(Received for publication, October 24, 1995; and in revised form, February 12, 1996)
From the
The precursor of matrix metalloproteinase 3 (MMP-3/stromelysin
1) is activated in vitro by proteinases or mercurial compounds
by stepwise processes which include the initial formation of
short-lived intermediates and the subsequent intermolecular cleavage of
the His-Phe
bond to generate the fully
activated mature MMP-3 (Nagase, H., Enghild, J. J., Suzuki, K., and
Salvesen, G.(1990) Biochemistry 29, 5783-5789). To study
the enzymatic properties of the intermediates we have mutated either
His
or Phe
to Arg to obtain a stable MMP-3
intermediate. The mutant proteins were expressed in Chinese hamster
ovary K-1 cells using a mammalian expression system. The proMMP-3(H82R)
mutant was activated by chymotrypsin, elastase, and
4-aminophenylmercuric acetate to the 45-kDa MMP-3 with similar
mechanism and kinetics as the wild-type. In contrast, the activation of
the proMMP-3(F83R) mutant by proteinases or 4-aminophenylmercuric
acetate resulted in 46-kDa forms, which retained 13, 14, or 15 amino
acids of the pro-domain depending on the activators. The
proteinase-activated MMP-3(F83R) intermediates exhibited little
enzymatic activity, but they were partially active after treatment with
SH-reacting reagents. These molecules could bind to the tissue
inhibitor of metalloproteinases-1 and
-macroglobulin.
However, the SH group of Cys
of the intermediates was not
modified by SH-reagents, indicating that the enzymatic activity
generated by SH-reagents resulted from molecular perturbation of the
enzyme rather than their interaction with Cys
. When
gelatin and transferrin were digested with the 46-kDa intermediates the
products were different from those generated by the wild-type MMP-3,
suggesting an alteration in substrate specificity. The treatment of
proMMP-3 with trypsin resulted in the formation of a 45-kDa MMP-3 with
an NH
-terminal Thr
, whose activity and
substrate specificity were similar to those of the 46-kDa MMP-3(F83R)
obtained from the proMMP-3(F83R) mutant. These observations indicate
that the correct processing at the His
-Phe
bond is critical for expression of the full activity and the
specificity of MMP-3.
Matrix metalloproteinase 3 (MMP-3), ()also designated
stromelysin 1, is a member of the matrixin family which plays a pivotal
role in the degradation and remodeling of the extracellular matrix.
MMP-3 degrades a number of extracellular matrix constituents such as
aggrecan core protein, fibronectin, laminin, collagen types IV (Okada et al., 1986; Murphy et al., 1991) IX, X, and XI
(Okada et al., 1986; Wu et al., 1991) and the large
tenasein C (Siri et al., 1995). The overproduction of MMP-3
has been implicated in connective tissue diseases such as rheumatoid
arthritis and osteoarthritis (Okada et al., 1989; 1992;
Harris, 1990; Gravallese et al., 1991; McCachren, 1991;
Firestein et al., 1991; Walakovits et al., 1992) and
tumor cell invasion and metastasis (see Stetler-Stevensen et
al. (1993)). The importance of MMP-3 in tissue matrix catabolism
is further substantiated by its role in generating the fully active
interstitial collagenase (MMP-1) (Suzuki et al., 1990) and
neutrophil collagenase (MMP-8) (Knäuper et
al., 1993), and its ability to activate progelatinase B (MMP-9)
(Ogata et al., 1992).
Like other matrixins, MMP-3 is
secreted from cells as an inactive zymogen (Okada et al.,
1986, 1988). ProMMP-3 comprises an NH-terminal propeptide
of 82 amino acids, a catalytic domain of 165 amino acids, and a
COOH-terminal hemopexin/vitronectin-like domain of 213 amino acids (see
Woessner (1991)). Latent proMMPs can be activated in vitro by
proteolytic and non-proteolytic pathways (Okada et al., 1988;
Nagase et al., 1990). The ``cysteine-switch'' model
has been proposed to explain non-proteolytic activation of proMMPs by
SH-reacting reagents (e.g. mercurial compounds, iodoacetamide, N-ethylmaleimide, oxidized glutathione) and chaotropic agents
(Springman et al., 1990; VanWart and BirkedalHansen, 1990).
