From the School of Biological Sciences, University of
East Anglia, Norwich, Norfolk NR4 7TJ, United Kingdom, the
§ Department of Biomedical Engineering, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, and the
¶ Laboratory of Structural and Mechanistic Enzymology, Department
of Biochemistry, Queen Mary and Westfield College, University of
London, London E1 4NS, United Kingdom
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ABSTRACT |
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We compared the association constants of tissue
inhibitor of metalloproteinases (TIMP)-3 with various matrix
metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous
assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid
association with gelatinases A and B. Experiments with the N-terminal
domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A
makes an important contribution to the interaction with TIMP-3. The
exchange of portions of the gelatinase A hemopexin domain with that of
stromelysin revealed that residues 568-631 of gelatinase A were
required for rapid association with TIMP-3. The N-terminal domain of
gelatinase B alone also showed slower association with TIMP-3, again
implying significant C-domain interactions. The isolation of complexes
between TIMP-3 and progelatinases A and B on gelatin-agarose
demonstrated that TIMP-3 binds to both proenzymes. We analyzed the
effect of various polyanions on the inhibitory activity of TIMP-3 in
our soluble assay. The association rate was increased by dextran
sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or
hyaluronic acid. Because TIMP-3 is sequestered in the extracellular
matrix, the presence of certain heparan sulfate proteoglycans could
enhance its inhibitory capacity.
The tissue inhibitors of metalloproteinases
(TIMPs)1 are specific protein
inhibitors of the matrix metalloproteinases (MMPs), a group of
zinc-dependent enzymes that include collagenases,
gelatinases, and stromelysins. Four forms of human TIMP have been
cloned: TIMP-1 (1), TIMP-2 (2), TIMP-3 (3-6), and, more recently,
TIMP-4 (7). TIMP-1 and TIMP-2 are secreted by many cell types in
culture and are found in body fluids and tissue extracts. TIMP-3 is
unique in that it appears to be a component of the extracellular matrix (8-10) and occurs in relatively small amounts, possibly being expressed during specific cellular events (11).
The TIMPs have comparable abilities to inhibit the active forms of the
MMPs when assessed using macromolecular substrates (12, 13) and have
been shown to make tight binding noncovalent complexes with active MMPs
with a 1:1 stoichiometry (14-17). The inhibitors have related primary
and secondary structures, consisting of an N-terminal subdomain of
three disulfide bonded loops and a smaller C-terminal region also
containing three loops (18-20). The N-terminal domain of TIMP-1 and
TIMP-2 can act as a functional inhibitor (19, 21, 22), interacting with
the catalytic domain of the enzymes such that competition with low
molecular weight substrate analogue inhibitors can be observed
(23).2 Using peptide
substrate assays, it has been possible to demonstrate that TIMP-MMP
complexes interact with Ki values of
10 In this study, we have assessed the ability of TIMP-3 to associate with
active MMPs using a kinetic method, and we have compared this with
TIMP-1 and TIMP-2. We have also investigated the contribution of the
C-terminal domains of both gelatinase A and gelatinase B to the
interaction with TIMP-3, because this has important implications for
the regulation of proenzyme activation. We have tested the effect of
heparin and other polyanions on TIMP-3 activity in our soluble kinetic
assay to determine whether interaction with similar components of the
extracellular matrix could affect the capacity of TIMP-3 to inhibit MMPs.
Materials--
All chemicals and reagents were purchased from
Sigma, ICN Flow, or Pierce unless stated otherwise. Quenched
fluorescent peptides (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) and
(7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Ala-Norval-(3-(2, 4-dinitrophenyl)-L-2,3-diaminopropionyl)-Ala-Arg-NH2
(Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2) were made
by Dr C. G. Knight (Biochemistry Department, University of
Cambridge, Cambridge, United Kingdom). The following polyanions were
purchased from Sigma: heparin (porcine intestinal mucosa, H3149);
de-N-sulfated heparin (porcine intestinal mucosa, D4776); heparan
sulfate (bovine kidney, H7640; bovine intestinal mucosa, H7641);
hyaluronic acid (human umbilical cord, H1504); dermatan sulfate
(chondroitin sulfate B; bovine mucosa, C0320); dextran sulfate (average
Mr 10,000; D6924).
