(Received for publication, August 5, 1996, and in revised form, December 16, 1996)
From the Department of Cell and Molecular Biology,
Strangeways Research Laboratory, Worts' Causeway, Cambridge CB1 4RN,
United Kingdom, ¶ Celltech Limited, 216 Bath Road, Slough SL1 4EN,
United Kingdom, the Departamento de Bioquimica y Biologia
Molecular, Universidad de Oviedo, 33006 Oviedo, Spain, and the
** Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genoa, Italy
Recombinant human procollagenase-3
and a C-terminal truncated form (249-451
procollagenase-3) have been stably expressed in myeloma cells and
purified. The truncated proenzyme could be processed by
aminophenylmercuric acetate via a short-lived intermediate form
(N-terminal Leu58) to the final active form (N-terminal
Tyr85). The kinetics of activation were not affected by
removal of the hemopexin-like C-terminal domain. The specific
activities of both collagenase-3 and
249-451
collagenase-3 were found to be similar using two quenched fluorescent
substrates, but
249-451 collagenase-3 failed to cleave
native triple helical collagens (types I and II) into characteristic
one- and three-quarter fragments. It was noted, however, that the
1,2(I) chains of type I collagen were susceptible to
249-451 collagenase-3, which indicates that the
catalytic domain displays telopeptidase activity, thereby generating
1,2(I) chains that are slightly shorter than those in native
type I collagen. It can be concluded that the C-terminal domain is only
essential for the triple helicase activity of collagenase-3. Binding of
procollagenase-3 and active collagenase-3 to type I collagen is
mediated by the C-terminal domain. Both collagenase-3 and
249-451 collagenase-3 hydrolyzed the large tenascin C
isoform, fibronectin, recombinant fibronectin fragments, and type IV,
IX, X, and XIV collagens; thus, these events were independent from
C-terminal domain interactions. In contrast, the minor cartilage type
XI collagen was resistant to cleavage. Kinetic analysis of the
mechanism of inhibition of wild-type and
249-451
collagenase-3 by wild-type and mutant tissue inhibitors of
metalloproteinase (TIMPs) revealed that the association rates for
complex formation were influenced by both N- and C-terminal domain
interactions. The C-terminal domain of wild-type collagenase-3 promoted
increased association rates with the full-length inhibitors TIMP-1 and
TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33. In
contrast, the association rates for complex formation with TIMP-2 and
N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain
interactions.
The matrix metalloproteinases (MMPs)1 are a family of zinc-dependent enzymes that have the capacity to degrade most protein components of the extracellular matrix. Their uncontrolled activity contributes to the tissue destruction that is observed during such diverse pathologies as arthritis and cancer. Four main subfamilies of MMPs have been defined according to their substrate specificity, primary structures, and cellular localization: the collagenases, stromelysins, gelatinases, and membrane-type matrix metalloproteinases.
Human procollagenase-3 (MMP-13) is a new member of the collagenase
subfamily of MMPs, which consists of three members showing an overall
sequence homology of 55% (1-4). Human collagenase-3 expression has
been demonstrated in breast tumors and in osteoarthritic cartilage (1,
5), indicating that the enzyme plays a role in degradation of collagen
during disease progression. Furthermore, its expression in cartilage
was strongly induced at both the message and protein levels by
interleukin-1. Biochemical characterization of human collagenase-3
has shown that it is a powerful collagenolytic enzyme, preferentially
cleaving type II collagen, while it is five or six times less efficient
at hydrolyzing type I or III collagen (5, 6). Type II collagen was
initially hydrolyzed at the same peptide bond as by MMP-1, but this was
then followed by a secondary cleavage event, thereby removing three
amino acids from the initial N terminus of the one-quarter fragment
(5). Furthermore, denatured collagens and small synthetic peptide
substrates were more efficiently cleaved than by the two other
homologous human collagenases, MMP-1 (fibroblast collagenase) and MMP-8
(neutrophil collagenase). In addition, we recently demonstrated that
collagenase-3 cleaved human cartilage aggrecan at three sites within
the interglobular G1-G2 domain (7), which indicates that this enzyme
may play a role in aggrecan loss during arthritis. Analysis of the
activation mechanism of human procollagenase-3 revealed that APMA,
stromelysin, trypsin, plasmin, gelatinase A, and MT1-MMP were able to
activate the proenzyme, which led to the removal of the complete
propeptide domain in each case (6, 8). It was thereby established that the conserved PRCGVPD sequence motif of the propeptide domain was
responsible for the latency of the proenzyme (9), which was
earlier demonstrated for the two other human collagenases, MMP-1 and
MMP-8 (10-13). Cellular activation of procollagenase-3 could also be
demonstrated using concanavalin A-stimulated fibroblast monolayers,
which was shown to be dependent on the activity of MT1-MMP and/or
gelatinase A. This mechanism is unique within the collagenase
subfamily of MMPs since MMP-1 and MMP-8 were not affected (8).