This model suggests that activation occurs when the cysteinyl residue
in the conserved propeptide sequence PRCG[V/N]PD transiently
dissociates from the zinc atom at the active site and reacts with
SH-reacting reagent, thereby preventing the reassociation of the
cysteine-zinc complex. Studies on proMMP-3 activation with
organomercurials and proteinases have indicated that the zymogen is
processed in a stepwise manner (Nagase et al., 1990):
treatment with APMA results in the initial formation of a 46-kDa
intermediate by cleavage of the Glu
-Val
bond;
and proteinases also produce an intermediate by cleaving a peptide bond
in a short segment (residues 34-39), referred to as
``bait'' region, located near the middle of the pro-domain.
The intermediates are then converted into the 45-kDa active form by a
bimolecular reaction cleaving the His
-Phe
bond. Stepwise activation has been also shown for interstitial
collagenase (MMP-1), gelatinase B (MMP-9), and matrilysin (MMP-7)
(Suzuki et al., 1990; Ogata et al., 1992; Crabbe et al., 1992), but the enzymatic properties of the transiently
generated intermediates are not known. We have hypothesized that the
intermediates generated during activation may bind to endogenous MMP
inhibitors such as TIMP-1 and
M, so that this unique
activation system provides an additional regulatory mechanism in
matrixin activities.
In this study, we aimed to generate a stable
intermediate of MMP-3 by mutating residues involved in the final
activation site. Such a mutant was obtained by substitution of
Phe with Arg in proMMP-3. The intermediates generated from
the proMMP-3(F83R) mutant have limited proteolytic activity but they
interact with TIMP-1 and
M. In addition, these
intermediates exhibit an altered substrate specificity when compared
with the fully processed wild-type MMP-3. Our studies suggest that
intermolecular processing of the His
-Arg
bond
is critical for the expression of the full enzymatic activity and the
specificity of MMP-3.
ProMMP-3 and its mutant cDNA constructs were stably transfected into CHO K-1 cells using Lipofectin according to the manufactures description. Transfected cells were initially grown in glutamine-free Dulbecco's modified Eagles's medium in the presence of dialyzed fetal bovine serum and 25 µM methionine sulfoximine for 2 weeks. After colony formation, cells were harvested by treating with trypsin, split into 24-well plates, and grown for additional 2 weeks in the same medium in the presence of increasing concentration of methionine sulfoximine (100-400 µM). Expression of proMMP-3 and its mutants was confirmed by Western blot analysis.
Figure 1:
Activation of recombinant proMMP-3 and
proMMP-3 mutants by APMA. A, proMMP-3 (), proMMP-3(H82R)
(
), and proMMP-3(F83R) (
), 4 µg/ml each, were
activated with 1.5 mM APMA at 37 °C for the indicated time
period and the proteolytic activity of the samples was assayed against
[
H]Cm-Tf. B, SDS-PAGE analysis of the
APMA-activated proMMP-3 and its mutants. Each MMP-3 precursor was
treated with 1.5 mM APMA at 37 °C for the indicated
periods of time. The reaction was stopped by 20 mM EDTA, and
the samples were subjected to SDS-PAGE (7.5% acrylamide) under reducing
conditions. Lanes 1, 4, 7, and 10, proMMP-3; lanes 2, 5, 8, and 11,
proMMP-3(H82R) mutant; and lanes 3, 6, 9, and 12,
proMMP-3(F83R) mutant.
