From the Departamento de Bioquímica y
Biología Molecular, ¶ Biología Funcional, and
** Morfología y Biología Celular, Facultad de Medicina,
Instituto Universitario de Oncología, Universidad de Oviedo,
33006-Oviedo, Spain and the
School of Biological Sciences,
University of East Anglia, Norwich NR4 7TJ, United Kingdom
Received for publication, August 23, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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Remodeling of fibrillar collagen in
mouse tissues has been widely attributed to the activity of
collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the main
collagenase identified in this species. This proposal has been largely
based on the repeatedly unproductive attempts to detect the presence in
murine tissues of interstitial collagenase (MMP-1), a major collagenase
in many species, including humans. In this work, we have performed an
extensive screening of murine genomic and cDNA libraries using as
probe the full-length cDNA for human MMP-1. We report the
identification of two novel members of the MMP gene family which
are contained within the cluster of MMP genes located at murine
chromosome 9. The isolated cDNAs contain open reading frames of 464 and 463 amino acids and are 82% identical, displaying all structural
features characteristic of archetypal MMPs. Comparison for sequence
similarities revealed that the highest percentage of identities was
found with human interstitial collagenase (MMP-1). The new proteins
were tentatively called Mcol-A and Mcol-B (Murine
collagenase-like A and
B). Analysis of the enzymatic activity of the recombinant
proteins revealed that both are catalytically autoactivable but
only Mcol-A is able to degrade synthetic peptides and type I and II
fibrillar collagen. Both Mcol-A and Mcol-B
genes are located in the A1-A2 region of mouse chromosome 9, Mcol-A
occupying a position syntenic to the human MMP-1 locus at
11q22. Analysis of the expression of these novel MMPs in murine tissues
revealed their predominant presence during mouse embryogenesis,
particularly in mouse trophoblast giant cells. According to their
structural and functional characteristics, we propose that at least one
of these novel members of the MMP family, Mcol-A, may play roles as
interstitial collagenase in murine tissues and could represent a
true orthologue of human MMP-1.
Controlled degradation of the extracellular matrix is an essential
event in a variety of physiological conditions involving connective
tissue remodeling such as embryonic growth and development, uterine
involution, ovulation, bone growth and resorption, and wound healing
(1, 2). In addition, excessive breakdown of connective tissue plays an
important role in a number of pathological processes such as rheumatoid
arthritis, atherosclerosis, pulmonary emphysema, and tumor invasion and
metastasis (1, 2). Among the diverse proteolytic enzymes potentially
involved in these physiological and pathological processes, many
studies have focused on matrix metalloproteinases
(MMPs),1 a family of
structurally related endopeptidases collectively capable of degrading
the major protein components of the extracellular matrix and basement
membranes. At present, 20 different human MMPs have been characterized
at the amino acid sequence level (3). According to structural and
functional characteristics, these human MMPs can be classified into at
least six different subfamilies of closely related members:
collagenases, gelatinases, stromelysins, matrilysins, membrane-type
MMPs (MT-MMPs), and other MMPs.
The collagenase subfamily of human MMPs consists of three distinct
members: fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13). An additional collagenase called
collagenase-4 has been identified in Xenopus laevis (4), but
to date the putative orthologues of this enzyme in other vertebrate species have not been described. Biochemical characterization of all
these collagenases has revealed that they share the ability to cleave
fibrillar collagens at a specific peptide bond, resulting in the
generation of fragments of about three-fourths and one-fourth the size
of the intact molecule. Then, the resulting fragments denature
spontaneously to gelatin in physiological temperature and become
susceptible to degradation by other MMPs (5-8). Interestingly, kinetic
studies have revealed that each human collagenase shows distinct
substrate preferences toward the diverse fibrillar collagens. Thus,
MMP-1 degrades preferentially type III collagen (6), MMP-8 prefers type
I collagen (7), and MMP-13 degrades type II collagen 6-fold more
effectively than type I and type III collagens (8). It is also
remarkable that MMP-13 displays about 40-fold stronger gelatinolytic
activity than MMP-1 and MMP-8 (8). On the basis of these data, we have
previously proposed that the different human collagenases have evolved
as specialized enzymes to participate in the remodeling of tissues with
different collagen composition (8). The observation that the three
human collagenases exhibit distinct tissue distribution and are
subjected to different regulatory mechanisms (9, 10) is also consistent
with the idea that they may play different functional roles in both
physiological and pathological processes. To provide further
experimental support to this proposal, it is essential that animal
models be available in which the activity of the different enzymes can
be selectively manipulated. However, these studies have been seriously
hampered by the inability to detect the murine orthologue of MMP-1. In fact, to date only murine MMP-8 and MMP-13 have been identified and
characterized at the amino acid sequence level (11-13), whereas all
attempts from many different groups to isolate murine MMP-1 have been
repeatedly unsuccessful. These data have suggested that MMP-1 may be
functionally substituted in murine tissues by other enzymes with
collagenolytic activity such as MMP-8 and MMP-13. Nevertheless, the
possibility that additional as yet unidentified murine enzymes could be
structurally or functionally related to human MMP-1 cannot be
definitively ruled out. To evaluate this possibility, we have performed
an extensive screening of murine genomic and cDNA libraries using
as probe the full-length cDNA for human MMP-1. As a direct result
of this work, we report herein the identification of two novel members
of the MMP gene family originally selected by their positive
hybridization with the human MMP-1 probe, and contained within the
cluster of MMP genes located at murine chromosome 9. We also describe
the expression of the genes in Escherichia coli and perform
an analysis of the enzymatic activity of the recombinant proteins.
Finally, we analyze the expression of these novel MMPs in murine
tissues with the finding of their predominant presence at sites of
embryo implantation.