The catalytic domain of human collagenase-3, which contains the
catalytic zinc-binding site, has 55% sequence similarity to the
catalytic domains of human MMP-1 and MMP-8. The catalytic domains of
these enzymes are all known to consist of a five-stranded -sheet
structure containing three
-helices in a typical sequential order
(14-17). The C-terminal domain of MMPs shows homology to hemopexin and
vitronectin and is of particular interest in the case of the
collagenases MMP-1, MMP-8, and MMP-13 since this domain is essential
for their specific triple helicase (collagenolytic) activity (6,
18-23). In the case of MMP-1 (17) and MMP-13 (24), x-ray
crystallographic data have shown that this domain displays a
four-bladed
-propeller structure that is linked via a short hinge
sequence motif to the catalytic domain.
Detailed analyses of fibroblast and neutrophil collagenases in relation
to their domain organization are well advanced (18, 21, 22), but there
are currently no data available regarding the functions of the
collagenase-3 domains. We have expressed full-length procollagenase-3
and a C-terminal deletion mutant (249-451
procollagenase-3) and assessed the role of the C-terminal domain of
collagenase-3 in relation to activation of the proenzyme, substrate
specificity, and TIMP binding.
The digoxigenin antibody labeling kit was purchased from Boehringer (Mannheim, Germany). All other chemicals were obtained from Sigma and were the purest grade available. Collagens were generously donated by Drs. M. Barnes, K. Kühn, E. Aubert-Foucher, J.-J. Wu, and D. R. Eyre.
Generation ofThe
procollagenase-3 cDNA in pSP64 was cleaved with XcmI and
EcoRI, which removed the complete C-terminal domain. The
purified cleaved plasmid was ligated to the oligonucleotides
5-TCT
GTTAAG-3
and 5
-AATTCTTAAC
AGAG-3
,
thereby introducing a stop codon following the codon for glycine 248, which was followed by an EcoRI site. The
249-451 procollagenase-3 cDNA was sequenced using
the dideoxy method, which conformed its identity, except for
replacement of the C-terminal coding sequences with a C-terminal stop
codon. The construct was subcloned into the HindIII and
EcoRI sites of the mammalian expression vector pEE12. The
NaeI-linearized
249-451 procollagenase-3
cDNA in pEE12 (50 µg) was transfected into NSO mouse myeloma
cells by electroporation. Stable clones were selected through growth in
glutamine-free medium essentially as described for other members of the
matrix metalloproteinase family (6, 18). Clones producing high amounts
of
249-451 procollagenase-3 were grown in bulk in
serum-free defined medium. Cell-conditioned culture medium containing
249-451 procollagenase-3 was dialyzed against 20 mM Tris-HCl (pH 7.3), 5 mM CaCl2,
and 0.05% NaN3 (buffer A) and applied to a DEAE-Sepharose
fast flow column (10 × 3 cm).
249-451
procollagenase-3 bound to the column under these conditions and was
eluted in buffer A containing 100 mM NaCl. The partially
purified enzyme solution was dialyzed against buffer A and applied to
an S-Sepharose fast flow column (10 × 1.4 cm), followed by
gradient elution (0-0.5 M NaCl). About 4.5 mg of pure
249-451 procollagenase-3 were obtained from 500 ml of
culture medium.
The expression and purification of wild-type procollagenase-3 have been described in detail elsewhere (6). Activation of the proenzymes was achieved by treatment with 1 mM APMA at 37 °C for 30 min (6).
Construction and Expression of Chimeric TIMPs and Purification of Recombinant Wild-type and Mutant TIMPsAn N.TIMP-1/C.TIMP-2
chimeric cDNA was prepared by polymerase chain reaction using one
mutagenic oligonucleotide to the sequence VGCEE126/128CKITR
(5-GCGCGTGATCTTGCATTCCTCACAGCCAAC-3
) and an N.TIMP-2/C.TIMP-1 chimeric cDNA using oligonucleotides to the sequence
LNHRYQMGCE127/127CTVFPCLSIPC
(5
-GCAGGGGATGGATAAACAGGGAAACACTGTGCACTCGCAGCCCATCTGGTACCTGTGGTTCAGGC-3
and
5
-GCCTGAACCACAGGTACCAGATGGGCTGCGAGTGCACAGTGTTTCCCTGTTTATCCATCCCCTGC-3
) in the presence of vector oligonucleotides and the respective wild-type
cDNAs in pEE12 using overlap extension mutagenesis. The sequences
of the constructs were confirmed using the dideoxy method. The
linearized cDNAs in pEE12 were transfected into NSO mouse myeloma
cells by electroporation, and stable clones were selected through
growth in glutamine-free medium as described above for
249-451 procollagenase-3.
TIMP-1, TIMP-2, TIMP-3, 127-184 TIMP-1,
187-194 TIMP-2, and
128-194 TIMP-2 were
prepared as described previously (25-27). The chimeric inhibitors
N.TIMP-1/C.TIMP-2 and N.TIMP-2/C.TIMP-1 were purified from the
conditioned serum-free culture medium of transfected NSO mouse myeloma
cells using an S-Sepharose fast flow column.
