From the Strangeways Research Laboratory, Worts'
Causeway, Cambridge CB1 4RN and School of Biological Sciences,
University of East Anglia, Norwich NR4 7TJ, United Kingdom,
§ InVitek GmbH, Robert-Roessle-Strasse 10, D-13125
Berlin-Buch, Germany, ¶ Chugai Pharmaceuticals Co. Ltd., 135 Komakado, 1 Chome, Gotemba-Shi, Shizuoka 412, Japan,
Celltech
Therapeutics Ltd., 216 Bath Road, Slough SL1 4EN, United Kingdom,
** British Biotech Pharmaceuticals Ltd., Watlington Road, Oxford OX4
5LY, United Kingdom, and
INSERM U296,
Faculté de Médecine, Avenue du Général Sarrail,
94010 Créteil, France
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ABSTRACT |
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We have used C-terminal domain mutants to further define the role of interactions of progelatinase A and membrane type 1 matrix metalloproteinase (MT1 MMP) in the binding of TIMP2 and in the cell-associated activation of progelatinase A. Soluble constructs of MT1 MMP were used to demonstrate that binding with TIMP2 occurs primarily through N-terminal domain interactions, leaving the C-terminal domain free for interactions with progelatinase A. The rate of autolytic activation of progelatinase A initiated by MT1 MMP cleavage could be potentiated by concentration of the proenzyme by binding to heparin. Residues 568-631 of the progelatinase A C-terminal domain are important in formation of the heparin binding site, since replacement of this region with the corresponding stromelysin-1 sequence abolished binding to heparin and the potentiation of activation. The same region of gelatinase A was required for binding of latent and active enzyme to TIMP2, but residues 418-474 were not important. A similar pattern was seen using cell membrane-associated MT1 MMP; residues 568-631 were required for binding and activation of progelatinase A, whereas residues 418-474 were not. Neither region was required for activation in solution. The addition of TIMP2 to HT1080 membrane preparations expressing MT1 MMP, but depleted of endogenous TIMP2, resulted in potentiation of progelatinase A activation. This effect was dependent upon TIMP2 binding to MT1 MMP rather than at an independent membrane site. Together, the data suggest that TIMP2 forms a receptor with MT1 MMP that regulates the concentration and efficient generation of functionally active gelatinase A.
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INTRODUCTION |
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The activation of MMPs1 by sequential proteolysis of the propeptide blocking the active site cleft is regarded as one of the key levels of regulation of these proteinases (1, 2). The activation of progelatinase A (MMP2) has proved particularly enigmatic since early studies indicated that it cannot be efficiently activated by plasmin, in contrast to other MMPs including stromelysin 1, collagenase 1, and gelatinase B (3). Treatment of progelatinase A with organomercurials initiates autolytic cleavages, leading to activation, as does proteolysis by either matrilysin or collagenase (4-6). At high concentrations, progelatinase A also self-activates in a process that is concentration-dependent, and the rate of activation can be enhanced by the presence of heparin at low ionic strength (7). Truncated forms of progelatinase A lacking the C-terminal domain are also activated by all the above mechanisms, but when no C-terminal domain is present, self-cleavages are not accelerated in the presence of heparin (7, 8). This implies a role for the C-terminal domain in the concentration of proenzyme by binding to heparin.
Searches for potential physiological activation mechanisms for progelatinase A have implicated a role for a membrane-mediated process involving certain types of activated cell (9, 10). This has led to the identification of the membrane-associated MT MMPs as potential mediators of progelatinase A activation (11-14). Many laboratories noted that activation of progelatinase A at the cell surface requires the C-terminal domain of progelatinase A (8, 15, 16). It was shown that MT1 MMP expressed at the surface of cells lacks the propeptide and is largely associated with TIMP2 (17, 18). The TIMP2 has no inhibitory capacity, which is likely to be due to an interaction of the N-terminal domain with the catalytic domain of MT1 MMP. Progelatinase A can bind to TIMP2 by interactions between the C-terminal domain of the enzyme and the C-terminal three loops and charged tail of the inhibitor (19). The problem has arisen as to how to unequivocally identify this mechanism of progelatinase A binding to the cell surface as a prerequisite for its efficient activation. Data suggest that TIMP2 is critical for the cellular activation process; too little and activation is compromised, but in the presence of excess TIMP2, there is no free MT1 MMP available to initiate progelatinase A activation. It has been clearly established, however, that MT1 MMP can cleave the propeptide of progelatinase A at Asn-Leu38, a process that is not inhibited by TIMP1 (15, 20). The efficiency of this activation process depends upon the concentration of gelatinase A present, since this effects the final cleavage at Asn-Tyr81. Soluble forms of MT1 MMP have been prepared, and their ability to initiate this two step activation of progelatinase A has been confirmed (20, 21). In solution, this process is apparently not dependent upon TIMP2, which merely functions as an inhibitor if it is introduced into the system, nor does it require the presence of the C-terminal domain of progelatinase A. Since most cell-based studies of activation, namely propeptide processing, have been carried out using zymography as an assay, it is not clear to what extent functionally active gelatinase A is being generated. To resolve the differences in the data derived from the study of progelatinase A activation by membrane-associated versus soluble MT1 MMP, we have addressed the questions of the precise kinetics and concentration dependence of these events. We have also assessed the role of the C-terminal domain of progelatinase A in both the activation process and TIMP2 binding to determine whether they are related.
