B), with Cysm and Cys'j5forming a disulfide bridge, were incubated with the membrane fraction inthe presence or absence of the protease inhibitors, and the hydrolysis products were subjected to HPLC separation and sequence determination. The major hydrolysis product had the amino-terminal residue instead of Leu3', and the reaction was not affected by the presence of EDTA, suggesting that membrane-associated proteases are notcapable of faithfully reproducing the cleavage leading to the activation of 72T4C1 usingsyntheticpeptides as substrates (Fig. 2). In addition, these peptides failed to competitively inhibit membrane-dependent activation of the enzyme even when present at 2 X lo4 M excess relative to theenzyme (Fig. 4). These resultsindicate thatthe second mechanism, i.e.

Membrane Activation of 72-kDA Type IV Collagenase A

6

94 67 A3

66,l

7 ;:1

30 20.1

'

14037

3

peptide: 1 2 3 4 5 6 7 8 9 IO I 1 12 13 14 #I666 (ug) 12 e1667 (vel 12 # 1666A (pg) 12 #16668 ( p a ) 12 26kD0 CT (ne) 50 100200 400 26kDa CT (ng)** 100200400800

FIG.4. Inhibition of plasma membrane activation by 72T4CLderived peptides. A, 72T4C1 was activated with 1 mM APMA for 1 h a t 37 "C; the mixture was adjusted to a 0.1% final concentration of trifluoroacetic acidand dialyzed against 20 mM TrisHC1 (pH 7.5) containing 5 mM CaCI2, 150 mM NaCI, and 0.005% Brij 35. The resulting digestion products (lane I ) were separated on gelatin-Sepharose (lane 2) and reactive red-agarose (lane 3 ) affinity columns. The isolated 26-kDa carboxyl-terminal (CT)fragment was further reduced with dithiothreitol, alkylated with iodoacetamide as described under "Materials and Methods," and purified on an Aquapore RP-300 microbore column (7 pm)using an Applied Biosystems microbore separation system (lane 4 ) . B,72T4Cl (15 ng; lane I ) was activated with 2 pgof plasma membranes as described for Fig. lA (lanes 2-14) for 2 h a t 37 "C. 12 pg of peptides 1666, 1667, 1666A, and 1666B (lanes 3-6) (see Fig. 2); 50-400 ng of the 26-kDa carboxylterminal fragment (lanes 7-10);and 100-800 ng of the reduced and alkylated 26-kDa carboxyl-terminal peptide (**) (see A ) (lanes 1I1 4 ) were added to the enzyme prior to activation as indicated.

I

10 20 30 FRACTION NUMBER

FIG. 5. Inactivation of 26-kDa carboxyl-terminal peptide by CNBr cleavage. The 26-kDa carboxyl-terminal (CT) fragment was cleaved with CNBr (see "Materials and Methods"), andthe resulting peptides were separated on an Aquapore RP-300 column (7 pm, 2-mm inner diameter) using an Applied Biosystems microbore separation system developed with a 5-70% acetonitrile gradient in 0.1% trifluoroacetic acid. Inset, 72T4Cl (lane 1 ) was activated with plasma membranes as described for Fig. 4 in the absence (lane 2) or presence of 200 ng of the 26-kDa carboxyl-terminal fragment (lane 3 ) , 1200 ng of peptide from peak 4 (lane 4 ) , or 600 ng of peptide from peak 2 (lane 5 ) .