The fluorescent substrate
Mca-PLGL-DpaAR-NH2 was used throughout. Routine
assays were performed at 25 °C at a substrate concentration of 1-3
µM in assay buffer containing 0.1 M Tris-HCl,
10 mM CaCl2, 150 mM NaCl, and
0.05% (v/v) Brij 35 (pH 7.5) (28). Under these conditions, progress
curves were first-order in substrate, fulfilling the requirements [S]
Km and allowing the direct determination of
kcat/Km. Collagenolytic and
gelatinolytic activities were assayed using 14C-labeled
substrates as described (29).
Several extracellular matrix proteins (10 µg each)
such as the large and small isoforms of tenascin and recombinant
fibronectin fragments as well as intact fibronectin were incubated with
active wild-type collagenase-3 (100 ng) and 249-451
collagenase-3 (45 ng) for 24 h at 37 °C, followed by analysis
of the degradation products by silver-stained SDS-PAGE. Three different
type IV collagen preparations (10 µg each) were cleaved with 200 ng
of wild-type collagenase-3 or 90 ng of
249-451
collagenase-3 for 16 h at 25 °C, followed by analysis of the
reaction products under nonreducing and reducing conditions by
SDS-PAGE. Bovine tendon type XIV collagen (4.2 µg) was cleaved with
400 ng of wild-type collagenase-3 or 180 ng of
249-451
collagenase-3 for 16 h at 25 °C. Bovine or rat type IX, X, and
XI collagens (10 µg each) were incubated with 400 ng of wild-type
collagenase-3 or 180 ng of
249-451 collagenase-3 for
16 h at 25 °C. The reaction products were analyzed by SDS-PAGE
under nonreducing (types IX and X) or reducing conditions and stained
with Coomassie Blue.
The concentrations
of the active enzymes were estimated by titration against TIMP-1 of
known concentration (25). The initial rate of substrate hydrolysis
(y axis) was plotted versus TIMP-1 concentration
(x axis), and the active enzyme concentration was determined
from the x axis intercept. The kinetics of the inhibition of
active full-length collagenase-3 and active 249-451 collagenase-3 by wild-type and mutant TIMPs were analyzed by evaluation of the second-order rate constant (kon)
(30).
Full-length procollagenase-3 and 249-451
procollagenase-3 were labeled with digoxigenin using a
digoxigenin-3-O-succinyl-
-aminocaproic acid-N-hydroxysuccinimide ester according to the
manufacturer's instructions (DIG antibody labeling kit, Boehringer),
except that phosphate-buffered saline was replaced with 50 mM HEPES (pH 7.6), 10 mM CaCl2, 150 mM NaCl, and 0.04% Brij 35. It was verified that labeling
had no effect on latency and enzymatic activity following APMA
activation using the quenched fluorescence substrate assay. The binding
of these labeled enzymes to type I collagen was determined using a
modification of the enzyme-linked immunosorbent assay method described
by Murphy et al. (18). Rat skin type I collagen (0.5 mg/ml
in phosphate-buffered saline) was plated at 50 µl/well onto a 96-well
microtiter plate and allowed to form fibrils before drying at 37 °C.
The films were washed (50 mM Tris-HCl (pH 7.0), 150 mM NaCl, and 0.02% Tween 20) and then blocked in 100 mM Tris (pH 7.4), 150 mM NaCl, 1% bovine serum
albumin, 2% nonfat dry milk, and 0.1% Triton X-100 (blocking buffer)
for 20 min at room temperature. Latent or active enzymes were applied
in blocking buffer (100 µl/well) and incubated at 4 °C for 12 h, and then unbound enzymes were removed by washing. Bound enzymes were
detected and quantified using anti-digoxigenin polyclonal antibody Fab fragments conjugated to alkaline phosphatase (Boehringer; 1:2000 in
blocking buffer for 1 h at 15 °C) followed by
p-nitrophenyl phosphate substrate. The absorbance at 405 nm
was measured after stopping the reaction with NaOH.
Studies of the stability of active collagenase-3 at
37 °C showed that fragmentation occurred, as observed for other
members of the MMP family. The enzyme was cleaved into two major
fragments, a doublet of Mr 29,000, which is due
to N-glycosylation, and Mr 27,000 (Fig.
1). These represent the catalytic domain as revealed by
gelatin zymography (data not shown; doublet of
Mr 29,000) and the C-terminal domain
(Mr 27,000) of collagenase-3. N-terminal sequence determination showed that the
Ser245-Leu246 peptide bond had been
autoproteolytically hydrolyzed, thereby releasing the C-terminal domain
(Fig. 2). The initial specific collagenolytic activity
of 100 µg/min/nmol was lost completely, indicating that the
C-terminal domain was essential for the collagenolytic activity of the
enzyme.