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EXPERIMENTAL PROCEDURES |
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Materials-- All chemicals were obtained from Sigma, ICN, or Pierce. Molecular biology enzymes and buffers were obtained from Promega or Stratagene. CT1746 (N1-(1-(S)-carbamoyl-2,2-dimethylpropyl)-N4-hydroxy-2-(R)-[3-(4-chlorophenyl)propyl]succinamide) was kindly donated by Dr. A. Docherty (Celltech Ltd., Slough, United Kingdom (UK)). The quenched fluorescent peptide (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-[3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-Ala-Arg-NH2 and standard (7-methoxycoumarin-4-yl)acetyl-NH2 were made by Dr. G. Knight (Strangeways Research Laboratory, UK).
Production of Enzymes and Inhibitors--
The catalytic domain
of MT1 MMP (269-559; MT1 MMPcat) was prepared as
described previously (20). A version of MT1 MMP with the transmembrane
and cytoplasmic domains deleted (
501-559;
TM MT1 MMP) was
produced.2 Progelatinase A,
the N-terminal domain of progelatinase A (
418-631; N-GLA) and
N-GL.C-SL (gelatinase A residues 1-417 fused with stromelysin-1 residues 248-460) were prepared as described previously (8, 22). The
C-terminal domain of gelatinase A (
1-414; C-GLA) was purified as
before (16). TIMP2, (
186-194)TIMP2, and (
128-194)TIMP2 were
prepared as described (23). The catalytically inactive mutant of
gelatinase A, E375A-progelatinase A, was prepared as detailed (24).
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Preparation of Membrane-associated Active MT1 MMP-- HT1080 fibrosarcoma cells were transfected with wild-type MT1 MMP using the HCMV/gpt vector, pGWIHG (26), and overexpressing clones were selected with mycophenolic acid. The highest MT1 MMP-expressing clone was expanded and cultured in the presence of 1 µM CT1746, a peptide hydroxamate inhibitor of metalloproteinases, to deplete endogenous TIMP2 binding to cell membrane-associated MT1 MMP. Crude plasma membranes were prepared as described (10), but with extra washes to remove the CT1746. Membranes were resuspended in buffer (20 mM Tris-HCl, pH 7.8, 10 mM CaCl2, 0.025% Brij 35, 0.02% sodium azide), containing proteinase inhibitors, phenylmethanesulfonyl fluoride (100 µM), pepstatin (1 µg/ml), and L-transexpoxysuccinic acid (1 µg/ml).
NS0 mouse myeloma cells were stably co-transfected with MT1 MMP and TIMP2 using the method described previously for progelatinase A (25). Positive clones were identified by reverse gelatin zymography for the presence of TIMP2 and for their ability to activate exogenously added progelatinase A. Crude plasma membranes were prepared from the positive clones using the method described previously (10).Binding of Proenzymes to TIMP2-- Two µg of each proenzyme was incubated for 1-2 h at room temperature with 1 µg of TIMP2 in TCAB buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaC12, 0.025% Brij 35, 0.02% sodium azide) containing 0.15 M NaCl (TCABN). Samples were then loaded onto gelatin-Sepharose, and columns were washed with the same buffer. Bound material was eluted using TCABN containing 15% v/v dimethyl sulfoxide and analyzed by SDS-PAGE with silver staining, and activity was measured using the rabbit collagenase diffuse fibril assay (27).
Activation of MT1 MMP--
The catalytic domain of MT1 MMP (MT1
MMPcat) was activated at 30 µg/ml with 5 µg/ml trypsin
(20). TM MT1 MMP was activated with 1:20 w/w active MT1
MMPcat in FAB buffer (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM CaCl2, 0.05%
Brij 35) for 16 h at 25 °C.
Titrations-- TIMP2 preparations were titrated against gelatinase A immediately following titration of the enzyme against a TIMP1 standard of known concentration (23). All fluorimetric analyses were carried out in a Perkin Elmer LS50B spectrofluorimeter using 1 µM quenched fluorescent peptide substrate (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-[3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-Ala-Arg-NH2 (28). The machine was calibrated using a known concentration of the standard (7-methoxycoumarin-4-yl)acetyl-NH2.