Preparation of TIMPs and Gelatinases--
TIMP-1, TIMP-2, and
TIMP-3 were expressed from NS0 myeloma cells and purified as described
previously (13, 19, 20). Progelatinase A, ( Kinetic Studies--
Active enzymes were active site titrated
against a standard preparation of TIMP-1 (20). TIMP-2 and TIMP-3 were
active site titrated with stromelysin-1 that had been titrated against
the standard TIMP-1. Assays were performed at 25 °C for gelatinase A
and gelatinase B or at 37 °C for stromelysin-1 and matrilysin in a
buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM CaCl2, and 0.05%
Brij 35 (fluorometry buffer). Hydrolysis of 1 µM
substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 for the
gelatinases and matrilysin or
Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2 for stromelysin-1 was
followed using a Perkin Elmer LS 50B fluorescence spectrometer (20, 25,
38). Inhibition of the matrix metalloproteinases by TIMPs was analyzed
under pseudo-first-order conditions using suitable ratios of
enzymes:inhibitors as described previously (20, 25). Association rate
constants (kon) were estimated from the progress
curves using published equations (20, 25) and the Enzfitter (Biosoft)
or Grafit (Erithacus Software) program. The effect of ionic strength
was analyzed by increasing the concentration of NaCl in the standard
buffer from 0.1 M to 0.25 M and 0.5 M. For competition assays, various concentrations of
( Binding of TIMP-3 to Progelatinases--
TIMP-3 was incubated in
the presence or absence of progelatinases in TCABN for 1-2 h at
25 °C. Complexes with progelatinases were isolated on
gelatin-Sepharose that had been blocked with 0.2 mg/ml bovine serum
albumin in TCABN. The column was washed with TCABN, and bound material
was eluted with TCABN containing 15% dimethyl sulfoxide. Eluates were
analyzed by rabbit collagenase diffuse collagen fibril assays (39) and
reverse zymography (40).
Binding of TIMPs to Heparin-Agarose--
Approximately 1 µg of
each TIMP was applied to heparin-agarose (blocked with 0.2 mg/ml bovine
serum albumin) in TCABN buffer. Columns were washed with TCABN, and
proteins were eluted stepwise with the same buffer containing 0.5 M NaCl and then 2 M NaCl. Bound and unbound
fractions were analyzed for TIMP content by SDS-polyacrylamide gel
electrophoresis and silver staining and by rabbit collagenase diffuse
collagen fibril assay (39).
Deglycosylation of TIMPs--
5 µg of TIMP-3 or TIMP-1 were
incubated for 4 h at 37 °C in the presence or absence of 1250 units of PNGase F (New England Biolabs). TIMPs were diluted in
fluorometry buffer and used in assays as above.
We analyzed the inhibition of active gelatinase A, gelatinase B,
stromelysin-1, and matrilysin by TIMP-3 using continuous fluorometric
assays with the appropriate fluorescent peptide substrate (see
"Experimental Procedures"). As discussed previously for TIMP-1 and
TIMP-2 (20, 25), we were unable to obtain accurate values of
Ki (<200 pM). Our measurements were
therefore limited to the association rate constants
(kon) at low reagent concentrations, over a
range where the observed rate was linear with TIMP concentration. In
Table I, the data are compared with
kon values for TIMP-2 that were re-assayed at
the same time and kon values for TIMP-1 derived
from our previous work (25, 26). All three TIMPs bound relatively
slowly to stromelysin-1 and matrilysin. In general, we found that
TIMP-3 was more like TIMP-2 than TIMP-1, showing rapid binding to
gelatinase A and slower association with gelatinase B. The contribution
of the C-terminal domains of gelatinase A and gelatinase B to TIMP-3
binding was assessed by measuring the association rate of the isolated
catalytic domains, (
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9 to 10
12 M (24). Comparative
studies of the association rates of TIMP-1 and TIMP-2 with different
members of the MMP family in our laboratory have shown exceptionally
strong C-terminal domain interactions between TIMP-1 and gelatinase B
and between TIMP-2 and gelatinase A, suggesting that complexes between
the respective pro forms of these enzymes, the active sites of which
are inaccessible, and inhibitors can also occur (20, 25, 26). This
supports other biochemical studies of these complexes (27-30).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-414)gelatinase A,
pro(
418-631)gelatinase A, and the catalytically inactive mutant
proE375A-gelatinase A were prepared as described previously (20,
31-33). Progelatinase B and pro(
426-688)gelatinase B were prepared
as described in Ref. 26. Stromelysin-1 and matrilysin were prepared as
recombinant pro forms as described previously (34, 35). N-GL.C-SL was prepared as described previously (25). The preparation of gelatinase A
mutants N-G.C-SGG and N-G.C-SGS was as described previously (36).