Figure 2:
Sites cleaved in the propeptide of
proMMP-3 and the proMMP-3(F83R) mutant after APMA or proteinases
treatment. Cleavage sites identified by NH-terminal
sequence analysis of the products generated by APMA and proteinases are
shown by arrows. The His
-Phe
bond is
cleaved by MMP-3 (Nagase et al., 1990). X indicates
that the cleavage did not occur due to the substitution of Phe
with Arg. The residue in brackets indicates the NH
terminus of the individual MMP-3 species generated after a
proteinase or APMA treatment. Molecular masses of individual enzyme
species were estimated by SDS-PAGE. The cleavage sites in the boxed bait region are taken form Nagase et al.(1990). The
conserved cysteine-switch sequence is boxed by dotted
lines. CT, chymotrypsin; T, trypsin. Dotted arrows indicate sites cleaved by autolysis in the presence of
APMA.
Since the treatment of the proMMP-3(H82R) mutant with proteinases also processed this precursor in a similar manner and kinetics as the wild-type (data not shown), further studies were carried out with the F83R mutant.
Figure 3:
Activation of proMMP-3 and proMMP-3(F83R)
mutant by HNE. A, proMMP-3 () and proMMP-3(F83R)
mutant (
) (4 µg/ml) were treated with 10 µg/ml HNE at 37
°C for the indicated period of time. After inhibition of HNE by 2.5
mM DFP the MMP-3 activity was measured against
[
H]Cm-Tf. B, SDS-PAGE analyses of
proMMP-3 and proMMP-3(F83R) mutant activated with HNE. ProMMP-3 (40
µg/ml) (lanes 1, 3, 5, and 7) and
proMMP-3(F83R) (40 µg/ml) (lanes 2, 4, 6, and 8) were activated by HNE (10 µg/ml) for 30
min (lanes 3 and 4), 1 h (lanes 5 and 6), and 4 h (lanes 7 and 8). After
terminating the reaction with 2.5 mM DFP and 20 mM EDTA, the samples were subjected to SDS-PAGE (7.5% acrylamide)
under reducing conditions.
Figure 4:
Activation of proMMP-3 and proMMP-3(F83R)
mutant by chymotrypsin. A, proMMP-3 () and
proMMP-3(F83R) (
) were treated with 10 µg/ml chymotrypsin at
37 °C for the indicated period of time, and MMP-3 activity was
measured as described in the legend to Fig. 3A. B, processing of proMMP-3 and the proMMP-3 mutant by
chymotrypsin. Proteins (40 µg/ml) were activated with chymotrypsin
(10 µg/ml) for 30 min (lanes 3 and 4), 1 h (lanes 5 and 6), and 4 h (lanes 7 and 8) and processed as described in Fig. 3B. Lanes 1, 3, 5, and 7, proMMP-3; lanes 2, 4, 6, and 8,
proMMP-3(F83R) mutant.
Figure 5:
Activation of proMMP-3 and proMMP-3(F83R)
mutant by trypsin. A, the wild-type proMMP-3 () and
mutant proMMP-3(F83R) (
) (4 µg/ml) were treated with 5
µg/ml trypsin at 22 °C for the indicated period of time, and
the proteolytic activity of the samples was assayed against
[
H]Cm-Tf after termination of trypsin activity
with 2.5 mM DFP. B, SDS-PAGE analysis of the
trypsin-treated proMMP-3 and its mutant. The wild-type and the mutant
proMMP-3 were treated with trypsin as above and the samples were
processed as described in the legend to Fig. 3B. Lanes 1, 3, 5, and 7 wild-type
proMMP-3; lanes 2, 4, 6, and 8,
proMMP-3(F83R).