Estimation of Association Rate Constants-- Association rate constants (kon) were estimated from progress curves obtained after the addition of TIMP2 to the enzyme-substrate reaction with appropriate concentrations of enzyme and inhibitor at 25 °C for gelatinase and 37 °C for MT1 MMP using published equations (22, 23) and either Enzfitter (Biosoft) or Grafit (Erithacus Software).
Activation of Progelatinase A and C-terminal Domain Mutants in
Solution--
Proenzyme concentrations were estimated by activating
each enzyme at 100 µg/ml for 4 h at 25 °C with 2 mM APMA, ensuring complete activation (confirmed by
SDS-PAGE and silver staining). The rate of cleavage of 1 µM fluorescent peptide was measured, and the enzyme
concentration was calculated using a
kcat/Km value of 6.29 × 105 M1·s
1 (28)
for each of the gelatinase constructs (since the catalytic domain is
identical in each and the hemopexin domain is not involved in the
hydrolysis of peptide substrates; Ref. 8). Equimolar amounts of each
proenzyme were activated at 37 °C in FAB buffer, either with 2 mM APMA or with MT1 MMP at molar ratios detailed in the
text. Heparin was included in some cases and is described under
"Results." Aliquots of the activation mixture were removed at
intervals and diluted into ice-cold FAB buffer. Activity was measured
against the fluorescent peptide substrate at 37 °C, or aliquots were
used for SDS-PAGE analysis.
Binding of Enzymes to Heparin-Agarose--
One to 2 µg of each
proenzyme was loaded onto heparin-agarose in TCAB, and columns were
washed extensively using the same buffer. Bound material was eluted
using a higher salt buffer and analyzed by SDS-PAGE (gelatin zymography
or silver stain). Binding of both pro and active forms of TM MT1 MMP
or MT1 MMPcat was tested in TCAB. Protein was detected by
Western blotting with a sheep polyclonal antibody to MT1
MMP.3
Activation of Progelatinase A and C-terminal Domain Mutants by
Membrane-bound MT1 MMP--
The amount of active MT1 MMP present on
the membranes was assessed using the quenched fluorescent peptide assay
and the concentration was calculated using a
kcat/Km value of 1.9 × 105 M1·s
1
obtained for soluble
TM MT1 MMP (this study). The activity could be
inhibited by TIMP2, but not by TIMP1, and was attributed to MT1 MMP
alone. NS0 cells do not produce detectable levels of endogenous TIMP2.
Endogenous levels of MT1 MMP are also negligible (0.03 pmol/mg of
membrane protein). Cotransfection with MT1 MMP and TIMP2 resulted in
approximately 0.10 pmol of active MT1 MMP/mg of membrane protein.
Membranes from HT1080 cells overexpressing MT1 MMP and depleted of
TIMP2 were found to contain no bound gelatinase A by zymographic
analysis, and TIMP2 was below the level of detection of Western
blotting (data not shown). Membrane preparations were incubated with
either progelatinase A or the various mutants at 37 °C, and aliquots
of the incubation were removed at intervals for analysis by gelatin
zymography or in fluorescent peptide assays.
Potentiation of Progelatinase A Activation by TIMP2-- Various molar ratios of TIMP2 were incubated for approximately 1 h at room temperature with TIMP2-depleted HT1080 membranes, containing known amounts of MT1 MMP, in TCAB buffer. Progelatinase A was added, and the reaction was incubated at 37 °C. Aliquots of the reaction were removed at intervals and diluted into ice-cold FAB buffer. Samples were assayed at 37 °C in duplicate for activity against the fluorescent peptide substrate. For zymographic analysis, aliquots of progelatinase A were incubated with HT1080 cell membranes that had been preincubated with TIMP2 as before. After 18 h at 37 °C, the reactions were terminated with non-reducing sample buffer and the samples were analyzed by gelatin zymography.
SDS-PAGE, Western Blots, and Zymography-- These techniques were carried out as described previously (29).
Immunolocalization of Progelatinase A and C-terminal Domain Mutants on Cell Surfaces-- Chinese hamster ovary (CHO) cells were transfected with the cDNA for MT1 MMP under transient expression conditions (29) and incubated with exogenous progelatinase A or the domain mutants; 18 h post-transfection, the cells were washed with serum-free Dulbecco's modified Eagle's medium and then incubated on ice for 30 min in Hanks' balanced salt solution (HBSS) containing 0.5% BSA. After three washes with HBSS/0.5% BSA, the cells were incubated with 5 µg/ml progelatinase A or the domain mutants in HBSS/5% BSA for 1 h on ice. Cell monolayers were extensively rinsed and then fixed with 4% paraformaldehyde prior to immunolocalization of surface-bound progelatinase using a sheep polyclonal antibody to gelatinase A (30). It had previously been established that this antibody recognized the mutant enzyme forms. The distribution of endogenous TIMP2 on the transfected CHO cells was assessed using a sheep polyclonal antibody to TIMP2 (31) and a monoclonal antibody specific to the C-terminal charged "tail" sequence of TIMP2, QEFLDIEDP (kindly donated by Dr. Kazushi Iwata, Fuji Chemical Industries Ltd., Toyama, Japan).