The catalytic domain of collagenase-3 was also further processed
internally, releasing 79 amino acid residues from the initial N-terminal Tyr85. This second fragmentation product
displayed the N-terminal sequence LLAHAFPPG, which was the result of
the hydrolysis of the Gly164-Leu165 peptide
bond (Fig. 2). During autoproteolysis, the enzymatic activity
versus Mca-PLGL-Dpa-AR-NH2 decreased to 20% of
the initial value (after 16 h) and further declined upon prolonged
incubation (>24 h) until no proteolytic activity was measurable,
which we deduced was due to cleavage of the
Gly164-Leu165 peptide bond within the
catalytic domain. It was therefore not possible to use the original
fragmentation products for analysis of the enzymatic and TIMP binding
properties of the catalytic domain of collagenase-3; hence, we
produced an intact C-terminal deletion mutant (249-451
procollagenase-3) by protein engineering.
249-451 procollagenase-3 was expressed
by NSO cells and purified from the resulting conditioned medium. The
recombinant deletion mutant (
249-451 procollagenase-3)
was analyzed by SDS-PAGE and shown to occur as a single band of
Mr 35,000 (Fig. 3A,
lane 1) relative to the Mr 60,000 (Fig. 3B, lane 1) for full-length procollagenase-3. The discrepancy between the calculated
Mr of
249-451 procollagenase-3
of 25,000 and the observed Mr of 35,000 is due
to N-glycosylation as earlier demonstrated for the
full-length proenzyme (6). No proteolytic activity was detectable prior
to
249-451 procollagenase-3 activation, indicating that the proenzyme was correctly folded. Furthermore, N-terminal sequence analysis confirmed that
249-451
procollagenase-3 displayed the sequence LPLPSGGD, which was
earlier demonstrated for the wild-type proenzyme (6).
Activation by APMA treatment at 37 °C for 30 min resulted in
autoproteolytic processing of 249-451 procollagenase-3 and wild-type procollagenase-3 through one detectable intermediate of
Mr 28,000 (Fig. 3A, lane
2) and 50,000 (Fig. 3B, lane 2),
respectively, to the final active forms of ~24,000 (Fig.
3A, lane 3) and 48,000 (Fig. 3B,
lane 3). N-terminal sequence data confirmed that
249-451 procollagenase-3 was processed via a
short-lived intermediate form displaying the sequence
58LEVTGK that was converted to the final active form
through hydrolysis of the Glu84-Tyr85 peptide
bond. These data confirm that
249-451 procollagenase-3 was processed in an identical manner as earlier described for wild-type
procollagenase-3 (6). Our sequence analysis of active
249-451 collagenase-3 furthermore confirmed that the
enzyme was N-glycosylated at Asn98 due to a lack
of a signal during amino acid sequencing.
Activity measurements using the synthetic peptide substrate
Mca-PLGL-Dpa-AR-NH2 demonstrated the rapid generation of
enzymatic activity, which reached a plateau after 30 min and declined
to 20% of the initial maximal activity after 16 h at 37 °C
(data not shown). This suggests that the secondary cleavage at
Gly164-Leu165 described for wild-type
collagenase-3 also occurred in the case of active
249-451 collagenase-3. The
kcat/Km values of optimally
activated enzymes (30 min at 37 °C) were determined after the enzyme
concentrations had been determined by active-site titration using
TIMP-1 and gave similar values for both enzymes (Table
I). In contrast,
249-451 collagenase-3
showed no detectable triple helicase activity when incubated with type
I or II collagen (Fig. 4A, lanes 2 and 5). It is noteworthy, however, that
249-451 collagenase-3 cleaved the
1,2(I) chains of type I collagen, generating
1,2(I) chains that were somewhat smaller
than the
1,2(I) chains of the native substrate (Fig. 4B,
lane 1). This indicates that the catalytic domain of
collagenase-3 is an efficient telopeptidase that is not dependent on
the presence of the C-terminal domain and does not require binding of
the enzyme to the substrate (see below). This is a unique feature of
collagenase-3 since MMP-1 and MMP-8 do not display any significant
telopeptidase activity. In the case of type III collagen, partial
hydrolysis (10%) by
249-451 collagenase-3 was
observed, which generated fragments corresponding to one- and
three-quarters in size (data not shown). This was due to nonspecific
susceptibility of a part of the type III collagen preparation to
hydrolysis since identical products were generated by gelatinase B
(data not shown). However, if the collagens were heat-denatured prior
to cleavage,
249-451 collagenase-3 degraded these with
the same efficiency as the full-length enzyme (data not shown).
|
Collagen binding experiments revealed that labeled procollagenase-3 and
active collagenase-3 bound to type I collagen films, while
249-451 procollagenase-3 and active
249-451 collagenase-3 did not bind (Fig.
5). Thus, binding was clearly promoted by the C-terminal
domain of the enzyme. The binding experiments were performed over a
wide range of concentrations, and saturation was reached at ~200
nM enzyme binding to the substrate. The assay revealed that
the active enzyme bound marginally better than procollagenase-3. The
binding of the proform of collagenase-3 to type I collagen was somewhat
unexpected since the two other human collagenases bind only as active
enzymes to type I collagen. To confirm these data, the amount of bound
enzymes was also quantitated by activity measurements using the
quenched fluorescence substrate Mca-PLGL-Dpa-AR-NH2 after
the proenzyme had been activated by APMA treatment (data not shown),
and the results showed that both the active enzyme and the proenzyme
bound with comparable efficacy. However, the amount of bound active
enzyme was marginally higher compared with the proenzyme, and this was
consistent over a wide range of bound enzyme concentrations (50-400
nM[R)) (SEE FIG. 5).