binding of the enzyme to a membrane component alters the enzyme conformation, leading to activation by autoproteo52 lysis, may be a plausible pathway for enzyme activation in 44 this system. Consequently, we have attempted to identify a protein moiety within the 72T4C1enzyme that canpotentially 1 2 3 4 5 6 7 8 910111213 mediate such binding. The purified TIMP-2-free proenzyme TIMP-2 ( p g ) - .2 .2 .2 .2 - - .2 - .2 .2.2 .2 was subjected to activation withAPMA, and furtherautopro26kDa CT ( p g ) - - .15 45 1.3 .35.05.05 - - - - teolysis was induced by exposure to acid pH and slow neu26kDo CT CNBr (pg) - - - - - - - - .4 .I5 45 1.3 2.7 tralization by dialysis (10). This treatment generated several FIG. 6. Dimerization of 26-kDa carboxyl-terminal peptide large peptides that were fractionated on gelatin- and reactive and interaction TIMP-2. The 'Z51-labeled26-kDa carboxylred-agarose affinity columns. The fractions eluted from the terminal fragmentwith (1.6 X lo5cpm/pl, 10' cpmlpg; see "Materials and gelatin-agarose column containing residual uncleaved enzyme Methods") alone (lane 1 ) or in the presence of the indicated amounts and the peptides containing the fibronectin-like collagen- of TIMP-2 and/or the unlabeled 26-kDa carboxyl-terminal (CT) binding domain (18,29) failed to inhibitactivation of 72T4C1 fragment or the CNBr-cleaved 26-kDa carboxyl-terminal fragment when added to the membrane activation reaction (data not (Fig. 5, peak 4 ) was cross-linked using 2 mM bis(sulfosuccinimidy1) shown). Two large fragments withapparent molecular masses suberate (Pierce Chemical Co.) for 1 h at 0 "C in 25 mM HEPES/ KOH (pH 7.5) containing 150 mM KCl. of 31 and 26 kDa that were not retained onthe gelatin affinity column were purified further ona reactive red-agarose affinity of 72T4C1 (inset). These results suggest that the secondary column and were eluted withbuffer containing 2 M NaCl (Fig. structure of the 26-kDa carboxyl-terminal domain fragment 4A).These fragments were derived from the carboxyl-termi- is important for its inhibitory activity. These results also nal hemopexin-like domain of the enzyme, and both have indicate that the interaction between the carboxyl-terminal amino-terminal amino acid sequence starting at Leu444.The domain of the enzyme and the membrane-associated compo26-kDa fragment contains 2 Cys residues and, upon reduction, nent is essential for membrane-dependent activation of the co-migrates with the 31-kDa fragment, demonstrating that enzyme. The fact that proenzyme-inhibitor complex formathe latter represents a reduced version of the former. The tion with TIMP-2 is also mediated through the carboxylresults presented in Fig. 4B show that the mixture of these terminal domain of the enzyme (23,30) is consistent with the fragments inhibits membrane activation of 72T4C1 in a dose- model in which interaction of TIMP-2 with the carboxyldependent fashion (lanes 7-10). The activation is inhibited terminal domain of the proenzyme prevents its interaction at theinitial stage of the formation of the 64-kDa intermedi- with the membrane activator, thus blocking an initial step in ate, which is similar to the effect observed upon addition of activation. TIMP-2. Reduction of the disulfide bridge C y ~ ~ ~ ~ - C in y s " j ~To provide additional support for this interpretation of the the 26-kDa peptide (see "Materials and Methods") leads to results, we have studied the interaction of the 26-kDa fraginactivation of its inhibitory activity (lanes 11-14). A similar ment with TIMP-2. Cross-linking experiments demonstrate result was obtained when the 26-kDa fragment was subjected that the'251-labeled26-kDa fragment (present inall reactions topartial cleavage with CNBr (Fig. 5) andthe resulting presented in Fig. 6) forms a 44-kDa complex with purified fragments were subjected to HPLC separation, amino acid TIMP-2 ( l a n e 2). This interaction canbe effectivelycompeted sequence analysis, and mass spectrometry.The internalfrag- in the presence of the unlabeled 26-kDa fragment (lanes 3ment produced by cleavage a t Metso4and Met5'* (peak 2) and 5 ) . The increase in the final concentration of this fragment a fragment containing a single cleavage a t Met'" (peak 4 ) leads to the formation of a 52-kDa dimer (lanes 4-7). The lacked the ability to inhibit membrane-dependent activation dimerization of the 26-kDa fragment does not require the

14038

Membrane Activation of 72-kDA TypeIV Collagenase

presence of TIMP-2 (lanes 6 and 7), but excess TIMP-2 prevents the dimerization (lane 8).Addition of the 26-kDa fragment cleaved with CNBr failed to promote dimerization of the labeled fragment (compare lanes 6 and 9 ) or to compete the formation of the complex with TIMP-2 (lanes 10-13). These results demonstratethat purified TIMP-2 iscapable of complex formation with the 26-kDa carboxyl-terminal fragment and that this interaction is abolished upon cleavage of the fragment with CNBr (Fig. 5, peak 4 ) or reduction of the disulfide bridge (data not shown). Thus, modifications of the 26-kDa carboxyl-terminal fragment that abolish its inhibitory activity in the membrane-dependent reaction of 72T4C1 activation also abolish its ability to interact with TIMP-2.