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RESULTS |
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Binding of Progelatinase A and C-terminal Domain Mutants to
TIMP2--
Progelatinase A, proN-GLA and the gelatinase A-stromelysin
hybrids, proN-G.C-SGS and proN-G.C-SGG, each bound to
gelatin-Sepharose. TIMP2 alone did not bind to gelatin-Sepharose;
neither did TIMP2 that had been preincubated with proN-GLA or
proN-G.C-SGS, nor (128-194)TIMP2 that was preincubated with
progelatinase A. Only TIMP2 that was preincubated with progelatinase A
or proN-G.C-SGG was detected in the bound fraction by silver staining
(not shown) and TIMP activity assays (Table
I).
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Association Constants for TIMP2 and Active Gelatinase A or
C-terminal Domain Mutants--
Fluorimetric assays demonstrated that
TIMP2 bound rapidly to gelatinase A (kon
38.0 × 106
M1·s
1; Table
II). The mutant N-G.C-SGG was the only
construct to show a comparable rate of binding (33.2 × 106 M
1·s
1).
Mutants N-G.C-SGS, N-GLA, or N-GL.C-SL bound to TIMP2 between 45 and
200 times more slowly than wild-type gelatinase A. (
186-194)TIMP2, which lacks the charged tail, interacted 5-7-fold
more slowly with wild-type gelatinase A or the mutant N-G.C-SGG.
Removal of the charged tail of TIMP2 had no effect on the already poor
association rate of N-G.C-SGS.
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Association Constants for TIMP2 and MT1 MMP--
The
kcat/Km for TM MT1 MMP
cleavage of the fluorescent peptide at 37 °C and pH 7.5 was
1.92 × 105
M
1·s
1, which is comparable
with the kcat/Km for MT1
MMPcat (20). Estimates of the association constant for wild
type TIMP2, or mutant forms lacking the charged tail (
186-194) or
the C-terminal domain three loops (
128-194) with soluble MT1 MMP
(
TM or catalytic domain) were similar, ranging from 2.8 to 4.2 × 106 M
1·s
1
(Table III).
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Activation of Progelatinase A and C-terminal Domain Mutants in
Solution--
In agreement with previous data comparing the activation
of wild-type progelatinase A and proN-GLA (8) or proN-GL.C-SL (22),
APMA activation of equimolar concentrations of progelatinase A and
C-terminal domain mutants proN-G.C-SGS, proN-G.C-SGG, and proN-GLA was
similar, showing rapid and immediate activation, which was complete by
60 min (Fig. 2). When progelatinase A or the domain mutants were activated under the same conditions with a 10:1
molar ratio of proenzyme:TM MT1 MMP, activation occurred over a 6-h
period and each of the enzymes showed a similar activation profile,
with a lag phase of 1 h and then rapid activation followed by a
plateau (Fig. 3). Similar results were
obtained using MT1 MMPcat (data not shown).
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The Effect of Local Concentrations of MT1 MMP and Progelatinase
A--
We have shown previously that the concentration of
progelatinase A determines its rate of activation by MT MMPs in
solution (32). Here, we investigate the effect of MT1 MMP concentration on progelatinase A activation (Fig. 4).
In the absence of MT1 MMP, there was no detectable activation of the
progelatinase A in the 7-h period studied. With a 6:1 molar ratio of
progelatinase A:TM MT1 MMP, a characteristic sigmoidal activation
curve resulted, where a 30-60-min lag was followed by a phase of rapid
activation and then a plateau. As the molar ratio of progelatinase
A:
TM MT1 MMP was increased to 30:1, 60:1, and 125:1, the rate of
activation decreased, so that after 7 h, the amount of substrate
cleaved was only 43%, 35%, 24% of that cleaved by gelatinase A
incubated in a 6:1 molar ratio with
TM MT1 MMP. The rate of
activation of progelatinase A was similarly dependent upon the relative
concentration of MT1 MMPcat (data not shown), suggesting
that C-terminal domain binding did not contribute to enzyme
interactions.