Materials--
A high density gridded mouse P1 artificial
chromosome (PAC) genomic library was supplied by the Human Genome
Mapping Resource Center (Cambridgeshire, UK). Restriction endonucleases
and other reagents used for molecular cloning were from Roche Molecular Biochemicals (Mannheim, Germany). Oligonucleotides were synthesized in
an Applied Biosystems (Foster City, CA) model 392A DNA synthesizer. Double-stranded DNA probes were radiolabeled with
[ Screening of a Mouse Genomic Library--
The mouse PAC genomic
library was hybridized with a [ cDNA Cloning of Mouse Mcol-A and
Mcol-B--
Oligonucleotides derived from the coding yexons of the
previously isolated genomic DNA sequences were used as primers for RT-PCR amplification of RNA from mouse embryos using the RNA-PCR kit
from PerkinElmer Life Sciences. All PCR assays were carried out in a
GeneAmp 2400 or 9700 PCR system from PerkinElmer Life Sciences.
Full-length cDNA of Mcol-A and Mcol-B was obtained by RT-PCR
amplification and further assembly of two overlapping fragments of each
gene, covering from the ATG sequence to the stop codon of the
previously identified genomic fragments.
Nucleotide Sequence Analysis--
DNA fragments of interest were
sequenced by the dideoxy chain termination method, using the Sequenase
Version 2.0 kit (U.S. Biochemicals, Cleveland, OH), and the ABI-Prism
DNA sequencer (Applied Biosystems). Computer analysis of DNA and
protein sequences was performed with the GCG software package of the
University of Wisconsin Genetics Computer Group. A phylogenetic tree
directed to examine the evolutionary relationships between human and
mouse MMPs clustered in human chromosome 11 and mouse chromosome 9 was constructed on-line at the United Kingdom Human Genome Mapping Project Resource Center, using PIE, which provides a
www interface to programs included in the PHYLIP software package.
Fluorescent in Situ Hybridization on Mouse
Chromosomes--
Labeling of the probes was performed by using 2 µg
of PAC or BAC DNA in a nick translation reaction with biotin-16-dUTP.
Biotinylated probes were hybridized to mouse male metaphase chromosomes
and detected using two avidin-fluorescein layers. Chromosomes were diamine-2-phenylindole dihydrochloride-banded, and images were captured
in a Zeiss axiophot fluorescence microscope equipped with a
charge-coupled device camera (Photometrics). The specific probe for mouse chromosome 9 was the telomeric probe BAC 55J6 corresponding to the marker D9Mit152 (14).
Northern Blot Analysis--
Nylon filters containing 20 µg of
RNA of murine tissues were prehybridized at 42 °C for 3 h in
50% formamide, 5× SSPE (1× = 150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA,
pH 7.4), 10× Denhardt's solution, 2% SDS, and 100 µg/ml denatured
herring sperm DNA, and then hybridized for 20 h under the same
conditions. Filters were washed with 0.1× SSC, 0.1% SDS for 2 h
at 50 °C and exposed to autoradiography. RNA integrity and equal
loading was assessed by hybridization with an actin probe.
In Situ RNA Hybridization--
Digoxigenin-11-UTP-labeled
single-stranded RNA probes were prepared with digoxigenin RNA-labeling
mix and the corresponding T3 or T7 RNA polymerase (Roche Molecular
Biochemicals) according to the manufacturer's instructions. Mcol-A
probe was a 770-bp BamHI fragment, Mcol-B probe was a 700-bp
BamHI/HindIII fragment, and MMP-9 probe was a
1353-bp BamHI fragment, and all of them were subcloned in
pBluescript (Stratagene) vector. In situ hybridization was
performed on paraffin-embedded tissue sections from 9.5-day postcoitum
(dpc) mouse embryos or 10.5-dpc rat embryos, essentially as described
(15).
Construction of Expression Vectors for Mcol-A and Mcol-B and
Expression in Escherichia coli--
1.3-kbp fragments of the cDNAs
encoding the prodomain, catalytic domain, and hemopexin domains of
these proteins were generated by PCR amplification with primers
5'-ggctcgagaTTCCCTGTGATTCAGGAT-3' and
5'-ggaattcTTAGCAGTTGAACCAAGTATTAAT-3' for Mcol-A, and
5'-ggctcgagaTTCCCTGTGTTTCACAACG-3' and
5'-ggaattcTTTCCATTAACTTGATAAGG-3', for Mcol-B. The PCR-amplified product was cloned in the pRSETB expression vector and transformed into
BL21(DE3)pLysS-competent E. coli cells. After induction with isopropyl-1-thio- Enzyme Assays--
Enzymatic activity of purified recombinant
Mcol-A and Mcol-B against fibrillar collagens was followed by SDS-PAGE.
All assays were performed in 50 mM Tris/HCl, 5 mM CaCl2, 150 mM NaCl, and 0.05%
(v/v) Brij-35, pH 7.6, for 16 h at 37 °C (17). The
enzyme/substrate ratio (w/w) used in these experiments was 1/10.
Enzymatic activity was also analyzed using the synthetic fluorescent
substrates Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (QF-24),
Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2 (QF-35), and
Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 (QF-41)
(provided by C. G. Knight, University of Cambridge, UK). Routine
assays were performed at 37 °C at substrate concentrations of 1 µM in an assay buffer of 50 mM Tris/HCl, 5 mM CaCl2, 150 mM NaCl, 0.05% (v/v)
Brij-35, pH 7.6, with a final concentration of Me2SO of 1%
(17). The fluorometric measurements were performed using an LS50B
PerkinElmer Life Sciences spectrofluorometer. Enzyme concentrations
were determined by active site titration using a standard TIMP-1
solution following 4-h preincubation to allow complex formation (17).
