(Received for publication, July 14, 1995; and in revised form, October 24, 1995)
From the
The cDNA of a novel matrix metalloproteinase, collagenase-3
(MMP-13) has been isolated from a breast tumor library (Freije, J. M.
P., Diez-Itza, I., Balbin, M., Sanchez, L. M., Blasco, R., Tolivia, J.,
and López-Otin, C.(1994) J. Biol. Chem. 269, 16766-16773), and a potential role in tumor progression
has been proposed for this enzyme. In order to establish the possible
role of collagenase-3 in connective tissue turnover, we have expressed
and purified recombinant human procollagenase-3 and characterized the
enzyme biochemically. The purified procollagenase-3 was shown to be
glycosylated and displayed a M of 60,000, the
N-terminal sequence being LPLPSGGD, which is consistent with the
cDNA-predicted sequence. The proenzyme was activated by p-aminophenylmercuric acetate or stromelysin, yielding an
intermediate form of M
50,000, which displayed the
N-terminal sequence L
EVTGK. Further processing resulted in
cleavage of the Glu
-Tyr
peptide bond to
the final active enzyme (M
48,000). Trypsin
activation of procollagenase-3 also generated a Tyr
N
terminus, but it was evident that the C-terminal domain was rapidly
lost, and hence the collagenolytic activity diminished. Analysis of the
substrate specificity of collagenase-3 revealed that soluble type II
collagen was preferentially hydrolyzed, while the enzyme was 5 or 6
times less efficient at cleaving type I or III collagen. Fibrillar type
I collagen was cleaved with comparable efficiency to the fibroblast and
neutrophil collagenases (MMP-1 and MMP-8), respectively. Unlike these
collagenases, gelatin and the peptide substrates
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH
and
Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH
were efficiently
hydrolyzed as well, as would be predicted from the similarities between
the active site sequence of collagenase-3 (MMP-13) and the gelatinases
A and B. Active collagenase-3 was inhibited in a 1:1 stoichiometric
fashion by the tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2,
and TIMP-3. These results suggest that in vivo collagenase-3
could play a significant role in the turnover of connective tissue
matrix constituents.
The human matrix metalloproteinases (MMPs) ()comprise
a family of at least 11 homologous zinc-dependent endopeptidases that
degrade the macromolecular components of extracellular matrices. They
have been implicated in matrix remodeling processes associated with
normal mammalian development and growth and in the degradative
processes accompanying arthritis and tumor invasion. The MMPs can be
divided into three main subfamilies, collagenases, stromelysins, and
gelatinases, and other enzymes that do not belong to these groupings.
Three highly homologous human collagenases, fibroblast (MMP-1),
neutrophil (MMP-8), and collagenase-3 (MMP-13) have been identified by
analysis of their respective cDNAs (Goldberg et al., 1986;
Whitham et al., 1986; Hasty et al., 1990; Freije et al., 1994). Sequence comparison revealed that they share
more than 50% sequence identity and three functionally important
domains, namely the propeptide, catalytic, and C-terminal domains.
Procollagenase latency is due to the propeptide domain, which consists
of about 80 amino acids including a free cysteine residue within the
highly conserved PRCGVPD sequence motif. The catalytic domain of about
180 amino acids contains two or one calcium and two zinc binding sites
as revealed by x-ray crystallographic analysis of the catalytic domains
of fibroblast and neutrophil collagenases in the presence of synthetic
inhibitors (Borkakoti et al., 1994; Bode et al.,
1994; Lovejoy et al., 1994). The structure comprises a
five-stranded
-sheet, two bridging loops, and two
-helices.
The C-terminal domain is linked via a short hinge sequence motif to the
catalytic domain and shares sequence homology with vitronectin, being
essential for the triple helicase activity of fibroblast and neutrophil
collagenases (Murphy et al., 1992; Clark and Cawston, 1989;
Sanchez-Lopez et al., 1993; Hirose et al., 1993;
Knäuper et al., 1993a). The active enzymes
form tight binding noncovalent complexes with their natural inhibitors,
referred to as tissue inhibitors of metalloproteinases (TIMPs), in a
1:1 stoichiometric fashion. The interaction of the collagenases with
TIMPs is mainly regulated by the catalytic domain (Murphy et
al., 1992), but C-terminal domain interactions increase the
association rates of complex formation.
Biochemical studies on fibroblast and neutrophil collagenases describing their activation mechanism, substrate specificity, and inhibitor interaction in relation to their domain organization are well advanced (Murphy et al., 1987, 1992; Clark and Cawston, 1989; Hirose et al., 1993; Sanchez-Lopez et al., 1993; Knäuper et al., 1990a, 1990b, 1993a, 1993b), but there are currently no data available regarding the activation mechanism, substrate specificity, and inhibitor interaction of human collagenase-3. We have therefore expressed the human collagenase-3 cDNA in a mammalian expression system and characterized the purified recombinant enzyme in comparison to the fibroblast and neutrophil collagenases.