The membrane-dependent activation of 72T4C1is sensitive to the metalloprotease inhibitors EDTA and o-phenanthroline, but inhibitors of other protease classes have no effect. These results suggest that if plasma membranes contain a specific protease capable of activating 72T4C1, it is a metalloprotease. Alternatively, the enzyme caninteract with a membrane component causing a change in the secondary structure of the enzyme, which leads to autocatalytic processing of the amino-terminal domain. Since both membranedependent activation and autoproteolytic activation are sensitive to EDTA and o-phenanthroline, thesetwo possibilities could not be discriminated based on these results. Thus, we have attempted to identify a specific inhibitor of membranedependent 72T4C1 activation. Our results demonstrate that DISCUSSION synthetic peptides spanning the initial cleavage site are not Activation of type IV collagenases in solution, initiated by capable of competitive inhibition of the reaction even when treatment with organomercurials, results in proteolytic proc- added in large molar excess relative to theenzyme. Moreover, essing of the amino-terminal domain (6, 17, 18, 31). When these peptides failed to serve as specific substrates for the not complexed with TIMP-1, 92T4C1 can be efficiently acti- putative membrane-associated protease. vated by stromelysin (7,8). Here we demonstrate that recomFractionation of 72T4C1 into smaller peptides yielded a 26binant TIMP-2-free72T4C1 is resistant to activation by stro- kDa fragment derived from the carboxyl-terminal domain of melysin and plasmin. Although partial activation of 72T4C1 the enzyme that was able to competitively inhibit the memby urokinase-type plasminogen activator in the conditioned brane-dependent activation of the enzyme. Partial cleavage medium was recently reported (32), our results using purified with CNBr or reduction of a disulfide bridge C y ~ ~ ~ ~ - C y s ~ ~ ) enzymes suggest that urokinase-type plasminogen activator contained within this fragment led to a loss of its inhibitory is not capable of enzyme activation. To investigate a possible activity. The fragment was shown to interact with purified mechanism of 72T4C1 activation, we have used the observa- TIMP-2 and toform a dimer. Formation of the complex with tion that a substantial amountof activated enzyme is present the inhibitor and dimerization of the fragment are mutually consistently in the medium conditioned by the TPA-induced exclusive as demonstrated by the fact that an excess of the human tumor cell line HT1080. All fractions of conditioned unlabeled 26-kDa fragment is able to compete the formation medium from these cells, however, lacked an ability to activate of the complex with TIMP-2 with the concomitant appearexogenously added 72T4C1. We therefore fractionated TPA- ance of the 52-kDa dimer. The formation of this dimer is induced HT1080 cells on a discontinuous sucrose gradient abolished in thepresence of a molar excess of TIMP-2. and showed that plasma membrane-enriched preparations These results are in good agreement with the experiments from these cells were capable of activating the exogenously reported by Murphy et al. (23) demonstrating that carboxyladded recombinant TIMP-2-free 72T4C1 proenzyme. The terminal truncated72T4C1 cannot be activated by membranes “membrane-associated” activator was specific for 72T4C1 isolated from fibroblasts stimulated with concanavalin A and since a closely related enzyme (TIMP-1-free 92T4C1)was with mutagenesis experiments (30) demonstrating that proenresistant to activation under the same conditions. The acti- zyme-TIMP-2 complex formationis mediated by the carvator can be extracted from plasma membranes with the boxyl-terminal domain of 72T4C1. nonionic detergent Lubrol and is resistant to extraction with The fact that complex formation with TIMP-2 blocks memoctyl glucoside and salt. brane-dependent activation of the enzyme suggests that the The membrane-dependent activation of 72T4C1 results in physiological role of proenzyme-inhibitor complex formation amino-terminal cleavage of the proenzyme, generating the 64- is to block the initial step in the activation of 72T4C1 (19,261 kDa intermediate form with the amino-terminal Leu3*,which rather than tostabilize the proenzyme as was proposed earlier is subsequently converted into a 62-kDa activated enzyme. (10, 27). Similar results concerning the analogous complex of Formation of the 72T4C1 proenzyme complex with TIMP-2 92T4C1 with TIMP-1 (8) support this interpretation. blocks membrane-dependent activation of the proenzyme at Our results are consistent with the hypothesis that the the level ofthe initial Ad7-Leu cleavage. Addition of TIMP- interaction between the carboxyl-terminal domain of the en1 does not affect formation of the 64-kDa intermediate, but zyme and the membrane-associated component is essential prevents its further conversion into the62-kDa active enzyme. for membrane-dependent activation of the enzyme. Such an The effect of these two inhibitors on membrane activation interaction may cause a conformational change that leads to of the enzyme is of particular interest since they interactwith autoproteolytic processing of the amino-terminal domain. the enzyme via different mechanisms (3). TIMP-1 is unable This mechanism is similar to the one we observed earlier in to interact with the 72T4C1 proenzyme, but can inhibit its the case of organomercurial activation of interstitial collagenenzymatic activity and autoproteolytic conversion (19). ase (4, 33), in which enzyme activity was observed upon TIMP-2, however, forms a noncovalent stoichiometric com- addition of the organomercurial prior to theprocessing of the plex with 72T4C1 mediated through interaction with the car- amino terminus. Although our data favor this hypothesis, the boxyl-terminal domain of the proenzyme (19, 30). This com- involvement of a putative membrane-bound protease in the plex is analogous to the92T4C1. TIMP-1 proenzyme-inhibitor activation of72T4C1 cannot be completely excluded. The complex (17), the formation of which prevents proenzyme answer will have to await the reconstitution of the activation activation by stromelysin (8). Thus, the effect of TIMP-1 on reaction with purified components. the membrane-dependent activation is apparently due to the formation of an inhibitory complex with the partially actiAcknowledgments-We thank Dr. Arthur Eisen for critical review vated 64-kDa intermediate, which prevents its autocatalytic of the manuscript, Teresa Genrich for technical assistance, and Ginger Roberts for preparation of the manuscript. conversion.

Membrane Activation

of 72-kDA Type IV Collagenase

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