Gelatinase A and gelatinase A C-terminal domain mutants were activated
at 100 µg/ml with 2 mM 4-aminophenylmercuric acetate for
1 h at 25 °C. Stromelysin-1 was activated with trypsin using the standard method (37). Gelatinase B and (
426-688)gelatinase B
were activated at 2 µM with 0.1 µM active
stromelysin-1 at 37 °C for 2 h. Matrilysin was activated at 22 µg/ml with 1 mM 4-aminophenylmercuric acetate at 37 °C
for 1 h.
1-414)gelatinase A or proE375A-gelatinase A were added to the
cuvette with the gelatinase A before the addition of TIMP-2 or TIMP-3.
Because the Ki values for the TIMP:gelatinase A
interaction are unknown, the Ki and the
Kd for the TIMP:competitor interaction are expressed
as relative values using an arbitrary value of 1 for the
Ki. The relationship between the two dissociation
constants is given in Equation 1:
in which EI and Ef are the
TIMP:gelatinase A complex and free gelatinase A, respectively, whereas
FI and Ff are the TIMP:competitor complex and free competitor, respectively. Equation 1 can be rewritten as:
(Eq. 1)
in which Ft, Et,
and It are total reagent concentrations. In our
assays, Ft
(Eq. 2)
FI, and
If is negligible, so Equation 2 can be simplified to Equation 3, from which the relative Kd can be readily calculated.
The effect of various polyanions on the rate of association was
carried out using a constant amount of enzyme and inhibitor (concentrations similar to those used to calculate the
kon values listed in Table I) with increasing
concentrations of each test polyanion in the fluorometry buffer.
(Eq. 3)
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
418-631)gelatinase A and
(
426-688)gelatinase B. Whereas TIMP-2 binding was only affected by
the loss of the gelatinase A C-terminal domain, TIMP-3 association was
slower in the absence of the C-terminal domains of both
gelatinase A and gelatinase B (1400-fold and 12.5-fold, respectively).
Comparison of the rate constants for the interaction of TIMPs with
matrix metalloproteinases
The effect of ionic strength on the rate of association of gelatinase A
and TIMP-3 was analyzed at increasing NaCl concentrations. Similar to
TIMP-2 (20), there was a marked decrease in kon
from 16.0 × 106
M1·s
1 in 0.1 M
NaCl to 9.6 × 106
M
1·s
1 (0.25 M
NaCl) and 7.3 × 106
M
1·s
1 (0.5 M
NaCl), suggesting that ionic interactions are involved in the
association of gelatinase A and TIMP-3.
The contribution of the C-terminal domain of gelatinase A to TIMP-3
binding was assessed by measuring the effect of adding increasing
amounts of (1-414)gelatinase A (the isolated C-terminal domain) or
proE375A-gelatinase A (an inactive form of progelatinase A) to the
inhibition assay and observing the effect on the association rate for
active full-length gelatinase A. The effect on inhibition by TIMP-2 was
also measured for comparison. The increase in the final steady-state
velocity and the decreased rate of inhibition observed with increasing
concentrations of (
1-414)gelatinase A and proE375A-gelatinase A
were deduced to be due to an effective decrease in TIMP-3 concentration
by binding to the C-terminal domain, as was seen for TIMP-2 (20). The
data were analyzed as described under "Experimental Procedures" to
obtain an estimate for Kd, the dissociation
constant, relative to the Ki for the appropriate
TIMP:gelatinase A interaction (Table II). The interaction of TIMP-3 with (1-414)gelatinase A was significant but
was around 16-fold weaker than the interaction of TIMP-2. The
interaction between TIMP-3 and proE375A-gelatinase A was about five
times weaker than that for TIMP-2. In both cases, the interaction of
the TIMPs with proE375A-gelatinase A was stronger than that with the
isolated C-terminal domain, which suggests that additional sites of
interaction exist in the proenzyme-TIMP complex.