Comparison of the Proteolytic Activities of Collagenase-3 and
The large TN-C isoform was very susceptible to
proteolytic cleavage by both enzymes and was degraded into identical
fragments displaying Mr values of 190,000 and
120,000, respectively (Fig. 6, lanes 1 and
2). In contrast, the small TN-C isoform was resistant to
proteolytic attack (data not shown), indicating that the major collagenase-3-sensitive sites are located within the alternatively spliced FN type III repeats. The C-terminal domain of collagenase-3 had
no influence on the rate of TN-C hydrolysis, indicating that specificity is mediated by the catalytic domain alone.
Purified human plasma FN and recombinant FN fragments were hydrolyzed by both enzymes with equal efficiency (data not shown). Intact plasma FN was cleaved into four main fragments with Mr values of 100,000, 43,000, 35,000, and 29,000. In contrast, the recombinant Mr 120,000 FN fragment was hydrolyzed into smaller fragments with Mr values of 48,000, 46,000, 35,000, 32,000, and 28,000, while the Mr 110,000 FN fragment was cleaved into fragments with Mr values of 100,000, 38,000, and 36,000.
Wild-type and 249-451 collagenase-3 were able to
hydrolyze type IV collagen into fragments, and hydrolysis even
proceeded at 25 °C. Both the
2(IV) chain
(Mr 247,000) and the
1(IV) chain (Mr 217,500) were cleaved as observed by
analysis of the reaction products under reducing conditions. The major
cleavage products displayed Mr values of 240,500 and 80,000, respectively (Fig. 7). Under nonreducing
conditions, a single cleavage product of Mr
76,000 was visualized (data not shown). In comparison, human gelatinase
A was not able to hydrolyze type IV collagen under identical conditions
and cleaved only at elevated temperatures (data not shown and Ref. 31).
These data indicate that collagenase-3 may play a considerable role in
the dissolution of type IV collagen, a major component of the basement
membrane.
Furthermore, collagenase-3 and 249-451 collagenase-3
cleaved native type XIV collagen with comparable kinetics (data not
shown). When the cleavage products were compared with those generated
by gelatinase B, a different cleavage pattern was observed. The major
cleavage product generated by collagenase-3 or
249-451 collagenase-3 showed a Mr of 165,000, being
considerably smaller than the NC3 domain of type XIV collagen (Fig.
8). A minor smaller cleavage product of
Mr 110,000 was also generated; and furthermore, the type XIV collagen dimer was partially degraded, yielding a product
that retained the reduction-resistant disulfide bridge of the NC2
domain.
The minor cartilage collagens (types IX, X, and XI) were also
analyzed in cleavage experiments using collagenase-3 or
249-451 collagenase-3. Type X collagen was
susceptible to collagenase-3 and
249-451 collagenase-3,
generating a Mr 48,000 fragment (Fig.
9). Type IX collagen was also susceptible to cleavage by collagenase-3 and
249-451 collagenase-3, although the
differences between cleaved and noncleaved type IX collagens were only
very subtle and were dependent on the species source of the substrate under investigation. The intact
3(IX) and
1(IX) chains of bovine type IX collagen were reduced from Mr 175,000 to
170,000, while the low molecular weight Col1 domain, which is a result
of pepsin extraction, was resistant. In contrast, rat type IX collagen
showed reverse susceptibility, i.e. the high molecular
weight
1(IX) chain was resistant, while the low molecular weight
Col1 domain was cleaved (Mr 52,000 compared with
53,000 for the noncleaved material). It is furthermore noteworthy that
the higher molecular weight material (>175,000) in both type IX
collagen preparations was cleaved (Fig. 9). In contrast, the
1(XI),
2(XI), and
3(XI) chains were completely resistant to both
enzymes.
Kinetic Analysis of the Inhibition of Wild-type Collagenase-3 and
Active-site titrations of collagenase-3 and
249-451 collagenase-3 with wild-type and mutant TIMPs
revealed a stoichiometry of 1:1 (data not shown). It is currently not
possible to determine accurate Ki values for
collagenase-3 and
249-451 collagenase-3 interactions
with wild-type and mutant TIMPs due to the tight binding nature of
their interaction, with appropriate Ki values well
below 200 pm. This is due to the limitations in assay
sensitivity, substrate solubility, and fluorescence quenching at high
substrate concentrations. Furthermore, the enzymes are unstable at low
concentrations, which does not allow analyses during long-term assays.