Figure 6:
Substrate specificity of the wild-type
MMP-3, [Thr]MMP-3,
[Pro
]MMP-3, and the APMA-activated 46-kDa
[Val
]MMP-3. Type-1 gelatin (30 µg) (A) and Cm-Tf (30 µg) (B) were incubated with
various forms of MMP-3 (0.4 µg) at 37 °C. The reaction was
terminated by the addition of 20 mM EDTA, and the products
were analyzed by SDS-PAGE (10% acrylamide). Lane 1, substrate
without enzyme as control; lane 2, wild-type MMP-3; lane
3, [Thr
]MMP-3; lane 4,
[Pro
]MMP-3; lane 5, APMA activated
46-kDa [Val
]MMP-3. C, time-dependent
degradation of type-1 gelatin by MMP-3 and
[Thr
]MMP-3. Type-I gelatin (30 µg) was
digested with an equal amount (0.4 µg) of MMP-3 (lanes 2, 4, and 6) and of the trypsin-activated
[Thr
]MMP-3 (lanes 3, 5, and 7) at 37 °C for the indicated period of time. The
reactions were terminated by the addition of 20 mM EDTA and
the digestion products were analyzed by
SDS-PAGE.
Figure 7:
Treatment of MMP-3 with trypsin. A, MMP-3 was incubated in the presence of trypsin (5
µg/ml) at 23 °C for the indicated time periods. After
inactivation of trypsin with 2.5 mM DFP, the enzymatic
activity of MMP-3 was measured against [H]Cm-Tf.
MMP-3 samples treated with trypsin for various times were incubated
with type-I gelatin (B) or Cm-Tf (C), and the
digestion products were analyzed by SDS-PAGE as described in the legend
to Fig. 7. Lane 1, substrate with no enzyme; lane
2, substrate incubated with the 45-kDa MMP-3; lanes
3-5, substrate incubated with
[Phe
]MMP-3 after reaction with trypsin for 1, 3,
and 4 h.
Figure 8:
Interaction of the 46-kDa Intermediates
with TIMP-1. The fully active [Phe]MMP-3 and the
APMA-activated [Val
]MMP-3 were applied to a
TIMP-1 affinity column. The HNE-activated
[Met
]MMP-3 was applied to the column before and
after reacting with 1 mM APMA. The proteins bound were eluted
with 5% formic acid. Unbound (UB) and bound (B)
fractions were precipitated by 3.3% trichloroacetic acid and the
proteins were visualized by Western
blotting.
Figure 9:
Incorporation of
[C]iodoacetamide into Cys
of the
46-kDa intermediate. A, proMMP-3(F83R) (1 µM) was
treated with either HNE (10 µg/ml) or chymotrypsin (10 µg/ml)
at 37 °C. After inactivation of the activating proteinases with 2.5
mM DFP the samples were incubated with 1 mM [
C]iodoacetamide in the presence or absence
of 20 mM EDTA at 23 °C for 15 min. The reactions were
terminated by the addition of 5 mM cysteine and the samples
were subjected to SDS-PAGE (7.5% acrylamide) under reducing conditions,
and the incorporation of [
C]iodoacetamide to the
proteins was visualized by fluorography according to Laskey and
Mills(1975). Lanes 1 and 2, proMMP-3(F83R); lanes
3 and 4, the mature 45-kDa
[Phe
]MMP-3 that lack propeptide; lanes 5 and 6, proMMP-3(F83R) treated with HNE for 2 h; lanes
7 and 8, proMMP-3(F83R) treated with chymotrypsin for 30
min. B, proMMP-3(F83R) was activated either by 1 mM APMA (lanes 1 and 2) or HNE (10 µg/ml) at 37
°C for 1 h (lanes 3-6), and the samples were then
reacted with either TNC buffer (lanes 3 and 4) or 1
mM iodoacetamide (lanes 5 and 6) at 37
°C for 2 h. Under the conditions used HNE partially converted
proMMP-3(F83R) to the 46-kDa form. After dialysis against TNC buffer
the samples were reacted with [
C]iodoacetamide
in the presence or absence of 20 mM EDTA. The radioactivity
incorporated was visualized using a
PhosphorImager.