Collagenolytic activity was determined by incubating soluble rat type I
collagen (18) or acid-soluble bovine type II collagen (Biogenesis Inc.,
Poole, UK) with the recombinant enzymes at 25 °C, and the
degradation products were demonstrated by SDS-PAGE. Additionally, we
determined the specific collagenolytic activity of Mcol-A using
14C-labeled rat type I collagen in a fibrillar assay at
35 °C essentially as described (18). The activity of both murine
orthologues against 14C-labeled gelatin and casein was
determined by overnight incubation at 37 °C.
Homology Modeling--
Three-dimensional models of catalytic
domains of Mcol-A and Mcol-B were calculated using a semiautomated
modeling server (19) and analyzed with the Swiss-PdbViewer. Briefly,
the amino acid sequences of the respective catalytic domains were
compared with the sequences of the macromolecules deposited in the
Protein Data Bank to identify suitable templates. We chose nonredundant
proteins that had the highest structural quality, and high similarity
with Mcol-A and Mcol-B. The pdb files corresponding to these proteins are 2TCL (human MMP-1), 1JAN (human MMP-8), the B chain of file 830C
(human MMP-13), and 1SLM (human MMP-3). The templates were superimposed
and aligned structurally. Then, the target sequences were automatically
threaded over the structure, built with ProMod II, and energy-minimized
with Gromos96. The models were analyzed with Swiss-Pdb Viewer, whereas
the electrostatic calculations were performed with MolMol (20). Charges
of conserved ions were also included in the calculations: two
Zn2+ and two Ca2+ for the catalytic domain, as
present in 2TCL. The figures were modeled with MolMol and rendered with
Megapov and POV-Ray (from the POV-Ray site on the Web).
Identification, Cloning, and Sequence Analysis of Two Novel
MMPs--
To identify putative murine MMPs structurally related to
human interstitial collagenase (MMP-1), we screened a mouse PAC genomic library using as a probe a full-length cDNA coding for this human protease. After hybridization under low stringency conditions, several
PAC clones were selected on the basis of positive hybridization to the
probe. The inserts contained in these clones were characterized by
endonuclease restriction analysis and selected fragments showing hybridization with the MMP-1 cDNA probe were cloned and subjected to nucleotide sequencing. This analysis allowed the identification of
two DNA fragments, derived from PAC 528 C11, whose nucleotide sequences
were similar to those previously determined for other murine MMPs.
Further sequence analysis of these fragments and comparison with the
exon-intron distribution of other MMP genes led us to identify several
putative exons of a presumably novel MMP gene. To try to determine the
complete structure of this MMP, studies were undertaken to isolate a
full-length cDNA encoding this enzyme. To do that, two primers
covering the start and stop codons identified in the putative first and
last exons of the cloned MMP gene were synthesized and used for RT-PCR
amplification of total RNA obtained from mouse embryos. The
PCR-amplified product was cloned, and its identity was confirmed by
nucleotide sequencing. Computer analysis of the obtained sequence (Fig.
1A) revealed an open reading
frame coding for a protein of 464 amino acids with a predicted
molecular mass of 53.5 kDa, which was tentatively called Mcol-A
(Murine collagenase-like
A).
Further analysis, of additional clones obtained by RT-PCR amplification
of murine embryos RNA with oligonucleotides derived from the sequence
determined for Mcol-A, revealed the presence of sequences highly
related to but distinct from that determined for this novel MMP. A
full-length cDNA for this apparently distinct MMP was isolated
following the same strategy as above and then characterized by
nucleotide sequencing. Analysis of the resulting sequence (Fig.
1B) allowed the finding of an open reading frame encoding a
protein of 463 residues, with a calculated molecular mass of 53.5 kDa,
and tentatively called Mcol-B. Genomic clones for this second MMP gene
were also identified from DNA fragments obtained from PAC 519 F1 and
allowed to confirm the sequence determined by analysis of the cDNA
amplified by RT-PCR of murine embryos RNA. A comparison of the deduced
amino acid sequences determined for Mcol-A and Mcol-B showed that they
were closely related, exhibiting about 82% identities between them.
Pairwise comparisons for sequence similarities between the identified
amino acid sequences (Fig. 1C) and those determined for
other murine MMPs showed that the maximum percentage of identities was
found with mouse neutrophil collagenase (MMP-8) (48% and 45% with
Mcol-A and Mcol-B, respectively). Interestingly, a higher percentage of
identities (58% in amino acids and 74% in nucleotides) was found with
human interstitial collagenase (MMP-1). This comparative sequence
analysis also revealed that both Mcol-A and Mcol-B display all
structural features characteristic of archetypal MMPs, including signal
sequences, prodomain regions with the conserved Cys residues in the
conserved PRCGVPD motif (at positions 87-93), catalytic, and hemopexin
domains (Fig. 1C). The percentage of identities of each
domain of the murine proteins with human MMP-1 is 53% (prodomain),
63% (catalytic), and 59% (hemopexin) in the case of Mcol-A, and 53%
(prodomain), 58% (catalytic), and 61% (hemopexin) in the case of
Mcol-B. The amino acid sequence deduced for Mcol-A and McolB contains
three and two potential sites of N-glycosylation,
respectively, including the one at position 117 absolutely conserved in
the catalytic domain of collagenases, macrophage metalloelastases,
stromelysin-1 and -2, gelatinase B, and MT-MMPs.