It is possible, however, to determine the apparent first-order rate
constant (kobs) at low reagent concentrations (50 pM enzymes, 0.06-2 nM inhibitors) from the
analysis of the curvature in the progress of substrate cleavage. At
these low concentrations, inhibition can be treated as "slow
binding" as recently discussed (30). The second-order rate constant
(kon) can be calculated for the interaction of
TIMPs with collagenase-3 and
249-451 collagenase-3; the
data are summarized in Table II (30).
|
The data presented clearly demonstrate that the C-terminal domain of
collagenase-3 contributed significantly to the binding of wild-type
TIMP-1 and TIMP-3 and the chimeric inhibitor N.TIMP-2/C.TIMP-1 since
the association rates of TIMP-1, TIMP-3, and N.TIMP-2/C.TIMP-1 with
249-451 collagenase-3 were significantly reduced
(17-33 times slower). In contrast, TIMP-2, N.TIMP-1/C.TIMP-2, and
187-194TIMP-2 showed kon values
of 0.3-1.8 × 106 M
1
s
1 with either full-length collagenase-3 or
249-451 collagenase-3 and thus are not significantly
affected by C-terminal domain interactions with the enzyme. The
increase in the association rate of collagenase-3·TIMP complexes was
also shown to be mediated by the C-terminal domain of the inhibitor
since the kon values for the interaction of
full-length collagenase-3 with
127-184 TIMP-1 and
128-194 TIMP-2 were in the range of those obtained with
249-451 collagenase-3 and full-length TIMPs. In
contrast, the C-terminal domain of TIMP-2 does not contribute
significantly to complex formation between enzyme and inhibitor, which
is applicable to the charged tail deletion mutant
187-194 TIMP-2. The N-terminal domains of both enzyme
and inhibitor lead to association rates of 3-8.1 × 105 M
1 s
1; and it
can therefore be concluded that the N-terminal domain interactions are
the major contributors of complex formation between collagenase-3 and
TIMPs.
We have determined the autoproteolytic fragmentation sites in
collagenase-3 and assessed the function of the C-terminal domain of the
molecule in activation, substrate specificity, and inhibitor interaction. Since collagenase-3 has been implicated in the pathologies of breast cancer and osteoarthritis, we have also examined the ability
of collagenase-3 and 249-451 collagenase-3 to degrade different components of the extracellular matrix in order to evaluate the possible function of the enzyme in vivo.
Active collagenase-3 is not stable, and autoproteolytic cleavage was observed at the Ser245-Leu246 peptide bond, which is localized at the end of the catalytic domain, thereby releasing the C-terminal domain including the complete hinge sequence motif. Similar autoproteolytic cleavages have been observed earlier for the two other human collagenases (MMP-1 and MMP-8), although these underwent fragmentation farther downstream from the catalytic domain within the hinge sequence motif (19, 23). MMP-1 is hydrolyzed at the Pro250-Ile251 locus and MMP-8 at the Gly242-Leu243 or Pro247-Ile248 locus, indicating similarities between the three collagenases, but clearly demonstrating differences in their precise autoproteolytic processing. The peptide bonds hydrolyzed by all three enzymes resemble typical collagenase cleavage sites, and autoproteolytic fragmentation could be inhibited by complex formation with TIMPs.
The catalytic domain of collagenase-3 generated by autoproteolytic fragmentation was itself unstable, and further autoproteolysis was observed by cleavage of the Gly164-Leu165 peptide bond, which results in a decrease in the peptidolytic activity to 20 or 0% of the initial value, which was dependent on the incubation time employed. Similar processing has been observed in the case of gelatinase A, although it is not clear whether the activity of the enzyme was affected (32). We have earlier drawn attention to the similarities between collagenase-3 and the gelatinases, and the similarities in autoproteolytic fragmentation of the active enzymes underline our earlier results (6).
To assess the function of the C-terminal domain of collagenase-3, we
prepared a recombinant form lacking this domain. Comparison of the
activation of wild-type and 249-451 procollagenase-3 by
APMA revealed that the corresponding propeptides were processed in an
indistinguishable way. In addition, activation by the catalytic domain
of MT1-MMP was observed for both full-length and
249-451 procollagenase-3, indicating that these events
were not influenced by the C-terminal domain (data not shown). We have
previously shown that progelatinase A and a C-terminal deletion mutant
were equally well processed by the catalytic domain of MT1-MMP in
vitro, indicating that the biochemistry of the reaction is also
not influenced by C-terminal domain interactions in solution at high
concentrations (33).
We have extended our previous analysis of the substrate specificity of
collagenase-3 (6) using a wide range of extracellular matrix proteins
and compared the full-length enzyme with the C-terminal deletion mutant
in order to determine the role of the C-terminal domain in substrate
specificity. Comparison of the enzymatic activities of full-length and
249-451 collagenase-3 revealed that the collagenolytic
activity (triple helicase activity) versus interstitial
collagens was mediated by the C-terminal domain, while the
gelatinolytic and peptidolytic activities were unchanged. These results
confirm earlier observations that the triple helicase activities of the
two other human collagenases are dependent on the C-terminal domain
(18, 19, 23). We have furthermore established that human collagenase-3
is an efficient telopeptidase and demonstrated that this activity is
dependent on its catalytic domain and independent from C-terminal
domain interactions. This feature distinguishes collagenase-3 from the
two other human collagenases (MMP-1 and MMP-8) since these show no
detectable telopeptidase activity. However, an
N-telopeptidase activity has been described for the highly
homologous rat collagenase (34) using a mutant type I collagen that
cannot be cleaved into one- and three-quarter fragments. It was
suggested that the N-telopeptidase activity might be
sufficient for resorption of type I collagen during embryonic and early
adult life, while triple helicase activity is necessary during intense
tissue resorption such as observed in the postpartum uterus and in the
dermis later in life (34). The telopeptidase activity of collagenase-3
does not require binding of the enzyme to the substrate since
249-451 collagenase-3 does not bind to triple helical
type I collagen (see below), and this activity might be important
during bone resorption and cartilage turnover since collagenase-3 is
expressed at high levels in these tissues in the human.