The involvement of matrixins in extracellular matrix
degradation is controlled, in part, by the activation of their zymogens
and the inhibition of the activated enzymes by their endogenous
inhibitors. Promatrixins are activated in vitro by several
proteinases, SDS, HOCl, chaotropic agents (see Woessner, 1991), low pH
(Davis and Martin, 1990), and elevated temperature (Koklitis et
al., 1991). Our previous work, demonstrating that the activation
of proMMP-3 occurs in a stepwise manner (Nagase et al., 1990),
led us to investigate enzymatic properties of the intermediates and
their ability to interact with endogenous inhibitors, TIMP-1 and
M. A stable intermediate was generated by mutating
Phe
to Arg, whereas the proMMP(H82R) mutant was converted
to the active 45-kDa species. These results are in agreement with the
substrate specificity of MMP-3 reported by Niedzwiecki et
al.(1992); i.e. the activity of MMP-3 decreased more then
a 100-fold when phenylalanine at the P
` site was replaced
by arginine.
The stable intermediates generated from proMMP-3(F83R)
could bind TIMP-1 and M, but this occurred only in the
presence of SH-reacting reagents. Since the activity of these
intermediates against the synthetic substrate NFF-3 was very low, their
binding constants with TIMP-1 could not be determined. Nonetheless, our
observations are in good agreement with those by Ward et
al.(1991) reporting that TIMP-1 was able to bind to MMP-3
intermediates during activation by APMA. TIMP-2 also interferes with
the proMMP-1 processing during activation (DeClerck et al.,
1991).
Little activity was detected with the proteinase-activated
MMP-3(F83R) intermediates, whereas the APMA-activated intermediate
expressed about 15-20% of the full MMP-3 activity against
[H]Cm-Tf and the synthetic substrate. The lack of
activity with the former is likely to be due to the retention of the
Cys
-Zn
interaction since Cys
did not react with [
C]iodoacetamide unless
the sample was treated with EDTA. This contrasts with our previous
studies with the wild-type proMMP-3 whose intermediates underwent
autoprocessing, indicating that those intermediates have proteolytic
activity (Nagase et al., 1990). This discrepancy may be
explained by the different length of the remaining propeptide. The
molecular mass of the major intermediate generated from the wild-type
proMMP-3 by proteinases is 53 kDa, whereas that of the stable
intermediate form the proMMP-3(F83R) is 46 kDa. It is, therefore,
speculated that the longer propeptide moiety of the 53-kDa species may
be more favorable than a shorter propeptide to create an open active
site, which allows a rapid intermolecular cleavage of the
His
-Phe
bond. A similar open structure may be
generated with the initial proteolytic cleavage in the bait region of
the proMMP-3(F83R) mutant, but the cleavage of the
His
-Phe
does not take place in this case due
to the mutation at the P
` site. Instead, the action of
activator proteinases further pruned the propeptide to the 13-15
amino acid length, which apparently interact with the catalytic site of
MMP-3 more tightly, possibly with the aid of the
Cys
-Zn
interaction. However, the 46-kDa
MMP-3(F83R) intermediate was converted to a 45-kDa form by trypsin and
the trypsin-treated form expressed about 20% of the MMP-3 activity
(data not shown), suggesting that the removal of a small stretch of the
propeptide by cleaving the Arg
-Thr
bond
generates an active MMP-3.
The
[Val]MMP-3(F83R) intermediate generated by APMA
expressed partial activity. It is notable, however, that the Cys
of this intermediate was not modified by APMA (Fig. 9B). This result was unexpected, because the
activation of proMMPs is considered to require the disruption of the
Cys-Zn interaction (VanWart and Birkedal-Hansen, 1990; Springman et
al., 1990; Chen et al., 1993) and the resulting free SH
group would react with APMA. Our observation, however, re-emphasizes
the notion that the activation of proMMP-3 by APMA and other
SH-reagents occurs not through their binding with Cys
but
rather by induction of conformational changes in the enzyme molecule
(Chen et al., 1993). The proteinase-activated intermediate
exhibited little enzymatic activity, but an increase in activity was
observed in the presence of APMA, iodoacetamide, or DTNB. Again, under
these conditions the SH group of Cys
did not react with
these agents, but it did in the presence of EDTA. This suggests that
the zinc atom at the active site still remains bound to the Cys
in the partially activated intermediates, possibly forming a
pentadentate coordination. Alternatively, the side chain of Cys
may be interacting with another moiety of the enzyme molecule.