Figure 1: SDS-PAGE of purified recombinant human procollagenase-3 and determination of molecular mass changes during activation by APMA, stromelysin and trypsin. Lane 1, purified procollagenase-3; lane 2, deglycosylated procollagenase-3; lane 3, procollagenase-3 in the presence of 1 mM APMA after 0 min; lane 4, as lane 3 after 5 min; lane 5, as lane 3 after 43 min; lane 6, APMA activation of procollagenase-3 in the presence of TIMP-1 after 0 min; lane 7, as lane 6 after 171 min; lane 8, APMA-activated collagenase-3 after 171 min; lane 9, procollagenase-3 in the presence of 2.8 µg of active stromelysin after 0 min; lane 10, as lane 9 after 200 min; lane 11, as lane 9 after 440 min; lane 12, procollagenase-3 in the presence of 400 ng TPCK-treated trypsin after 0 min; lane 13, as lane 12 after 7 min; lane 14, as lane 12 after 180 min. Molecular mass markers are indicated on the left. The position of the C-terminal domain of collagenase-3 is indicated by an arrow.
Figure 2: N-terminal sequence determination of procollagenase-3 and activated collagenase-3. N termini of APMA or stromelysin activated collagenase-3 are indicated by arrows.
In
contrast, autoactivated collagenase-3 displayed a M of 48,000 when analyzed by SDS-PAGE, and its proteolytic activity
could not be enhanced by APMA treatment. N-terminal amino acid analysis
revealed the sequence YNVFPRTLKWSKMXL demonstrating the
complete loss of the propeptide domain and assigning Tyr
as the first amino acid of the active enzyme. The Asn
residue was clearly glycosylated due to the lack of a signal
during amino acid sequencing.
Figure 3:
A, activation of procollagenase-3 by APMA.
Procollagenase-3 was incubated at a concentration of 626 nM in
the presence of 1 mM APMA at 37 °C. At the indicated time
points, aliquots were removed and assayed using
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH. Results are presented
as rates of substrate hydrolysis (M
s
). B, activation of procollagenase-3
by stromelysin. Procollagenase-3 was incubated with 1.4 or 2.8 µg
of active stromelysin at 37 °C. At the indicated time intervals,
aliquots were removed and assayed for activity using
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH
. Results are presented
as rates of substrate hydrolysis (M
s
).
, procollagenase-3 activated by 1.4
µg of stromelysin;
, procollagenase-3 activated by 2.8 µg
of stromelysin;
, procollagenase-3 in the presence of
buffer.
N-terminal amino acid
sequencing showed the initial generation of the sequence LEVTGKL after
a 4-min activation of procollagenase-3 by APMA, which is due to the
cleavage of the Gly-Leu
peptide bond (Fig. 2). During the progress of activation, the initial
intermediate form (M
50,000) was converted to the
final fully active enzyme by hydrolysis of the
Glu
-Tyr
peptide bond leading to the
release of the complete propeptide domain (Fig. 2).
The
gelatinolytic activity of collagenase-3 and its homologous counterparts
were determined using [C]gelatin (Table 1). Collagenase-3 displayed the highest specific activity,
90.7 µg/min/nmol, respectively. Thus the enzyme was 44 times more
efficient than fibroblast and 3-8 times better than neutrophil
collagenase.
The rapid proteolytic degradation of two different
serpins (-antichymotrypsin and plasminogen activator
inhibitor 2) by highly purified active collagenase-3 was demonstrated
by SDS-PAGE, while antithrombin III was resistant to degradation (not
shown). Further analysis of the
-antichymotrypsin
cleavage products by N-terminal amino acid sequence determination
revealed that collagenase-3 hydrolyzed the
Ala
-Leu
peptide bond within the
extended reactive site loop of the serpin, two amino acid residues
downstream from the reactive site center. The cleavage of the
Ala
-Leu
peptide bond of
-antichymotrypsin coincides with its inactivation as
recently demonstrated by Mast et al.(1991) for collagenase
(MMP-1) and stromelysin (MMP-3).
Figure 4:
Inhibition of active collagenase-3 by the
three homologous TIMPs. Active collagenase-2 (2 nM) was
incubated with increasing concentrations of either TIMP-1 (),
TIMP-2 (
) or TIMP-3 (
).
Human collagenase-3 is a novel member of the matrix metalloproteinase superfamily and has been cloned from a breast tumor cDNA library (Freije et al., 1994). The enzyme is expressed in the surrounding endothelia of the tumor and may be involved in tumor progression and metastasis. Consequently, biochemical analysis of the activation mechanism, substrate specificity, and inhibition profile of collagenase-3 is of vital importance in order to understand its possible role in vivo. We have, therefore, expressed and purified recombinant human procollagenase-3 and analyzed its biochemical properties in detail and compared these with the homologous human collagenases and gelatinase A.
