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INTRODUCTION |
Matrix metalloproteinases
(MMPs)1 or matrixins are a
family of zinc-dependent endopeptidases that degrade the
different extracellular matrix proteins at a neutral pH. These enzymes
are produced by many cell types, usually in response to disease
processes associated with inflammation or tumor progression. In
addition, they have been implicated in the connective tissue remodeling
occurring in normal processes such as embryonic development, bone
growth, or wound healing (1-4). In recent years, the number of known members of the family has grown after the discovery of a series of new
family members identified in both normal or pathological conditions. To
date, 16 human MMPs have been cloned and characterized at the amino
acid sequence level (3, 5). They can be classified into at least four
main subfamilies, according to their substrate specificity, primary
structures, and cellular localization: the collagenases, gelatinases,
stromelysins, and membrane-type MMPs (MT-MMPs). However, there are some
recently described enzymes like macrophage metalloelastase (6),
stromelysin-3 (7), MMP-19 (8), and enamelysin (5), which do not appear
to fall into any of these subfamilies. In addition to all these MMPs
identified in human tissues, distinct MMPs have been also cloned from
Xenopus laevis (9, 10), embryonic sea urchin (11), green
alga (12), soybean leaves (13), chicken (14), and Caenorhabditis
elegans (15). Biochemical characterization of the diverse MMPs has
opened new views on the role of these enzymes in connective tissue
remodeling processes, and evidence is accumulating that MMPs are not
exclusively involved in the proteolytic degradation of extracellular
matrix components. Thus, MMPs have been reported to play direct roles in other essential cellular processes such as differentiation, proliferation, angiogenesis, or apoptosis (16). Some of these functions
are mediated by the ability of MMPs to catalyze hydrolysis of a variety
of substrates including membrane-bound precursors of cytokines, growth
factors, or hormone receptors (17, 18), serum-amyloid A (19),
insulin-like growth factor-binding proteins (20, 21), proteinase
inhibitors (22-25), or interleukin-1
(26).
The identification of expanding roles for MMPs in a wide variety of
biological processes has stimulated the search for new family members
by using improved cloning strategies. Recently, we have utilized
PCR-based methods with degenerate oligonucleotides, and expressed
sequence tag (EST)-based approaches for cloning different human MMPs
from both normal or tumor tissues (5, 8, 27, 28). In this work, we have
examined the possibility that additional yet uncharacterized MMPs could
be produced by human tissues, with the finding of a novel family member
tentatively called MMP-23. We describe the molecular cloning and
complete nucleotide sequence of a cDNA coding for this proteolytic
enzyme. We also report the expression of the gene in Escherichia
coli and perform a preliminary analysis of the enzymatic activity
of the recombinant enzyme. Finally, we report the chromosomal location of the MMP-23 gene in the human genome and analyze its expression in
human tissues.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction endonucleases and other reagents used
for molecular cloning were from Boehringer Mannheim (Mannheim,
Germany). Synthetic oligonucleotides were prepared in an Applied
Biosystems (Foster City, CA) model 392A DNA synthesizer.
Double-stranded DNA probes were radiolabeled with
[32P]dCTP (3000 Ci/mmol) purchased from Amersham
International (Buckinghamshire, UK) using a commercial random-priming
kit from the same company. A human ovary cDNA library constructed
in
DR2 and two Northern blots containing polyadenylated RNAs from
different human tissues were from . A panel
of monochromosomal somatic cell hybrid DNAs and a human P1 artificial
chromosome (PAC) genomic library were provided by the Human Genome
Mapping Resource Center (Cambridgeshire, UK).
Probe Preparation and Screening of a Human Ovary cDNA
Library--
A search of the GenBankTM data base of human
ESTs for sequences with homology to proteases of the MMP family,
allowed us to identify a sequence (W76631; deposited by R. K. Wilson, Washington University-Merck EST Project) derived from a fetal
heart cDNA clone, and showing significant similarity with sequences
of known human MMPs. To obtain this DNA fragment, we performed PCR
amplification of a panel of cDNAs (Quick Screen,
) with two specific primers
5'-CTGGGTCCTGGGCCCCACG (primer 1) and 5'-CCATCGGGCACCAAGCC TG (primer
2) derived from the W76631 sequence. The PCR reaction was carried out
in a GeneAmp 2400 PCR system from Perkin-Elmer/Cetus for 40 cycles of
denaturation (94 °C, 15 s), annealing (61 °C, 20 s),
and extension (72 °C, 20 s). The 384-bp PCR product, amplified from human ovary cDNA, was phosphorylated with T4 polynucleotide kinase and cloned into a SmaI-cut pUC18 vector. The cloned
cDNA was sequenced and found to be virtually identical (97%
identities) to the W76631 sequence. This cDNA was then excised from
the vector, radiolabeled, and used to screen a human ovary cDNA
library according to standard procedures (29). Following plaque
purification, the cloned insert was excised by
BamHI/XbaI digestion and the resulting fragments
subcloned into pUC19.