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To further characterize the region of gelatinase A responsible for the
C-terminal domain interaction, we used two C-terminal domain mutants:
regions of the C-terminal domain of gelatinase A were exchanged for the
corresponding regions of the C-terminal domain of stromelysin-1, which
does not interact significantly with the TIMPs (25). As was the case
for TIMP-2 (36), replacement of residues 418-474 in N-G.C-SGG did not
affect the rate of association with TIMP-3 (kon = 17.0 × 106
M1·s
1, compared with
16.5 × 106
M
1·s
1 for gelatinase A).
However, the additional substitution of residues 568-631 in N-G.C-SGS
reduced the rate of association of TIMP-3 with gelatinase A by a factor
of 100 to 0.1 × 106
M
1·s
1, suggesting that
residues 568-631 of gelatinase A are crucial for the interaction with
TIMP-3.
Because the kinetic data suggested that TIMP-3 has significant
interactions with the hemopexin domains of gelatinase A and gelatinase
B, we assessed the ability of TIMP-3 to bind to various pro form
constructs of gelatinases A and B, in which normal catalytic domain
interactions are precluded due to the presence of the propeptide domain
(Table III). A small amount of TIMP-3
alone bound to the gelatin-Sepharose matrix. Enhanced retention of
TIMP-3 was observed after preincubation with progelatinase A or
progelatinase B, suggesting that TIMP-3 shows significant binding to
both proenzymes. TIMP-3 was recovered in the unbound fraction after
incubation with pro(418-631)gelatinase A or
pro(
426-688)gelatinase B. TIMP-3 bound to gelatin-Sepharose after
preincubation with proN-G.C-SGG but did not bind if proN-G.C-SGS or
proN-GL.C-SL were used. TIMP-2 was retained on the gelatin-Sepharose after incubation with progelatinase A but not after incubation with
progelatinase B.
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The Effect of Heparin on the Rate of Association of TIMPs with
Gelatinase A--
Increasing concentrations of heparin in the
fluorometry buffer reproducibly resulted in a bell-shaped distribution
for the association rate of TIMP-3 with gelatinase A (Fig.
1a). As the heparin
concentration was increased to 100 µg/ml, the association rate
increased 3.7-fold compared with the kon
measured in the absence of heparin. Further increases in the amount of
heparin resulted in a decrease in the rate of association to levels
approaching that observed in the absence of heparin. The addition of
heparin to TIMP-2 and gelatinase A had a negligible effect on the
association rate. The association rate of TIMP-1 and gelatinase A was
increased by 4.6-fold with 800 µg/ml heparin, but the distribution
was not bell-shaped, as it was for TIMP-3. Although TIMP-1 appears to be more dramatically affected than TIMP-3 due to the manner in which
the data is presented, the kon for TIMP-3
increased to 108
M1·s
1 and exceeds the maximum
rate accurately measurable using this system, whereas values for TIMP-1
plateaued at around 2 × 107
M
1·s
1. Preincubation of
either TIMP-3 or gelatinase A with heparin or the addition of heparin
to the buffer did not affect the association rate obtained. Increasing
amounts of heparin did not affect the association rate of
(
418-631)gelatinase A and TIMP-3 (data not shown).
SDS-polyacrylamide gel electrophoresis and silver staining (data not
shown) and a rabbit collagenase diffuse collagen fibril assay revealed
that TIMP-1 and TIMP-2 did not bind at all to heparin-agarose in 0.15 M NaCl, whereas TIMP-3 did bind, and 95% was eluted by 0.5 M NaCl, and 5% was eluted by 2 M NaCl.