To further explore the structural relationship between human MMP-1
and murine Mcol-A and Mcol-B, we next performed a more detailed
sequence analysis with special emphasis aimed at comparing a series of
residues conserved in all collagenases described to date and proposed
as essential determinants of collagenase specificity. These residues
include Tyr-210, Asp-231, and Gly-233 according to human MMP-1
numbering (5, 21). The equivalent residues at these three positions in
Mcol-A are Phe-208, Asp-229, and Gly-231, whereas in Mcol-B these
residues are Phe-208, Asp-229, and Glu-231, respectively (Fig.
1C). Therefore, it seems that Mcol-A is more related to
collagenases than Mcol-B at least in terms of occurrence of residues
important for this activity. This structural analysis also revealed
that both Mcol-A and Mcol-B contain an RGD (Arg-Gly-Asp) motif in the
catalytic domain. This motif is present at equivalent position in the
MMP-1 sequence from all species in which this protein has been
characterized, but not in other MMPs, providing additional evidence on
the structural relationship between MMP-1 and the newly identified
family members Mcol-A and Mcol-B. By contrast, both enzymes lack the
nine-residue insertion present in the hinge region of all stromelysins.
They also lack the fibronectin-like domain present in gelatinases, the
C-terminal extension rich in hydrophobic residues characteristic of
MT-MMPs, and the furin activation motif (RX(R/K)R)
mediating the intracellular activation of MT-MMPs and stromelysin-3
(22, 23). In summary, and taking collectively all these structural
comparisons, most data point to the inclusion of Mcol-A and Mcol-B as
members of the collagenase subfamily, although they cannot be
unequivocally classified within this group on the exclusive basis of
their amino acid sequence characteristics.
Physical Mapping of Mcol-A and Mcol-B Genes--
To determine the
chromosomal location of murine genes encoding Mcol-A and Mcol-B,
metaphase spreads from a male mouse were hybridized with the
biotinylated PACs 528 C11 and 519 F1 enclosing these genes and with the
telomeric marker of chromosome 9, BAC 55J6. After single- and
double-fluorescent in situ hybridization experiments with both
probes, fluorescent signal corresponding to Mcol-A and
Mcol-B genes was located to the A1-A2 region of chromosome
9 (Fig. 2). Other murine MMP genes
(MMP-7, -12, -13, and -20)
have been already mapped to this region (24-27), which is syntenic to
human chromosome 11q22-23 in which at least eight human MMPs are
clustered in a relatively small region (28, 29). To establish the
relative order of Mcol-A and Mcol-B loci within the cluster of MMP genes in mouse chromosome 9, DNA was isolated from
the YAC clone I139A1, which contains murine MMP-8 and
MMP-13 (11) as well as the new Mcol-A and
Mcol-B, as tested by PCR. DNA from this YAC was digested
with different rare-cutting restriction endonucleases (ClaI,
MluI, NarI, NruI, SacI,
SalI, SfiI, SpeI), and the resulting
fragments were separated by pulsed-field gel electrophoresis. After
blotting, DNA was successively hybridized with probes specific for
Mcol-A and Mcol-B, as well as with probes for
other MMP genes potentially contained within the analyzed YAC
(MMP-3, MMP-7, MMP-8, MMP-10, MMP-13). In addition,
hybridization with those probes was performed on the DNA of the seven
isolated PAC clones immobilized on a nylon filter (Table
I, and data not shown). The results
obtained after hybridization of the filters containing the YAC DNA
separated by pulsed-field gel electrophoresis and of those containing
the DNA from the different positive PACs were combined. Thus, a common
MluI band of about 200 kbp was detected by Mcol-A, Mcol-B,
MMP-3, MMP-8, and MMP-10 probes. MMP-10 and Mcol-A but not MMP-8 probes
shared 125-kbp NarI and 50-kbp ClaI bands. MMP-3
and Mcol-B but not Mcol-A probes detected a common NruI band
of 80 kbp. Taken collectively, these data show that the order of the
novel genes within the cluster would be compatible with the following:
MMP-8/MMP-10/Mcol-A/MMP-3/Mcol-B/MMP-12/MMP-13. The order and orientation of seven human MMP genes clustered on the
long arm of chromosome 11 is:
centromere/MMP-8/MMP-10/MMP-1/MMP-3/MMP-12/MMP-7/MMP-13/telomere (28). Therefore, we can conclude that Mcol-A is located in a position equivalent to that of MMP-1 in the human genome.
Nevertheless, further studies derived from the current human and mouse
genome projects will be required to confirm this possibility. In this regard, it should be mentioned that after submission of this manuscript two short genomic sequences (AZ349151 and AZ443051) containing partial
information for Mcol-A and Mcol-B have been
released to the Genome Survey Sequence data bank. According to our gene
structure analysis,2 AZ349151
would contain the exon 2 of Mcol-A, whereas AZ443051 would
contain information for exon 7 of Mcol-B.
Production of Recombinant Mcol-A and Mcol-B in E. coli and Analysis
of Their Enzyme Activity--
According to the above described data,
Mcol-A and Mcol-B have some structural features characteristic of
members of the collagenase subfamily of MMPs, and we therefore
expressed them in E. coli, refolded, and purified them as
described under "Experimental Procedures." Our protocol was
originally established to successfully refold procollagenase-3 and a
transmembrane deletion mutant of MT1-MMP and allows the correct folding
of collagenolytic MMPs (16). Therefore, we used this strategy to carry
out a preliminary analysis of the ability of both refolded Mcol-A and
Mcol-B to cleave triple-helical collagens. In addition, it's
remarkable that SDS-PAGE analysis of the refolded proteins under
reducing and nonreducing conditions revealed that they migrated faster
under nonreducing conditions, indicating correct folding of the
C-terminal domain, a prerequisite for our analysis of collagenolysis.