Procollagenase-3 showed a high
degree of N-linked glycosylation as demonstrated by enzymatic
deglycosylation (11.7% of its M corresponds to N-linked sugars). Amino acid sequencing revealed a lack of
signal for the Asn
residue, thus it can be deduced that
the glycosylation site N
LT carries N-linked
sugars. This glycosylation site is conserved between collagenase-3,
neutrophil collagenase (Knäuper et al.,
1990b), and gelatinase-B and is occupied in all three enzymes. The role
of the high levels of glycosylation observed for these three enzymes is
not quite clear to date. It has been speculated that glycosylation of
neutrophil collagenase and gelatinase-B might be important for
targeting these enzymes to the specific granules of neutrophils, where
they are stored prior to exocytosis. However, in the case of
collagenase-3 it is not clear where the enzyme might be produced in
vivo and why it carries a relatively high amount of N-linked sugars. It is most unlikely that the glycosylation
will cause any changes in the enzymatic properties, activation, or TIMP
interaction of collagenase-3, since studies on the natural and
recombinant catalytic domain of neutrophil collagenase have shown that
the unglycosylated recombinant protein has indistinguishable enzymatic
properties (Knäuper et al., 1993a;
Schnierer et al., 1993).
Activation of matrix metalloproteinases is one of the control mechanisms regulating extracellular connective tissue turnover. We have therefore studied the mechanisms leading to procollagenase-3 activation. Stromelysin activated procollagenase-3 by a two-step mechanism, which is similar to that observed for gelatinase-B (Shapiro et al., 1995; Ogata et al., 1992). In addition, neutrophil procollagenase was activated by stromelysin by a single-step mechanism (Knäuper et al., 1993b), while the fibroblast procollagenase cannot be directly activated by stromelysin (Murphy et al., 1987; Suzuki et al., 1990). The peptide bonds cleaved within procollagenase-3, neutrophil procollagenase and progelatinase-B seem to be readily accessible to stromelysin, while fibroblast procollagenase is resistant until proteolysis of upstream regions of the propeptide have been affected by combined trypsin-stromelysin treatment leading to ``superactivation'' (Murphy et al., 1987; Suzuki et al., 1990). In contrast, procollagenase-3 was very susceptible to either trypsin alone or trypsin in combination with stromelysin, which lead to the rapid loss of the C-terminal domain, thereby destroying the collagenolytic activity of the enzyme. Although relatively high amounts of stromelysin were needed to activate procollagenase-3 efficiently over 6 h, this activation pathway may still be of relevance in vivo, since very high levels of stromelysin have been observed under certain pathological conditions (Walakovits et al., 1992; Matrisian and Bowden, 1990).
Collagenase-3 can be assigned to the collagenase subfamily of matrix metalloproteinases, according to substrate specificity analysis, hydrolyzing the interstitial collagens I-III into 3/4 and 1/4 fragments preferentially cleaving type II collagen over type I and III. In contrast, fibroblast collagenase preferentially cleaves type III and neutrophil collagenase type I collagen (Welgus et al., 1981; Hasty et al., 1987). Thus the three collagenases show distinct collagen substrate specificities, which implies that they may have evolved as specialized enzymes in order to dissolve different connective tissues, which vary in their collagen composition. Collagenase-3 may especially be important in the turnover of articular cartilage, which is rich in type II collagen. The specific activities of the three collagenases against type I collagen were in the range of 100-120 µg/min/nmol enzyme with exception of ``superactive'' neutrophil collagenase, which cleaved 338 µg/min/nmol. By comparison of the ratios of collagenolytic/gelatinolytic activity (Table 3) or collagenolytic/peptidolytic activity (not shown) of the three enzymes, it becomes clear that fibroblast collagenase is the most specific collagenase within this group, although the specific collagenolytic activity of ``superactive'' neutrophil collagenase is 3 times higher.