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The TIMPs are differentially glycosylated by our NS0 cell expression system: TIMP-2 is nonglycosylated, TIMP-1 is glycosylated, and TIMP-3 is produced in glycosylated and nonglycosylated forms. The potential role of glycosylation in binding to the polyanions was investigated by comparing the effect of heparin on the inhibition of gelatinase A by TIMP-1 and TIMP-3 in their glycosylated and deglycosylated forms. After treatment of TIMP-1 and TIMP-3 with PNGase F, which cleaves off the carbohydrate at its link with asparagine, there was a decrease in the apparent molecular weight of both TIMP-3 and TIMP-1, giving a distinct band on a silver-stained 12% polyacrylamide gel, but no decrease in apparent molecular weight where the inhibitors were incubated under the same conditions without PNGase F (data not shown). This suggests that the carbohydrate had been removed. Using the collagenase fibril assay, we found that 98% of both glycosylated and deglycosylated TIMP-3 bound heparin-agarose in 0.15 M NaCl and both were eluted by 0.5 M NaCl, whereas neither form of TIMP-1 bound significantly. In the fluorimetric assay, PNGase F had no activity against Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, and neither gelatinase A activity nor the rate of inhibition of gelatinase A by TIMP-1 was affected by the addition of PNGase F (data not shown). Deglycosylation of TIMP-1 and TIMP-3 had no effect on the rate of inhibition of gelatinase A in either the presence or absence of heparin (data not shown). Hence, it appears that the carbohydrate component of TIMP-1 and TIMP-3 is not responsible for the effect seen with heparin.
To confirm that the effect of heparin is mediated by ionic interactions, the ionic strength of the fluorimetry buffer was increased, and the association rate of TIMP-3 and gelatinase A was measured. Increasing the NaCl concentration from 0.1 M to 0.25 M or 0.5 M in the presence of 10 µg/ml heparin abolished the effect of heparin on the association rate: in 0.1 M NaCl, heparin increased the kon 1.4-fold, whereas in 0.25 M NaCl and 0.5 M NaCl, the kon was identical in the presence and absence of heparin. TIMP-3 was also eluted from heparin-agarose by 0.5 M NaCl. Raising the ionic strength had an identical effect on deglycosylated TIMP-3 in the presence or absence heparin (data not shown). This indicates that the effect of heparin is mediated by ionic interactions, probably between its negatively charged sulfate groups and the positively charged residues in TIMP-3 and gelatinase A.
The Effect of Other Polyanions on the Inhibition of Gelatinase A by TIMP-3-- The effect of various polyanions on the rate of association of TIMP-3 with gelatinase A was tested using the standard fluorometric assay. Like heparin, dextran sulfate resulted in a bell-shaped distribution for the association rate over the concentration range studied, with an increase in kon of 4.4-fold at 50 µg/ml dextran sulfate (Fig. 1b). Heparan sulfate resulted in a slight increase in the association rate over the relatively small concentration range studied (Fig. 1b). Hyaluronic acid and dermatan sulfate had no effect, although the former did result in an increase in the steady-state rate, probably due to increasing viscosity (data not shown). There was no effect on the rate of association of TIMP-3 and gelatinase A when de-N-sulfated heparin was used (Fig. 1c).
The Effect of Heparin on the Rate of Association of TIMP-3 and
Other MMPs--
The association rate of TIMP-3 and stromelysin-1 was
not affected by heparin over the concentration range of 0-800 µg/ml
(data not shown), probably because stromelysin does not bind to heparin (34). The rate of interaction of TIMP-3 with matrilysin was increased
2-fold by heparin, but the rate did not decrease with high heparin
concentrations, as it did for gelatinase A, and de-N-sulfated heparin
also increased the association rate slightly (Fig.
2a). There was also a slight
increase in the rate of association with heparan sulfate, hyaluronic
acid (as well as an increase in the steady-state rate as for gelatinase
A), and dermatan sulfate (data not shown). Dextran sulfate increased
the association rate 15-fold, and the distribution was bell-shaped, as
it was for gelatinase A (Fig. 2b). However, the pattern of
these results differed from those of TIMP-3 and gelatinase A,
suggesting a different mode of action for the effect on TIMP-3 and
matrilysin.
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DISCUSSION |
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Our data show that TIMP-3 is able to associate with the MMPs
stromelysin-1 and matrilysin with rates similar to TIMP-1 and TIMP-2:
the association rate is relatively slow (105
M1·s
1), presumably because
stromelysin-1 has negligible C-terminal domain interactions with TIMPs
(17, 25), and matrilysin comprises solely a catalytic domain. The
interaction of TIMP-3 with active gelatinase A (107
M
1·s
1) and gelatinase B
(105 M
1·s
1) is
more similar to that of TIMP-2. The rate of association of TIMP-3 with
these gelatinases is enhanced by the hemopexin domains of the enzymes.