Mcol-A was autoproteolytically converted to the active enzyme form
(Mr 44,000) during the dialysis step
employed to remove the imidazole from the enzyme preparation after
purification using nickel-nitrilotriacetic acid-agarose (not shown). In
contrast, a large proportion of the Mcol-B preparation was still in the
proenzyme form (Mr 56,000 and
Mr 54,000) following purification and dialysis,
with minor bands at 46,000 and 43,000. It was, however, noted that
Mcol-B underwent autoproteolytic conversion to these two lower
molecular species (Mr 46,000 and
Mr 43,000) when incubated for 24 h at
25 °C, a process that was inhibited by EDTA (not shown). When both
enzyme preparations were analyzed for enzymatic activity, only Mcol-A
was able to hydrolyze triple-helical type I and type II collagen into
three-fourths and one-fourth fragments (Fig.
3), whereas Mcol-B remained inactive.
These data indicate that, although we were unable to show enzymatic
activity for Mcol-B versus macromolecular or quenched
fluorescent peptide substrates, the propeptide domain was
autoproteolytically removed, strongly suggesting that the enzyme is
active. Nevertheless, the possibility that Mcol-B is incompletely
folded cannot be ruled out. Activation trials for Mcol-B with either
trypsin alone or in combination with stromelysin revealed no change in
the ability of the enzyme to degrade macromolecular substrates. Thus
Mcol-B might have a very restricted substrate specificity, which will need further investigation in the future.
The enzymatic activity of Mcol-A was analyzed in more detail. The
enzyme was shown to hydrolyze three quenched fluorescent peptide
substrates with distinct
kcat/Km values, summarized in
Table II. The Mcol-A activity
versus these quenched fluorescent substrates was inhibited
by TIMP-1, thereby indicating that this enzyme is a typical MMP (not
shown). Comparison of the
kcat/Km values of Mcol-A with
human MMP-1 revealed that they were considerably reduced, indicating
that, although the active site may be different, it still allows
collagenolysis. Active site titrations were performed using the
quenched fluorescent substrates and a standard TIMP-1 solution of known
concentration that allowed us to determine the specific activity of
Mcol-A against 14C-labeled rat type I collagen,
14C-labeled rat gelatin, and 14C-labeled
Homology Modeling of Mcol-A and Mcol-B--
The homology models
deduced for the catalytic domains of Mcol-A and Mcol-B show a clear
superimposable pattern with the catalytic domain of human MMP-1,
consistent with the significant sequence similarity between them (Fig.
4, A-C). Likewise, the
molecular surfaces of these domains are also very similar (Fig.
4D). Analysis of specificity determinants further strengths
the close relationship between MMP-1, Mcol-A, and Mcol-B. An essential
factor for MMP specificity is the size of the S1' pocket (30-32). The
depth of this hydrophobic pocket is largely determined by the side
chain of the residue present at position 214 (MMP-1 numbering) (Fig. 4C). Most MMPs have a Leu residue at this position, and
consequently their S1' sites are very deep and form a channel across
the protein, allowing the digestion of substrates with large P1' side
chains. Interestingly, MMP-1 as well as the novel murine enzymes have large residues at this position (Arg and Tyr, respectively), occluding the S1' channel and leaving a cavity that can only accept middle-sized substrates (Fig. 4C) (33, 34). The character of the S1
subsite depends mainly on residue 180, which is hydrophilic in MMP-1, Mcol-A, and Mcol-B (Asn, Lys, and His, respectively) and hydrophobic in
the other MMPs. Taken together, these structural data support that the
novel murine enzymes are more closely related to MMP-1 than to other
MMPs. Furthermore, analysis of the molecular models depicted for Mcol-A
and Mcol-B provide some clues to explain the observed differences in
the catalytic activity of both enzymes. Thus, residue 181 is Leu in
most MMPs, including Mcol-A and MMP-1, but Mcol-B is unique by
possessing a Phe residue at this position. This bulky residue could
hamper the access of the substrate to the active site cleft (Fig. 4,
A and B). In addition, there is a series of
residues in Mcol-B that could be important in terms of catalytic
differences with M-colA. These include the Gly residue at position 233 (MMP-1 numbering) that is absolutely conserved in all collagenases but
in Mcol-B is replaced by Glu, as well as the acidic residues 194 and
201 involved in calcium binding in MMP-1, which are changed to amide
residues (Asn and Gln, respectively) in Mcol-B (Fig.
1C).
Analysis of Mcol-A and Mcol-B Expression during Murine
Embryogenesis--
To study Mcol-A and Mcol-B
expression in murine tissues, we first performed RT-PCR amplification
using specific oligonucleotides and RNA prepared from a variety of
tissues (uterus, kidney, ovary, lung, and placenta) and embryos at
different stages of development. These analyses revealed that
Mcol-A and Mcol-B were expressed during fetal
development. Mcol-A and Mcol-B expression during murine
embryogenesis was also confirmed by Northern blot analysis (Fig.
5). Specific hybridization with Mcol-A
probe was detected in the yolk sac and uterine tissue adjacent to mouse
embryos at 9.5 and 10.5 dpc but not in tissue from embryos of 13.5, 15.5, 17.5, and 19.5 dpc. This specific hybridization signal was also observed in the rat. In this case, bands were observed in the yolk sac
and uterine tissue adjacent to rat embryos of 11.5 and 12.5 dpc. These
days of gestation correspond to the beginning of development of the
chorioallantoic placenta. Hybridization was also detected in RNA
obtained from placenta at 13.5, 15.5, and 17.5 dpc. To examine the
identity of the cells responsible for the production of Mcol-A and
Mcol-B in the murine tissue during embryogenesis, we carried out an
in situ hybridization on tissue sections of rat and mouse
embryos and adjacent tissue from 8.5 to 16.5 dpc. A clear expression
for both Mcol-A and Mcol-B was found in a low
number of extra-embryonic cells located at the maternal interface (Fig.