Although we cannot exclude the possibility that this unexpected finding
is due to a subtle conformational change in the enzyme molecule
introduced by mutagenesis, this seems unlikely because the structure
and the stability of the proMMP-3(F83R) appear to be indistinguishable
from those of the wild-type proMMP-3 as demonstrated by a urea
denaturation curve and by activation by trypsin (Fig. 5).
Furthermore, recent studies by Shapiro et al.(1995) showed
that the activation of progelatinase B (proMMP-9) by APMA results in a
83- and 67-kDa species, both of which retained 13 residues of the
propeptide including the conserved cysteine. These forms of MMP-9
exhibited enzymatic activity only in the presence of APMA. No activity
could be detected after removal of APMA by dialysis, suggesting that
APMA does not covalently modify the cysteinyl residue in the propeptide
of proMMP-9.
The 46-kDa MMP-3(F83R) intermediates and the
[Thr]MMP-3 showed similar specific activity on
[
H]Cm-Tf and NFF-3, which was considerably lower
than that of the mature wild-type MMP-3. Kinetic parameters on NFF-3
and the cleavage patterns of Cm-Tf and gelatin indicate that these
enzyme species have similarly altered substrate specificity. Thus, the
reduced enzymatic activity associated with the intermediates is not
simply due to the presence of a part of the propeptide at the
NH
-terminal end of the enzyme. Although the altered
activity and specificity cannot be readily explained, our observations
are analogous to those for interstitial collagenase (MMP-1) and
neutrophil collagenase (MMP-8). Both collagenases express the full
collagenolytic activities when they possess Phe
and
Phe
at their NH
termini, respectively, but
[Val
]MMP-1 and [Met
]MMP-8
have only 40 to 20% of the full activities (Suzuki et al.,
1990, 1995; Knäuper et al., 1993).
Recently resolved crystal structures of the catalytic domains of
[Phe
]MMP-8 and [Met
]MMP-8
provides some insights into the different activities between the two
forms of MMP-8. In [Phe
]MMP-8 the ammonium group
of the NH
-terminal Phe
forms a salt linkage
with the side chain carboxylate of Asp
, but in the case
of [Met
]MMP-8 the NH
-terminal
hexapeptide was disordered (Bode et al., 1994; Reinemer et
al., 1994). It is postulated that a small structural and
rotational difference may result in different stabilization of the
active site or of the transition state, or that the mobile
NH
-terminal peptide may interfere with the substrate
(Reinemer et al., 1994). The changes in both k
and K
with the 46-kDa
intermediate, and the 45-kDa [Thr
]MMP-3 suggest
that both the enzymatic efficiency and the interaction with a substrate
are altered without a correct positioning of the
NH
-terminal Phe
.
In summary, our studies
have shown that intermolecular processing of the
His-Phe
bond is critical for the expression
of full enzymatic activity and specificity of MMP-3. The importance of
the correct NH
terminus may be related to other members of
the matrixin family. All matrixins identified to date have either Phe
or Tyr at this position. The activation of proMMP-1 by human and rat
mast cell chymases generates [Thr
]MMP-1 and
[Val
]MMP-1, respectively (Saarinen et
al., 1994; Suzuki et al., 1995), and reduced
collagenolytic activity was detected (Suzuki et al., 1995). In
the case of MMP-3, not only the reduction in activity, but also the
changes in substrate specificity occur when the
His
-Phe
bond is not correctly processed or
the Phe
is removed from the NH
terminus of the
mature enzyme. Although it is not known whether altered processing of
proMMP-3 occurs in vivo, a number of peptide bonds near the
activation site can become a target of various proteinases. Such
proteinases may arise not only from resident connective tissue cells
but also inflammatory cells, plasma, and opportunistic microorganisms
under certain pathological conditions.