5'-Extension of Isolated cDNAs--
The 5'-ends of cloned
cDNAs were extended by successive cycles of rapid amplification of
cDNA ends (RACE) using RNA from human ovary and the
MarathonTM cDNA amplification kit
(), essentially as described by the
manufacturer. Each cycle of RACE allowed the extension of approximately
60-100 bp of cDNA toward the 5'-end. After cloning and sequencing
the amplified products, new specific oligonucleotides were synthesized
and used for the next RACE experiment. Finally, the full-length
cDNA was obtained by PCR amplification using the Expand Long PCR
kit (Boehringer Mannheim). The PCR reactions were performed for 35 cycles of denaturation (15 s at 94 °C), annealing (15 s at
64 °C), and extension (2 min at 68 °C), with primers 5'-CTGCCCCATGCAGCCCTGAG and 5'-GAAAGTGCTTTATCAGCCGGGC. Following gel
purification, the amplification product was cloned and sequenced.
Nucleotide Sequence Analysis--
DNA fragments of interest were
cloned in the polylinker region of phage vector M13mp19 and sequenced
by the dideoxy chain termination method, using either M13 universal
primer or cDNA specific primers and the Sequenase version 2.0 kit
(U. S. Biochemical Corp.). All nucleotides were identified in both
strands. Computer analysis of DNA and protein sequences was performed
with the GCG software package of the University of Wisconsin Genetics
Computer Group (30).
Chromosomal Mapping--
DNA from a panel of 24 monochromosomal
somatic cell hybrids containing a single human chromosome in a mouse or
hamster cell line background was PCR-screened for the presence of the
genomic sequence flanked by the primers: 5'-CGAAACCACAGGCAGAC and
5'-CTGTAGGTGAGGTTGAAGTGGTC. Amplification conditions were as follows:
35 cycles of denaturation (94 °C, 15 s), annealing (51 °C,
15 s), and extension (68 °C, 20 s) using the Expand Long
PCR kit. Fluorescent in situ hybridization (FISH) mapping of
genomic DNA clones for MMP-23 was performed as described previously
(31). Briefly, genomic clones for MMP-23 were obtained from a human PAC
genomic library, screened by filter hybridization with the full-length
MMP-23 cDNA as probe. DNA from isolated PAC clones was obtained
with the standard alkaline lysis method using QIAGEN columns (QIAGEN
Inc., Chatsworth, CA), and nick-translated with biotin-16-dUTP. Then,
labeled probes were hybridized to normal male metaphase chromosomes
obtained from phytohemagglutinin-stimulated cultured lymphocytes, and
detected using two avidin-fluorescein layers (32). Chromosomes were
diamidine-2-phenylindole dihydrochloride (DAPI)-banded, and images were
captured in a Zeiss Axiophot fluorescent microscope equipped with a CCD
camera (Photometrics).
Northern Blot Analysis--
Nylon filters containing 2 µg of
poly (A)+ RNA of a wide variety of human tissues were
prehybridized at 42 °C for 3 h in 50% formamide, 5× SSPE (1×
SSPE = 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 with radiolabeled MMP-23 full-length cDNA
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.