6). By contrast, no transcripts were detected in either the embryo or the maternal decidua. Hybridizations with the two probes in adjacent serial sections demonstrated that both
enzymes were expressed in the same cells, although staining for Mcol-B
was much weaker. Expression of both genes was restricted to a network
of cells at the periphery of the embryo in contact with the adjacent
decidual cells. Positive cells were morphologically identified as
trophoblast giant cells. To further identify these positive cells,
adjacent serial sections were hybridized with antisense probes for
MMP-9, which is considered as a typical marker for terminally
differentiated trophoblast giant cells (35). Cells positive for
Mcol-A and Mcol-B showed high expression levels of MMP-9, although cells expressing MMP-9 but not
Mcol-A and Mcol-B were also found. Detection of
Mcol-A and Mcol-B transcripts was restricted to sections of 9.5 and
10.5 dpc in mice and 10.5 and 11.5 dpc in rat embryos. In both cases
the expression level significatively decreased with age of embryos and
virtually no expression was found in mouse embryos older than 10.5 dpc.
This work describes the identification of two murine
metalloproteases Mcol-A and Mcol-B, cloned as a result of their
positive hybridization with a probe for human MMP-1. According to their structural and functional characteristics, we propose that at least one
of these novel members of the MMP family, Mcol-A, may play roles as
interstitial collagenase in murine tissues and could represent a true
orthologue of human MMP-1.
Over the last years, the number and identity of collagenolytic enzymes
produced by murine tissues has been a debated question within the MMP
field. Studies performed by several groups have demonstrated the
existence in mouse and rat cells and tissues of the corresponding
orthologues of neutrophil collagenase (MMP-8) (11) and collagenase-3
(12, 13). However, and somewhat surprisingly, to date no evidence of
occurrence of murine interstitial collagenase (MMP-1) has been
reported. This is specially puzzling if we consider that MMP-1 was
likely responsible of at least part of the collagenolytic activity
discovered by Gross and Lapière in 1962 from the tail of the
metamorphosing tadpole (36), as well as the first human MMP cloned and
characterized at the amino acid sequence level (21). Furthermore, MMP-1
orthologues have been identified and cloned in a number of species
including Homo sapiens, Bos taurus, Sus
scrofa, Oryctolagus cuniculus, and Rana
catesbiana (21, 37-39). A partial cDNA sequence presumably
encoding a C-terminal fragment of the guinea pig MMP-1 has been also
reported, although it has been proposed that the guinea pig is more
closely allied with lagomorphs than with rodents (40). Our approach to
the identification of a putative murine orthologue of MMP-1 involved a
combined strategy based on screening of a mouse PAC genomic library
with a human MMP-1 cDNA probe, followed by RT-PCR amplification of
mouse embryo RNA with oligonucleotides derived from the sequence of
genomic clones hybridizing with the MMP-1 probe. This strategy led us
to identify two murine cDNAs coding for proteins with a series of
structural features present in MMPs, and more specifically in members
of the collagenase subfamily. Both Mcol-A and Mcol-B were most similar
to human interstitial collagenase (MMP-1). The overall identities (74%
in nucleotides and 58% in amino acids) are maintained throughout the
different domains of these proteins and are similar to those found in
the comparison of mouse and human MMP-12 (76% in nucleotides and 61%
in amino acids) but are lower than those shared by mouse and human
orthologues of most MMPs. Nevertheless, both Mcol-A and Mcol-B contain
a characteristic RGD motif present in MMP-1 from all species in which
this collagenase has been characterized but not in other MMPs.
Conversely, both enzymes are devoid of any structural features defining
members of other MMP subfamilies such as stromelysins, gelatinases, and MT-MMPs. An analysis of a phylogenetic tree constructed to evaluate the
evolutionary relationships of mouse Mcol-A and Mcol-B to other MMPs
also revealed that human MMP-1 was the most closely related to these
novel proteases (Fig. 7). Finally,
molecular modeling experiments based on the crystal structures known
for diverse MMPs have confirmed that the overall fold of the catalytic
domains of Mcol-A and Mcol-B is topologically very similar to that of MMP-1, including the small size of the S1' specificity pocket. Nevertheless, there are considerable differences at the amino acid
sequence level between Mcol-A and Mcol-B, which might be involved in
structural features that determine different enzymatic activities.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (3000 Ci/mmol) from Amersham Pharmacia
Biotech (Buckinghamshire, UK) using a commercial random-priming kit
purchased from the same company.
-32P]dCTP-labeled
cDNA probe corresponding to full-length human MMP-1 probe (ATCC
number 57684). Hybridization and washes were performed at 60 °C.
After autoradiographic exposure of the filters, 24 positive clones were
detected, and 7 of them were further analyzed by extensive Southern
blotting and DNA sequencing of isolated fragments.
-D-galactopyranoside (0.5 mM final concentration), inclusion bodies were prepared and
the enzymes were refolded as described previously (16). For comparative
purposes we refolded human MMP-13 and -14 using this protocol and
established that these enzymes displayed collagenolytic activity, as an
indication of correct folding of the C-terminal hemopexin-like domain,
which determines collagenolytic ability in all known human collagenases.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence and deduced amino acid
sequences of mouse Mcol-A and Mcol-B. The deduced amino acid
sequences for Mcol-A (A) and Mcol-B (B) are shown
below the nucleotide sequences. Potential sites for
N-glycosylation are underlined. C,
comparison of the amino acid sequences of mouse Mcol-A and Mcol-B with
mouse collagenases (MMP-8 and MMP-13), human MMP-1, and stromelysins.