We have also demonstrated that the C-terminal domain promotes binding of both procollagenase-3 and active collagenase-3 to type I collagen. Procollagenase-3 and active collagenase-3 bound nearly equally well to triple helical type I collagen, with the amount of active enzyme being marginally higher. Our data are in agreement with earlier data published on the highly homologous mouse collagenase-3 (35). In this case, the proenzyme could be eluted from a type I collagen-Sepharose column at lower salt concentrations than the active form (35), which might indicate that the interaction between the active enzyme and substrate is tighter and in part of ionic nature. The association and dissociation rates for proenzyme and active enzyme binding to triple helical collagen await further detailed analysis. Binding is essential for the triple helicase activity of active collagenase-3, but is not necessary for the telopeptidase activity of the enzyme. In contrast, the two other human collagenases bind only as active enzymes to collagen, while the latent counterparts do not bind to the triple helical substrates (18, 19). From binding data of chimeric N-terminal stromelysin-C-terminal collagenase and N-terminal collagenase-C-terminal stromelysin molecules to fibrillar type I collagen, it is known that binding is promoted by the C-terminal domain of both collagenase and stromelysin (18). However, stromelysin and the chimeric N-terminal collagenase-C-terminal stromelysin mutant also bound as proenzymes, which indicates that the binding motif within the respective C-terminal domain is unmasked in the respective proenzymes, which is also true for procollagenase-3. Furthermore, active stromelysin and the active chimeric proteinases were not able to cleave the triple helical substrates. From further data using a mutant neutrophil collagenase that contained the hinge sequence motif of stromelysin, which is nine amino acid residues longer than the collagenase hinge sequence, it became clear that collagenolysis only proceeds if the connecting "hinge" between the catalytic and C-terminal domains contains the correct number of amino acid residues (22). Thus, the capacity of the collagenases to cleave triple helical substrates obviously depends on the correct interplay between catalytic and C-terminal domains, which appear to be separately folded entities. Our recent x-ray crystallographic analysis of the C-terminal domain of collagenase-3 revealed that the overall structure of this domain shows more similarity to the C-terminal domain of fibroblast collagenase than to gelatinase A, which indicates that those structural features important for efficient triple helicase activity are conserved within the collagenases (24). The triple helicase activity of the collagenases might be coordinated by the hinge sequence motif, but it is not clear how this is mediated specifically. We have noted that none of the residues conserved within the C-terminal domains of the three human collagenases is unique to this subfamily. Recently, Bode (36) suggested that triple helical collagen might be bound between the catalytic and C-terminal domains in such a way that the substrate is bound like a waffle in a waffle iron. The triple helical substrate could be destabilized by this interaction, causing unwinding or relaxing around the cleavable bond. The unwound "single" strands would then be able to fit into the active-site cleft of the molecule, where hydrolysis of each strand would proceed. This hypothesis has been underlined by modeling experiments performed by De Souza et al. (37).
The analysis of the cleavage of the large TN-C isoform, FN, FN
fragments, and type IV, IX, X, and XIV collagens by wild-type collagenase-3 and 249-451 collagenase-3 revealed that
these substrates were equally well hydrolyzed, indicating that the
C-terminal domain had no affect on specificity. The cleavage products
of the large TN-C isoform revealed fragment sizes that were identical to those recently identified for gelatinase A hydrolysis of this extracellular matrix component (38). This indicates that collagenase-3 shows very similar specificity versus large TN-C, with the
major cleavage sites being located within the alternatively spliced FN
type III repeats, which is confirmed by the resistance of the small
TN-C isoform to proteolysis. The increased sensitivity of the large
TN-C isoform to degradation could modify its biological activities by
unmasking or abolishing specific functional sites. Thus, a more
suitable environment for cellular proliferation and migration may well
be established.
Interestingly, collagenase-3 and 249-451 collagenase-3
were able to hydrolyze type IV collagen at 25 °C, and these data
strongly indicate that collagenase-3 is an efficient type IV
collagenolytic enzyme. This observation may have important implications
for the pathophysiological role of collagenase-3 during breast cancer
pathology, allowing rapid dissolution of the basement membrane, leading
to increased ability of tumor cells to extravasate and metastasize.