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Activation of Progelatinase A and C-terminal Domain Mutants by Cell Membranes-- Gelatin zymography of the reaction products from incubations of progelatinase A or the C-terminal domain mutants with isolated NS0 cell membranes containing known amounts of active MT1 MMP (see "Experimental Procedures") revealed that only wild-type progelatinase A and mutant proN-G.C-SGG were activated in a dose-dependent manner by the membrane preparations. Mutant proN-G.C-SGS was hardly processed at all under these conditions (Fig. 7A). In fluorimetric assays, conducted using higher concentrations of progelatinase A, both progelatinase A and proN-G.C-SGG were activated rapidly by membranes derived from NS0 cells cotransfected with MT1 MMP and TIMP2, with maximal activity after 20 h (Fig. 7B). In contrast, after 20 h, proN-G.C-SGS was barely activated. A similar result was obtained using membranes from HT1080 cells transfected with MT1 MMP (data not shown). NS0 cell membranes from vector control transfections did not activate any of the proenzymes in the 24-h period studied.
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Potentiation of Activation by TIMP2--
Membrane preparations
from HT1080 cells were prepared essentially free from endogenous TIMP2
as described under "Experimental Procedures." Membranes from HT1080
cells transfected with vector alone contained 0.13 pmol of active MT1
MMP/mg of membrane protein, (estimated as described under
"Experimental Procedures"), whereas those transfected with MT1 MMP
cDNA contained approximately 1.52 pmol/mg of membrane protein.
Equal amounts of each membrane preparation were preincubated with 0, 1 nM, or 4 nM titrated TIMP2 (giving a molar
ratio of 1:0, 2:1, and 1:2 for MT1 MMP:TIMP2 for the membranes derived
from cells overexpressing MT1 MMP). Progelatinase A was added, and
activation was monitored using the fluorescent peptide assay. After
6.25 h, in the absence of added TIMP2, the membranes derived from
HT1080 cells transfected with MT1 MMP processed the progelatinase A to
give an activity of 12.1 pM product·s1
(Fig. 8A). When TIMP2 was
added in a 2:1 MT1 MMP:TIMP2 molar ratio, potentiation of gelatinase A
activation was observed, resulting in an activity of 18.3 pM product·s
1. Progelatinase A activation
was inhibited when TIMP2 was added in a ratio of 1:2 MT1 MMP:TIMP2.
Results from a similar experiment are also shown using zymography (Fig.
8B). The control membranes from HT1080 cells transfected
with vector DNA activated the progelatinase A to give 5.2 pM product·s
1, probably reflecting the
endogenous MT1 MMP activity. However, when 1 nM or 4 nM TIMP2 was added, inhibition of activation was observed
in both cases. Membrane preparations (data not shown) or progelatinase
A incubated alone had no detectable activity against the fluorescent
substrate. When (
128-194)TIMP2 was added to the membranes at the
same molar ratios, progelatinase A activation was inhibited (data not
shown); no potentiation of activation was seen at any
concentration.
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Immunolocalization of Progelatinase A and C-terminal Domain Mutants on Cell Membranes-- The binding of wild-type progelatinase A and the C-terminal domain mutants proN-G.C-SGG and proN-G.C-SGS to CHO cells, transiently transfected with vector alone or with vector containing MT1 MMP cDNA, was studied using immunolocalization techniques (Fig. 9). CHO cells synthesize negligible amounts of progelatinase A or MT1 MMP, but they normally secrete significant levels of TIMP2 into the culture medium. No TIMP2 was bound to the surface of CHO cells transfected with vector alone. However, when CHO cells were transiently transfected with MT1 MMP (10-20% of cells were positively transfected), TIMP2 was detected bound at the cell surface, using either a polyclonal antibody against TIMP2 or a monoclonal antibody raised against a C-terminal peptide of TIMP2. When wild-type progelatinase A or the mutant proN-G.C-SGG were added to the system, they could be detected, using a polyclonal antibody to progelatinase A, on the surface of the cells expressing MT1 MMP (panels A and B), but not on cells transfected with vector alone. ProN-G.C-SGS (or proN-GLA and proN-GL.C-SL; data not shown) did not bind to MT1 MMP-transfected cells (panel C). The binding of progelatinase A or proN-G.C-SGG to the cells abolished the signal from the antibody directed against the C-terminal epitope of TIMP2 (panels G and H), but the binding of the polyclonal antibody to the whole TIMP2 molecule was unaffected (panels D, E, and F).
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DISCUSSION |
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Previous studies have shown that the C-terminal domain of gelatinase A is required for binding of the proenzyme to TIMP2 (19, 22, 23, 33) and is also crucial for activation of progelatinase A by cell membranes (8, 15, 16). The C-terminal domains of gelatinase A and stromelysin-1 show >50% identity, but in stromelysin-1 this region does not interact significantly with TIMP2 (22). A mutant with the C-terminal domain of gelatinase A replaced with the corresponding region from stromelysin-1, N-GL.C-SL, was also not activated by cell membranes.4 To localize more precisely the regions of the C-terminal domain of gelatinase A involved in TIMP2 binding and cellular activation, we constructed further subdomain-exchange mutants.