The multiple alignment was performed with the PILEUP program of the GCG
package. Identical residues in all sequences are shadowed in
gray. RGD residues exclusive of MMP-1 are
underlined. Residues specific of collagenases are in
bold and marked with an asterisk.
Numbering refers to Mcol-A.
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Fig. 2.
Chromosomal mapping of mouse
Mcol-A. Fluorescent in situ
hybridization of mouse metaphase spreads was performed with the
biotinylated PAC 528 C11 and BAC 55J6. Telomeric hybridization signal
on chromosome 9 was obtained with the specific probe from BAC 55J6. PAC
528 C11 hybridized to the A1-A2 region of chromosome 9. Metaphase
cells were counterstained with diamidine-2-phenylindole
dihydrochloride.
Results of the dot blot hybridization of the indicated PACs with
different MMP probes
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Fig. 3.
Degradation of fibrillar type I and II
collagen by Mcol-A and Mcol-B. Lane 1, Mcol-B was
incubated with type I collagen for 24 h at 25 °C; lane
2, Mcol-A was incubated with type I collagen for 24 h at
25 °C; lane 3, type I collagen buffer control; lane
4, Mcol-B was incubated with type II collagen for 24 h at
25 °C; lane 5, Mcol-A was incubated with type II collagen
for 24 h at 25 °C; lane 6, type II collagen buffer
control. The reaction products were analyzed by SDS-PAGE and Coomassie
Blue staining. The positions of the respective three-fourths and
one-fourth fragments are indicated by arrows on the
left and right.
-casein. The results obtained are shown in Table
III, which also shows data for human
MMP-1. Our data revealed that, although 14C-labeled rat
type I collagen represents a substrate for Mcol-A, the enzyme was
unable to hydrolyze gelatin. This is a very surprising result, because
all known human collagenases hydrolyze gelatin and thus Mcol-A may
represent a more specific collagenase, needing the triple-helical
conformation for activity. Additionally, 14C-labeled
-casein was also cleaved by Mcol-A, but the specific activity
determined was extremely low (Table III).
Determination of kcat/KM for three quenched fluorescent
peptide substrates for Mcol-A and Mcol-B
Determination of the specific activity of Mcol-A and Mcol-B versus
macromolecular substrates (units) expressed as micrograms of collagen
fibril solubilized/min
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Fig. 4.
Homology models of the catalytic domains of
Mcol-A and Mcol-B. A, ribbon representations
of the models of Mcol-A and Mcol-B superimposed to the catalytic domain
of human MMP-1. The RO 31-4724 substrate analogue of MMP-1 and the
histidine side chains that coordinate the catalytic Zn are also shown.
B, detailed view of the interaction between RO 31-4724 and
residues Leu-181 of Mcol-A, and Phe-181 of Mcol-B. C,
cross-section of the catalytic domains of MMP-1, MMP-8, and modeled
Mcol-A showing the different shapes of the S1' pocket. The residues in
position 214, which determine the size of this pocket, are also shown.
MMP-1 and MMP-8 substrate analogs are shown in blue and
green, respectively. D, molecular surface of the
catalytic domains of MMP-1, Mcol-A, and Mcol-B. Standard view showing
the active site of the molecules. Electrostatic potentials lower than
1.8 V are in red, higher than 1.8 V are in
blue, and neutral is in white. Intermediate
values are interpolated.
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Fig. 5.
Expression of Mcol-A and
Mcol-B in mouse tissues. A, RT-PCR was performed
on 1 µg of RNA from whole embryos or placenta at the indicated days
of embryonic development, with specific oligonucleotides for
Mcol-A and Mcol-B as primers. 20 µl of the
final product were separated on a 2% agarose gel. Bl
lane shows RT-PCR performed without added template. The
standard lane is Marker V from Roche Molecular Biochemicals.
B, samples of 20 µg of total RNA from whole embryo
(W.E.), yolk sac (Y.S.), or placenta
(P.) at the indicated days of development were separated by
agarose gel electrophoresis under denaturing conditions, blotted onto
nylon filters, and analyzed by hybridization with full-length cDNA
for Mcol-A. Filters were exposed to autoradiography at 70 °C for 7 days with Kodak BIOMAX MS films and screens. The positions of the 28 S
and 18 S RNA are indicated.
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Fig. 6.
In situ hybridization for Mcol-A,
Mcol-B, and gelatinase B in mouse uterus and feto-maternal placental
tissues. Hybridization was performed on transverse serial sections
of an 8.5-dpc mouse uterus, including fetomaternal placental
tissues. As can be observed in A and B, signal
for Mcol-A (arrowheads) is found in a low number of cells
located at the periphery of the embryo (em) in contact with
adjacent decidual cells (de). B, a higher
magnification of cells positive for Mcol-A; C, the same
region in a parallel section stained with Gill's hematoxylin showing
that positive cells can be morphologically identified as trophoblast
giant cells (arrowheads). D, in situ
hybridization for Mcol-B showing a weaker but specific signal also in
trophoblast giant cells. E, in situ hybridization
with gelatinase B antisense probe showing intense labeling in a higher
number of trophoblast giant cells. Original magnifications:
A, ×40; B, C, and D,
×100; E, ×64.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Schematic illustration of evolutionary
relationships between human and mouse MMPs. The phylogenetic tree
includes the diverse MMPs clustered in human chromosome 11 and mouse
chromosome 9 and was constructed on-line at the United Kingdom Human
Genome Mapping Project Resource Center, using PIE, which
provides a www interface to programs included in the PHYLIP
software package.