Collagenase-3 cleaved gelatin and the two synthetic peptide substrates with highly improved efficiency when compared with fibroblast or neutrophil collagenase. Thus, it appears that collagenase-3 not only efficiently degrades type I collagen, but it might also act as a gelatinase to further degrade the initial cleavage products of collagenolysis to small peptides suitable for further metabolism. This is in agreement with results obtained earlier for rat collagenase, which shows relatively high levels of gelatinolytic activity (Welgus et al., 1985) and shares the highest degree of homology with human collagenase-3, as does mouse collagenase (Henriet et al., 1992; Quinn et al., 1990). According to the high degree of functional and sequence homology between human collagenase-3 and the rodent collagenases, these enzymes belong to the collagenase-3 subfamily (MMP-13) of matrix metalloproteinases and are distinct from human fibroblast collagenase (MMP-1). We therefore propose to introduce a revised nomenclature for the rodent collagenases to prevent further confusion in the literature assigning them as MMP-13. Indeed, it may be concluded that rat and mouse cells express only collagenase-3 (MMP-13), there being no evidence to date for a homologous MMP-1 in either rat or mouse. The relative distribution of fibroblast collagenase (MMP-1) and collagenase-3 (MMP-13) in human tissues awaits detailed studies, but initial observations suggest that MMP-1 is predominant.
Comparison of the ratios of gelatinolytic over
peptidolytic activity of collagenase-3 with those values obtained for
human gelatinase A revealed that collagenase-3 is 10 times less
efficient than wild-type gelatinase A (Murphy et al., 1994).
The high efficiency of wild-type gelatinase A against gelatin as a
substrate can be attributed to the fibronectin-like type II repeats,
since a gelatinase A deletion mutant
(gelatinase A) lacking these sequence motifs
has a similar ratio of gelatinolytic over peptidolytic activity to
collagenase-3 (Murphy et al., 1994). Thus collagenase-3 shares
some proteolytic characteristics with the gelatinase subfamily of
matrix metalloproteinases, which is reflected in common structural
elements shared by collagenase-3 and the gelatinases being localized
within the active site cleft as discussed below.
Sequence alignments
of the active site residues of the collagenases with the gelatinases
revealed that the Arg (Fig. 5, number 1) in fibroblast
collagenase is changed to Ile or Leu in collagenase-3, the rodent
collagenases, neutrophil collagenase, and in the gelatinases. It has
been noted by Stams et al. (1994) that the
S`-pocket in neutrophil collagenase is significantly larger
than the equivalent pocket in fibroblast collagenase and that we can
deduce that due to the presence of Leu within collagenase-3 and the
gelatinases that these have a similar enlarged S`
-pocket
and structure. Hence these enzymes should be able to hydrolyze a
broader range of substrates. Second, collagenase-3, neutrophil
collagenase, and the rodent homologues share a Pro residue (Fig. 5, number 3) with the gelatinases, while
fibroblast collagenase has an Ile residue in this position.
Furthermore, collagenase-3, the rodent enzymes, and the gelatinases
contain negatively charged residues just preceding the third His
residue of the catalytic zinc binding motif (either Asp or Glu; Fig. 5, number 2). In contrast, this residue
corresponds to Ser or Ala in fibroblast or neutrophil collagenase. The
presence of a negatively charged residue in collagenase-3 and the
gelatinases might well have implications on the polarization of the
zinc-bound water molecule within these enzymes, possibly increasing its
nucleophilic nature (Fig. 6). This would certainly account for
the increased proteolytic efficiency of collagenase-3 and the
gelatinases, as indicated by our experimental results, but it remains
to be confirmed by site-directed mutagenesis.
Figure 5: Sequence alignment of the active site residues of the collagenases with the gelatinases A and B from various species. Key residues specifically conserved between the gelatinases, collagenase-3 (and partially MMP-8), which may be of importance for gelatinolytic specificity are indicated in boldface italics.
Figure 6: Schematic display of the secondary structure elements of the active site of collagenase-3. The active site residues of collagenase-3 have been superimposed on the secondary structure elements of the active site (zinc environment) of the metzincins (Stöcker et al., 1995). Those features of potential functional importance (see Fig. 5) are marked with arrows.
Analysis of the
inhibition profile of collagenase-3 by the three homologous TIMPs
revealed that all react in 1:1 stoichiometry by forming noncovalent
tight-binding complexes, which is in agreement with earlier published
data on other matrix metalloproteinases (for review see, Murphy and
Willenbrock(1995)). Comparison of the efficacy of two synthetic
hydroxamate inhibitors against collagenase-3 confirmed the structural
similarity to the gelatinases. CT1399, which has a K of less than 10 pM for gelatinase A and 16 pM for gelatinase B, had an approximate K
of
4 pM for collagenase-3 and a K
of
385 nM for MMP-1. Similarly, CT1847, which has a K
of 1.55 nM against gelatinase A and 2.1
nM against gelatinase B had K
values of
0.54 nM against collagenase-3 and of 2.9 nM against
MMP-1. (
)It may be concluded that inhibitors directed
against gelatinases will also be efficient in the control of
collagenase-3.
Our studies have indicated that human collagenase-3 is a potent proteinase with a broad spectrum of activity against extracellular matrix proteins (data not shown) as well as collagenolytic and high gelatinolytic activity. The regulation and location of its expression relative to the more specific fibroblast collagenase will be a matter of great importance for future study.