The binding of TIMP2 to progelatinase A required the C-terminal region of both the inhibitor and proenzyme as demonstrated previously (8) and substitution of the C-terminal domain of gelatinase A with that of stromelysin-1 abolished binding of proN-GL.C-SL to TIMP2. TIMP2 was able to bind to proN-G.C-SGG, where residues 418-474 of gelatinase A have been replaced with stromelysin-1 sequence, but was unable to bind to proN-G.C-SGS, where there is an additional replacement of residues 568-631. A similar effect of these C-terminal domain mutations was seen on the rate of association of TIMP2 with the active enzymes, i.e. only N-G.C-SGG showed a rate of binding to TIMP2 comparable to that for wild-type gelatinase A.
Our results agree with those of Fridman et al. (33), who
made deletion mutants of gelatinase A (426-631 and
455-631). The proform of these mutants did not bind TIMP2 and the active enzyme
showed decreased inhibition by TIMP2. However, our domain-swap mutants
not only preserve the C-terminal disulfide bond (data not shown), which
is thought to be essential for integrity (16), but also allow us to
more precisely define the site of interaction with TIMP2. Our results
suggest that the region between residue 568 and the C-terminal residue
631 is required for formation of the TIMP2 binding site, whereas
residues 418-474 do not interact significantly with TIMP2. The x-ray
crystal structure of the C-terminal domain of gelatinase A predicts a
disc-shaped four-bladed propeller (34, 35). The region implicated here
(residues 568-631) consists of part of blade 3 and all of blade 4. Electrostatic interactions between the C-terminal domain of TIMP2 and
that of gelatinase A are believed to be responsible for an initial
docking, which aligns the inhibitory domain of TIMP2 with the catalytic
domain of the enzymes, increasing the rate of inhibition (22, 23). A
surface patch of positively charged residues around lysine residues 566, 567, and 568 (the latter is replaced in mutant N-G.C-SGS by a
serine residue) may be involved in binding TIMP2, in particular, the
negatively charged C-terminal tail. Our immunolocalization studies
support this proposal, since the binding of gelatinase A to cell-bound
TIMP2 blocked binding of the anti-TIMP2 C-terminal peptide monoclonal
antibody.
We then compared the association of TM MT1 MMP and MT1
MMPcat with TIMP2 and C-terminally truncated forms of
TIMP2, to determine which interactions are likely to occur in
vivo. The similarity of the association rates for all combinations
of full-length and truncated proteins suggests that only N-terminal
domain interactions occur between soluble MT1 MMP and TIMP2. Other
evidence suggests that this interaction is important at the cell
surface; binding of TIMP2 to HT1080 cell membranes is abolished by
excess N-terminal domain of TIMP2, and the activation of progelatinase
A is abolished by antibodies against the N-terminal domain of TIMP2
(17, 36). The role of the C-terminal domain of MT1 MMP is unclear;
however, in vitro, it has been shown to be important for
specific cleavage of type I collagen.2 This domain appears
to be responsible for the binding of active
TM MT1 MMP to
heparin.
The data presented here suggest that interactions occur between the C-terminal domain of TIMP2 and the C-terminal domain of progelatinase A and between the N-terminal domain of MT1 MMP and the N-terminal domain of TIMP2. This is consistent with the theory that a ternary complex between MT1 MMP, TIMP2, and progelatinase A can form on the cell surface (17, 18, 37). In agreement with this, an E375A-progelatinase A·TIMP2 complex was able to inhibit MT1 MMPcat in a fluorimetric assay,5 presumably due to the formation of a ternary complex as described by Kolkenbrock and colleagues (37-39).
In these experiments, we attempt to establish whether TIMP2 is involved
in the activation of progelatinase A at the cell surface by MT1 MMP. In
solution, the activation of progelatinase A and C-terminal domain
mutants by APMA was virtually identical. This is in accordance with
previous studies, which suggest that the C-terminal domain of
gelatinase A is not involved in catalysis (16, 22, 33). Similarly,
progelatinase A and the mutants were activated equally well by soluble
TM MT1 MMP or MT1 MMPcat. Thus, in solution, the
activation of progelatinase A by MT1 MMP does not require the
C-terminal domain of the proenzyme or of MT1 MMP and is efficient even
in the absence of TIMP2.