In addition to the above features that suggest that both murine enzymes are structurally related to MMP-1, we have also provided functional evidence that at least Mcol-A exhibits the ability to act as a collagenolytic enzyme. In fact, recombinant Mcol-A displays proteolytic activity against type I and type II fibrillar collagens, although its specific activity versus fibrillar type I collagen is much lower than that described for human MMP-1 or MMP-13. Mcol-B is apparently devoid of collagenolytic activity, although it can autoactivate when incubated for 24 h at 25 °C or stored for prolonged periods of time at 4 °C. On the other hand, genomic studies have indicated that both Mcol-A and Mcol-B exhibit the same exon-intron distribution as human MMP-1, and their proximal promoter region is significantly similar to that of human MMP-1.2 Furthermore, fine chromosomal mapping of the region containing these murine genes has revealed that Mcol-A seems to be located at a position syntenic to the MMP-1 locus in the human genome. On these bases, together with the above enzymatic analysis, we can conclude that Mcol-A is closer to MMP-1 than Mcol-B in both structural and functional terms. Thus, Mcol-A could be a homologue of MMP-1 in murine tissues, Mcol-B being the result of a specific gene duplication event that has retained a number of MMP-1 features but that has also accumulated some changes resulting in an impairment of its ability to act as a collagenolytic enzyme. As mentioned above, sequence comparisons and molecular modeling of the catalytic domains of these enzymes have suggested some specific features of Mcol-B that could contribute to the observed catalytic differences with Mcol-A and human MMP-1. Nevertheless, further studies involving site-directed mutagenesis experiments will be required to elucidate the molecular basis for the differential activities among all these closely related members of the MMP family.
To investigate the functional role of these novel MMPs, we have also examined the tissue distribution of both Mcol-A and Mcol-B in murine tissues. According to RT-PCR and Northern blot analysis, these enzymes are mainly produced in yolk sac and uterine tissue adjacent to mouse embryos at early times during implantation. In situ hybridization demonstrated that expression of both genes is restricted to trophoblast giant cells present at the embryo/maternal interface, although in all cases, the expression level of Mcol-A is higher than that of Mcol-B. According to these expression analysis, it is tempting to speculate that these novel murine proteases may participate in embryo implantation. The implantation of the mammalian embryo into the uterine stroma is a highly controlled process of tissue invasion that involves extensive remodeling of extracellular matrix components to accommodate the growing embryo as well as to establish the vascular structures necessary for transplacental exchange (41, 42). This process is initiated by the attachment of the blastocyst to the uterine epithelium on day 4.5 of mouse development and ceases on day 10.5, with placental function beginning on day 11. The observation that both Mcol-A and Mcol-B are produced by trophoblast cells on days 9.5-10.5 suggests that these enzymes may contribute to the final stages of the invasive process. Similarly, the observation that gelatinase B, whose expression peaks at 7.5 days, colocalizes with Mcol-A and Mcol-B could be indicative of the occurrence of a putative proteolytic cascade involving these proteases. Nevertheless, the possibility that Mcol-A and Mcol-B are involved in other processes distinct from blastocyst invasion, including angiogenesis regulation or collagen turnover accompanying decidualization, cannot be ruled out. It is also noteworthy in the context of the putative relationships between these novel murine MMPs and human MMP-1, that this interstitial collagenase has been also found to be produced by trophoblastic cells during human pregnancy (43, 44). These data suggest that the parallelisms between human MMP-1, and murine Mcol-A and Mcol-B, could be extended to their respective expression patterns. However, it is still unclear if the murine enzymes will share its wide distribution in processes such as wound healing or tumor progression, in which the presence of human MMP-1 has been repeatedly described (45, 46).
In conclusion, our structural and functional analysis suggest that
Mcol-A and Mcol-B can be considered as putative murine counterparts of
MMP-1, although it seems that they have diverged much more rapidly than
other mouse and human MMP orthologues. Furthermore, on the basis of
differences between both enzymes, it is tempting to speculate that
Mcol-A is functionally closer to MMP-1 than Mcol-B. Further studies
will be required to provide definitive evidence on the proposal that
the newly identified murine MMPs, and, more specifically, Mcol-A
represent structural and functional counterparts of MMP-1. It will be
also of interest to evaluate if the observed structural divergence
between them may underlie diverging functional roles for these
proteolytic enzymes.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. G. Velasco, A. M. Pendás, I. Santamaría, J. M. P. Freije, X. S. Puente, M. J. Jiménez, and J. A. Uría for helpful comments, and S. Álvarez for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants from the Plan Feder (1FD97-0214), EU-BIOMED II (BMH4-CT96-0017), the Arthritis and Rheumatism Council (to G. M.), and the Wellcome Trust (to V. K.). The Instituto Universitario de Oncología is supported by Obra Social Cajastur-Asturias.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.
Recipient of a predoctoral fellowship from Ministerio de
Educación, Spain.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ278461 and AJ278462.
§ To whom correspondence should be addressed: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Tel.: 34-985-104202; Fax: 34-985-103564; E-mail: mbf@sauron.quimica.uniovi.es.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M009586200
2 M. Balbín and C. López-Otín, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: MMP, matrix metalloproteinase; MT, membrane-type; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcriptase-polymerase chain reaction; TIMP, tissue inhibitor of metalloproteinases; PAC, P1 artificial chromosome; dpc, days postcoitum; bp, base pair(s); kbp, kilobase pair(s); Mca, (7-methoxycoumarin-4-yl)-acetic acid; Nva, norvaline; Dpa, L-dinitrophenyl-diamino propionic acid; Cha, cyclohexyl alanine.
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