The apparent Kd data suggest that significant interactions occur between the C-terminal domain of gelatinase A and
TIMP-3. The interaction between TIMP-3 and the C-terminal domain of
gelatinase A is slightly weaker than that of TIMP-2, probably due to
the absence of the highly negatively charged C-terminal tail in TIMP-3
that is present in TIMP-2 (these last 8 residues of TIMP-2 have been
shown to be highly significant in the interaction with the C-terminal
domain of gelatinase A (20)), but serves to increase the rate of
association of TIMP-3 and gelatinase A 1000-fold. As for TIMP-2 (36),
C-terminal domain interactions with residues 568-631 are particularly
important for rapid association of TIMP-3 and gelatinase A. The
decrease in the rate of inhibition of gelatinase A by TIMP-3 with
increasing ionic strength suggests the involvement of charged residues
in the interaction, as seen for TIMP-2 (20). We also reported
previously that TIMP-3 inhibition of the catalytic domains of MT1 MMP
and MT2 MMP was similar to that of TIMP-2 (41, 42). It is known that
TIMP-2 and TIMP-4 bind to progelatinase A via C-terminal domain
interactions (20, 43). Here we demonstrate that TIMP-3 is also able
bind to progelatinase A. Complex formation between TIMP-3 and
progelatinase A involves C-terminal domain interactions: the binding of
progelatinase A to TIMP-3 was reduced by removal of the hemopexin
domain or by replacement with the C-terminal domain of stromelysin-1.
As with the active enzyme, residues 568-631 but not residues 418-474
of the hemopexin domain play an important role in the association of
progelatinase A and TIMP-3. These residues constitute part of blade 3 and the whole of blade 4 in the four-bladed propeller structure
determined by x-ray crystallography (44, 45) and border a surface patch
of lysine residues (residues 566, 567, and 568) that may be important
for the electrostatic interaction. This region was also important for
the association of gelatinase A with TIMP-2 (36) and suggests that
TIMP-2 and TIMP-3 share common features of the binding site for
progelatinase A. Although TIMP-3 is able to bind progelatinase A and
MT1 MMP (41) like TIMP-2, we have been unable to convincingly
demonstrate its involvement in progelatinase A activation as we did for
TIMP-2 (36). It is unclear whether this is due to technical
difficulties caused by the adherent nature of TIMP-3 or an alternative
pericellular activation pathway involving TIMP-3 bound to the matrix or
whether the binding of TIMP-3 to progelatinase A is not strong enough to support the formation of a membrane receptor (36).
Removal of the C-terminal domain of gelatinase B significantly reduced
the rate of inhibition of this enzyme by TIMP-3. Hence, as for TIMP-1
(26), the hemopexin domain of gelatinase B is important for association
with TIMP-3, although it contributes little to the association with
TIMP-2. Like TIMP-1 (26), TIMP-3 can also bind to progelatinase B, but
not to pro(426-688)gelatinase B, indicating that these C-terminal
domain interactions are sufficient and necessary to yield a stable
proenzyme-inhibitor complex. The precise biological role of this
property of the TIMPs is not yet known, although a role in
progelatinase B activation is possible.
The assays of TIMP-3 inhibitory activity described above were carried out in solution. Although these studies are valuable from a comparative point of view, it must also be borne in mind that TIMP-3 is apparently largely extracellular matrix-bound in vivo, although the components to which it binds remain to be determined. Proteoglycans consist of core proteins with numerous attached glycosaminoglycan chains: the latter are negatively charged polysaccharides composed of repeating disaccharides (for reviews, see Refs. 46 and 47). TIMP-3 possesses 9 positively charged residues (8 lysines and 1 arginine) that are not present in TIMP-1 or TIMP-2 (see alignment, Fig. 3 in Ref. 3) and that are generally conserved in TIMP-3 from different species. It is likely that these charged residues may be involved in the interaction of TIMP-3 with cell surface or extracellular matrix glycosaminoglycans. We therefore tested the effect of some commercially available polyanions on the inhibition of various MMPs by TIMP-3.
The rate of inhibition of gelatinase A but not (418-631)gelatinase
A by TIMP-3 was increased by heparin. Both gelatinase A and TIMP-3 bind
to heparin, but there is no heparin binding site in
(
418-631)gelatinase A (48), suggesting that a heparin binding site
is required in both interacting proteins. The bell-shaped distribution
of the association rate over the heparin concentration range studied is
reminiscent of similar curves for the effect of heparin on
progelatinase A autoactivation (48) or for the inhibition of thrombin
by antithrombin III (49). The curve suggests a biomolecular mode of
binding to heparin that increases the local concentration of reactants,
thereby increasing their rate of interaction, rather than a
conformational effect.