The heparin binding site in the C-terminal domain of gelatinase A has
not previously been identified (7, 40). Other than C-GLA and
progelatinase A, N-G.C-SGG was the only mutant that bound to
heparin-agarose, suggesting that N-G.C-SGG retains the heparin binding
domain, whereas it is absent or non-functional in the other mutants,
N-G.C-SGS, N-GLA, and N-GL.C-SL. Hence removal of gelatinase A residues
568-631 disrupts the heparin binding site. TM MT1 MMP was
previously purified on heparin-Sepharose (21); in this study, pro and
active
TM MT1 MMP bound to heparin-agarose, whereas active MT1
MMPcat did not. Hence, there appears to be a heparin
binding site in the hemopexin domain of MT1 MMP. We have previously
demonstrated that activation of progelatinase A by MT1 MMP is dependent
upon the concentration of the proenzyme (32). In vitro, the
rate of activation of progelatinase A was also dependent upon the
concentration of MT1 MMP. At low ratios of MT1 MMP:progelatinase A,
e.g. 1:125, activation in solution was very slow, but as the
MT1 MMP concentration was increased, the activation rate increased. The
rate of activation of progelatinase A by MT1 MMP was increased by
heparin. Although both
TM MT1 MMP and progelatinase A bind to
heparin, experiments with MT1 MMPcat and
proE375A-gelatinase A suggest that the major effect of heparin was
exerted on the autolytic step. Hence, the two-step activation process
described by Will et al. (20) is likely to be critically dependent upon the concentration and colocalization of both
components.
In contrast to the activation of progelatinase A and the C-terminal
domain mutants in solution, there were differences when the mutant
proenzymes were exposed to membrane preparations from NS0 or HT1080
cells displaying MT1 MMP. Wild-type progelatinase A and proN-G.C-SGG
were efficiently activated by these membrane preparations, whereas the
mutant proN-G.C-SGS was not. An identical amount of membranes from NS0
cells transfected with vector DNA did not activate any of the
proenzymes, indicating a requirement for MT1 MMP for activation of
progelatinase A. We have demonstrated by immunolocalization that TIMP2
is bound to CHO cells transiently expressing MT1 MMP, but not to those
transfected with vector DNA alone, suggesting that TIMP2 binds to MT1
MMP in transiently expressing cells. Progelatinase A and proN-G.C-SGG,
but not proN-G.C-SGS, can bind to CHO cells that are transfected with
MT1 MMP, whereas none of the proenzymes bind to cells transfected with
vector alone. The pattern of binding of the gelatinase A C-terminal
domain mutants to CHO cells expressing MT1 MMP and their activation by
NS0 and HT1080 cell membrane preparations containing MT1 MMP correspond to their propensity to bind to TIMP2, i.e. the region
between residues 568 and 631 in gelatinase A is required, whereas
residues 418-474 do not play a significant role. Thus, it is likely
that the proenzymes are localized on the cell surface via TIMP2. These results indicate that, although TIMP2 in the HT1080 membranes was below
the level of detection by Western blotting, sufficient TIMP2 remained
to account for the differences in the efficiency of activation of the
mutants. It is also possible that activation occurs in the absence of
TIMP2 due to the presence of TIMP3 (which can bind to both MT1 MMP (20)
and progelatinase A)6 or
heparan sulfate proteoglycans which might localize progelatinase A at
the cell surface. The integrin, v
3, has
also been reported to act as a receptor for gelatinase A (41), although
in our laboratory no significant interaction has been detected in the cellular systems under
study.7 Progelatinase A
binding to and activation by membrane preparations or cells that were
transfected with vector DNA rather than MT1 MMP did not occur,
suggesting that MT1 MMP is the only membrane protein required for the
interaction with TIMP2 and progelatinase A. The involvement of both
TIMP2 and MT1 MMP in the activation process is further substantiated by
the potentiation of the activation of progelatinase A after the
addition of TIMP2 to TIMP2-depleted membranes. The addition of
increasing amounts of TIMP2 to a fixed concentration of MT1 MMP in a
membrane preparation derived from HT1080 cells overexpressing MT1 MMP
resulted in potentiation at low levels of TIMP2, but then inhibition of
progelatinase A activation at the highest concentrations of TIMP2. We
propose that this is due to the formation of additional receptors for
progelatinase A composed of MT1 MMP and TIMP2 and that activation is
inhibited when enough TIMP2 is added to bind and inhibit all the free
MT1 MMP. Membranes from HT1080 cells transfected with vector alone did
contain some endogenous MT1 MMP but the level was 10 times lower than
the MT1 MMP overexpressing cells. Progelatinase A activation was
inhibited in all cases by the same concentrations of TIMP2, which were
able to potentiate activation in the membranes from cells transfected
with MT1 MMP. Since the same concentrations of membrane proteins were
present in each experiment, the difference in progelatinase A
activation can be attributed directly to the difference in MT1 MMP
concentration. (
128-194)TIMP2 was inhibitory at all concentrations,
possibly reflecting its ability to inhibit MT1 MMP and active
gelatinase A but its failure to form a progelatinase A "receptor."