Taking into account that gelatinase A cleaves type IV collagen only at
elevated temperatures (
25 °C) (31), our findings might indeed
represent important data for the proteolytic turnover of type IV
collagen in vivo.
Type XIV collagen consists of two triple helical domains (Col1 and
Col2) interspersed by non-triple helical domains (NC1, NC2, and
NC3). Degradation by collagenase-3 and
249-451 collagenase-3 leads mainly to the
accumulation of two fragments of Mr 165,000 and
110,000, with minor products that retain the reduction-resistant
disulfide bridge of the NC2 domain. In contrast, gelatinase B, which
initially attacks the Col1 domain in a region of helix imperfections,
mainly generates the NC3 domain (39).
The minor cartilage collagens (types IX, X, and XI) showed different
susceptibility to collagenase-3 and 249-451
collagenase-3. While type XI was resistant to hydrolysis, types IX and
X were cleaved. Within intact cartilage, type IX and XI collagens
are cross-linked in a very specific manner to type II collagen either via lysyl oxidase-generated aldehyde residues or via
pyridinoline-cross-linking residues; therefore,stabilizing the tissue
and degradation of these minor collagens may be crucial in arthritic
disease. Until now, it was thought that stromelysin-1 (MMP-3) was the
only matrix metalloproteinase able to degrade type IX collagen into two
main triple helical fragments, Col1 and Col2,3, with the cleavage sites being located within the NC2 domain of all three chains (40, 41). Our
results suggest that collagenase-3 and
249-451 collagenase-3 act as "telopeptidases" on type IX collagen as well as on type I collagen (see above) and type II
collagen.2 Taking into account that
collagenase-3 is expressed at high levels in arthritic cartilage and
the high specific activity of collagenase-3 on type II collagen, it may
indeed be concluded that collagenase-3 contributes considerably to
cartilage damage during arthritis. This is furthermore underlined by
the fact that aggrecan is cleaved with four times the efficiency of
stromelysin-1, which demonstrates that collagenase-3 can hydrolyze the
two major cartilage proteins very
effectively.3
The assessment of the association rates for complex formation of
full-length and 249-451 collagenase-3 with wild-type and mutant TIMPs revealed that these were affected by N- and C-terminal domain interactions. C-terminal domain interactions were most pronounced in the case of the full-length inhibitors TIMP-3, TIMP-1, and N.TIMP-2/C.TIMP-1, which reacted up to 33 times faster with full-length collagenase-3 than with
249-451
collagenase-3. We have recently reported that the association rate
constants for complex formation between wild-type gelatinase A and
TIMP-1 were increased by a factor of 130 relative to the interaction with a C-terminal deletion mutant of gelatinase A (30). The importance
of C-terminal domain interactions in complex formation was underlined
using
127-194 TIMP-1, which reacted much more slowly
than wild-type TIMP-1 with wild-type collagenase-3. These results show
remarkable similarities to binding data obtained with wild-type TIMP-1
and gelatinase B (42), which were strongly affected by C-terminal
domain interactions. However, the association rates between gelatinase
B and wild-type TIMP-1 revealed a biphasic mechanism containing a fast
and slow binding phase. In the case of complex formation of TIMP-1 with
gelatinase B, the C-terminal domain of TIMP-1 increased the association
rate by a factor of 132. Inhibition of a gelatinase B C-terminal
deletion mutant (
426-688 gelatinase B) by wild-type
TIMP-1 revealed that the fast binding interaction was significantly
compromised, and in addition, the association rate constant for the
slow binding phase was further decreased by a factor of 17.
In contrast, the association rates for complex formation of
TIMP-2 and N.TIMP-1/C.TIMP-2 with collagenase-3 and
249-451 collagenase-3 were only marginally
affected by the C-terminal domain of TIMP-2 or collagenase-3, which has
been earlier demonstrated for the interaction between gelatinase B and
TIMP-2 (42). This is in marked contrast to the results described
earlier for the interaction between gelatinase A and TIMP-2, where
C-terminal domain interactions increase the association rates for
complex formation by a factor of 187 (30). In addition, the charged tail of TIMP-2 (
187-194 TIMP-2) contributed
significantly to the rate of complex formation with gelatinase A, which
is not observed during complex formation with collagenase-3. It has
been shown that the C-terminal domain and especially the charged tail of TIMP-2 (residues 187-194) are responsible for the formation of a
progelatinase A·TIMP-2 complex (30), and this interaction is thought
to be vital in the cellular activation of progelatinase A by MT-MMPs.
We have recently described the activation of procollagenase-3 in the
same system and can only conclude that TIMP-2 is unlikely to be
involved in the interaction of procollagenase-3 with the cell surface
during activation.
We are grateful to Mary Harrison for cell culture assistance. We thank Dr. Andy Docherty for helpful discussions; Dr. Graham Knight for use of the HPLC apparatus; Dr. Michael Barnes for type III collagen; Dr. Klaus Kühn for type IV collagen; Dr. Elisabeth Aubert-Foucher for type XIV collagen; and Drs. Jiann-Jiu Wu and David R. Eyre for type IX, X, and XI collagens.