The effects of the polyanions tested on the rate of inhibition of gelatinase A by TIMP-3 appear to correlate with negative charge density. The sulfated compounds, dextran sulfate, heparin, and heparan sulfate (4-5 O-linked, 2-3 O- and N-linked and 1 O- or N-linked sulfate per disaccharide, respectively (50)), enhanced the rate of interaction, whereas dermatan sulfate (1 O-linked sulfate per disaccharide), de-N-sulfated heparin, and hyaluronic acid (unsulfated) had no effect, suggesting that the interaction of enzyme and inhibitor with these polyanions is based on charge density as well as structure. A specific recognition domain in heparin has been described for basic fibroblast growth factor and antithrombin (51). The existence of such a domain for TIMP-3 would be compatible with a surface concentration mechanism like that of antithrombin and thrombin (51). TIMP-3 does not contain any of the reported linear heparin binding motifs, but a motif defined by the three-dimensional structure could exist (52). It is likely that TIMP-3 interacts with cell surface and extracellular matrix glycosaminoglycans via the large number of positively charged residues in TIMP-3, and that this is the basis for its location in the extracellular matrix both in vivo and in cell culture. Hence, colocalization of TIMP-3 with proenzymes in the pericellular environment may be a mechanism for increasing the rate of inhibition of MMPs and regulating extracellular matrix breakdown during morphogenetic processes.
Heparan sulfate proteoglycans such as perlecan (53) and syndecans (54)
are also implicated in binding growth factors that promote
angiogenesis. A recent study demonstrated that TIMP-3 can inhibit
endothelial cell migration and angiogenesis in response to the
angiogenic factors basic fibroblast growth factor and vascular endothelial growth factor (55). TIMP-2 had similar effects upon endothelial cell migration in vitro, but TIMP-1 was
ineffective (55, 56). This implicates MT1 MMP in the angiogenic
process, an enzyme that can degrade matrix components and initiate the autoactivation of gelatinase A (36, 40, 57). The study presented here
suggests that the effects of TIMP-3 and TIMP-2 might be due to the
specific ability of these inhibitors to bind to progelatinase A as well
as to inhibit MT1 MMP. Colocalization of TIMP-3 in the pericellular
environment via binding to the extracellular matrix, including heparan
sulfate proteoglycans, would place this inhibitor in a key position to
inhibit MMPs produced by endothelial cells, thus regulating degradation
of the extracellular matrix and release of the angiogenic factors
required for migration and angiogenesis.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tom Crabbe for the kind gifts of
(1-414)gelatinase A and (
418-631)gelatinase A and Dr. C. G. Knight for synthesis of the quenched fluorescent substrates.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust, the Arthritis and Rheumatism Campaign, United Kingdom, and The Arthritis Foundation, U.S.A.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.
An Arthritis and Rheumatism Campaign Senior Fellow.
** To whom correspondence should be addressed. Tel.: 44-1603-593811; Fax: 44-1603-592250; E-mail: g.murphy{at}uea.ac.uk.
2 J. O'Connell and G. Murphy, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
TIMP, tissue
inhibitor of metalloproteinases;
MMP, matrix metalloproteinase;
(1-414)gelatinase A, C-terminal domain of gelatinase A;
(
418-631)gelatinase A, N-terminal domain of gelatinase A;
proE375A-gelatinase A, catalytically inactive mutant of gelatinase A;
(
426-688)gelatinase B, N-terminal domain of gelatinase B;
N-G.C-SGG, gelatinase A mutant with residues 418-474 replaced with
residues 248-305 of stromelysin-1;
N-G.C-SGS, as N-G.C-SGG but with
the additional replacement of residues 568-631 of gelatinase A with
residues 400-460 of stromelysin-1;
N-GL.C-SL, gelatinase A residues
1-417 fused with residues 248-460 of stromelysin-1;
TCABN, 50
mM Tris, pH 7.5, 150 mM NaCl, 10 mM
CaCl2, 0.025% Brij 35, and 0.02% azide;
MT, membrane
type.
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REFERENCES |
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