Since the balance between the amount of "free" MT1 MMP and MT1
MMP·TIMP2 complex appeared to determine the degree of activation of
gelatinase A, we carried out a further experiment where the level of
free MT1 MMP remained constant and increasing levels of MT1 MMP
membranes:TIMP2 were added. As expected, molar ratios of MT1 MMP:TIMP2
to free MT1 MMP up to 3:2 resulted in a potentiation of progelatinase A
activation consistent with the idea of extra receptors being formed. As
a consequence, more progelatinase A is concentrated in the vicinity of
the free MT1 MMP; therefore, the two-step cleavage of progelatinase A,
initiated by MT1 MMP (15, 20), is enhanced. When higher molar ratios of
MT1 MMP membranes:TIMP2 were added to MT1 MMP, no potentiation was
observed. In this case, many of these receptors may be distant from
active MT1 MMP molecules so that an inactive ternary complex with
progelatinase A persists; this is in accordance with the findings of
Strongin et al. (17).
In contrast to previous studies that have been analyzed by zymography, this kinetic study has allowed us to precisely measure the amount of gelatinase A activity generated. The results described in this paper suggest that a crucial step in the cellular activation of progelatinase A is its concentration. Here, we have shown that the activation of progelatinase A by membrane-bound MT1 MMP is dependent upon binding of the C-terminal domain of the proenzyme to TIMP2. TIMP2 is able to bind to the N-terminal domain of MT1 MMP, suggesting that the C-terminal domain interaction with progelatinase A acts to colocalize and concentrate the progelatinase A in the vicinity of active MT1 MMP molecules. Until now, the role of TIMP2 in the activation of progelatinase A at the cell surface has been unclear, and the possibility that a multicomponent system was required in which MT1 MMP, TIMP2 and progelatinase A interact with other unknown membrane components had not been ruled out (42). We found no evidence for the involvement of membrane proteins other than MT1 MMP. Our studies suggest that the amount of TIMP2 present determines the balance between the level of activator (free MT1 MMP) and of receptor (MT1 MMP·TIMP2 complex), which regulates the degree of activation of progelatinase A. Hence, TIMP2 is novel in its dichotomous mode of action. On the one hand, TIMP2 acts as a specific inhibitor of metalloproteinases, while on the other, it is intricately involved in the mechanism of progelatinase A activation. It is anticipated that other members of the TIMP family such as TIMP3 and TIMP4 may also have as yet undiscovered functions.
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ACKNOWLEDGEMENTS |
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We thank Mary Harrison for technical assistance, Kazushi Iwata for antibodies, Ros Hembry and Heather Stanton for antibody preparation and characterization, Frances Willenbrock for valuable advice, and Julie Parsison and Linda Thrift for preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust, the Arthritis and Rheumatism Council, the Medical Research Council (United Kingdom), the Deutsche Forschungsgemeinschaft (Germany), and INSERM (France).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§§ To whom correspondence should be addressed: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. Tel.: 44-1603-456161; Fax: 44-1603-592250; E-mail: g.murphy{at}uea.ac.uk.
1
The abbreviations used are: MMP, matrix
metalloproteinase; MT1 MMP, membrane type 1 matrix metalloproteinase;
MT1 MMPcat, catalytic domain of MT1 MMP (269-559);
TM MT1 MMP, MT1 MMP lacking the transmembrane and cytoplasmic
domains (
501-559); TIMP, tissue inhibitor of metalloproteinases;
N-GLA, N-terminal domain of gelatinase A (
418-631); C-GLA,
C-terminal domain of gelatinase A (
1-414); N-GL.C-SL, gelatinase A
(residues 1-417) fused to stromelysin-1 (residues 248-460);
N-G.C-SGG, gelatinase A (residues 1-417) fused to stromelysin-1
(residues 248-305) then gelatinase A (residues 475-631); N-G.C-SGS,
gelatinase A (residues 1-417) fused to stromelysin-1 (residues
248-305) followed by gelatinase A (residues 475-567) and
stromelysin-1 (residues 400-460); E375A-gelatinase A, inactive mutant
with active site Glu mutated to Ala; (
128-194)TIMP2, N-terminal three loops of TIMP2; (
186-194)TIMP2, TIMP2 lacking the C-terminal charged tail QEFLDIEDP; APMA, 4-aminophenylmercuric acetate; PAGE, polyacrylamide gel electrophoresis; HBSS, Hanks' balanced saline solution; BSA, bovine serum albumin; CHO, Chinese hamster ovary.
2
d'Ortho, M.-P., Will, H., Atkinson, S., Butler,
G., Messent, A., Gavriloi, J., Smith, B., Timpl, R., Zardi, L.,
and Murphy, G. (1997) Eur. J. Biochem., in press.
3 M.-P. d'Ortho and R. M. Hembry, manuscript in preparation.
4 M. Cockett and G. Murphy, unpublished observation.
5 G. Butler and G. Murphy, unpublished results.
6 G. Butler and G. Murphy, unpublished observation.
7
A. J. Messent, G. Murphy, and J. Gavrilovi, unpublished observations.
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REFERENCES |
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