Construction of Expression Vectors for MMP-23 and Expression in
Escherichia coli--
A 973-bp fragment of the MMP-23 cDNA
containing the catalytic and C-terminal domains was generated by PCR
amplification with primers 5'-AGACAAGCTTACACGCTGACTCCAG and
5'-AGAGAAAAA GCTTTATCAGCCGG. The PCR amplification was performed for 20 cycles of denaturation (95 °C, 15 s), annealing (54 °C,
15 s), and extension (68 °C, 2 min), followed by 10 additional
cycles of denaturation (95 °C, 15 s), annealing (62 °C,
15 s), and extension (68 °C, 2 min) using the Expand Long PCR
kit and the GeneAmp 9700 PCR system. Due to the design of the
oligonucleotides, the amplified fragment could be cleaved at both ends
with HindIII and ligated in frame into the pRSETB E. coli expression vector (Invitrogen) previously cleaved with the
same restriction enzyme. In addition, an expression vector for a
chimeric enzyme consisting of the MMP-19 propeptide domain and the
MMP-23 catalytic was constructed by overlap extension mutagenesis using
the following strategy: the propeptide domain of MMP-19 was amplified
by PCR using a coding primer 5'-GGCGCCTGCAGACTACCTGTC, which introduced
a PstI site at the 5'-end, and a noncoding primer 5'-CAGCGTGTATTTAAGGGTCTTCTGGTTGAAG GG, complementary to the end of the
propeptide domain of MMP-19 and to the amino acid residues 89-91 of
MMP-23. The catalytic domain of MMP-23 was amplified using a coding
primer 5'-ACCCTTAAATACACGCTGACTCCAGCC AGGCTG, complementary to the last
three amino acid residues of the MMP-19 propeptide and to the amino
acid residues 89-96 of MMP-23, and a non-coding primer
5'-TCGAGGAATTCGTAGAGCCGGTGCAGCCC, complementary to the amino acid
residues 258-263 of the catalytic domain of MMP-23, and containing an
EcoRI site. The two fragments were isolated from agarose
gels and extended by PCR using the outermost primers. The PCR product
was cleaved with PstI and EcoRI and subcloned into the pRSETB expression vector. The different expression vectors were transformed into BL21(DE3)pLysS-competent E. coli cells
and grown on agar plates containing chloramphenicol and ampicillin. Single colonies were used to inoculate 2-ml cultures in 2YT medium supplemented with 33 µg/ml chloramphenicol and 50 µg/ml ampicillin. 500 µl of the corresponding culture was used to inoculate 200 ml of
2YT medium containing the above antibiotics. After culture reached an
A600 of 0.6, expression was induced by addition
of isopropyl-1-thio-
-D-galactopyranoside (IPTG) (0.5 mM final concentration) followed by further incubation for
3-20 h at 30 °C. Recombinant proteins obtained in inclusion bodies
were solubilized using 20 mM Tris buffer, pH 8.0, containing 6 M urea and 5 mM dithiothreitol. The solubilized proteins were purified using Ni-NTA-agarose. Fractions containing purified recombinant proteins were combined and refolded by
dilution (1:10) into refolding buffer 20 mM Tris/HCl, pH
8.0, 5 mM CaCl2, and 50 µM
ZnCl2. Precipitated proteins were removed by centrifugation.
Enzyme Assays--
Enzymatic activity of purified recombinant
MMP-23 or chimeric MMP19/MMP23 was detected using the synthetic
fluorescent substrates McaPLGLDpaARNH2,
McaPChaGNvaHADpaNH2, and McaPLANvaHADpaARNH2. Routine assays were performed at 37 °C at substrate concentrations of 0.5 and 1.5 µM in an assay buffer of 0.1 M
Tris/HCl, 10 mM CaCl2, 150 mM NaCl,
0.05% (v/v) Brij 35, pH 7.5 (33). Inhibition of enzymatic activity by
recombinant TIMP-1 was evaluated using the above assay.
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RESULTS |
Identification and Characterization of a Human Ovary cDNA
Encoding a New Member of the Matrix Metalloproteinase Family--
To
identify putative novel members of the MMP family expressed in human
tissues, we screened the GenBankTM data base of ESTs
looking for entries with sequence similarity to previously described
family members. This analysis allowed us to identify a 419-bp EST that,
when translated, generated an open reading frame with significant amino
acid sequence similarity to the C-terminal region of the catalytic
domain characteristic of MMPs. A cDNA containing part of this EST
was obtained by PCR amplification of total
-phage DNA prepared from
a human ovary cDNA library. The 384-bp PCR-amplified product was
cloned and its identity was confirmed by nucleotide sequence analysis.
Then, the cloned fragment was radiolabeled and used as a probe to
screen the same ovarian cDNA library used for the previous PCR
amplification experiment. Upon screening of approximately 1 × 106 plaque forming units, two positive clones named 1.1 and
1.2 were identified and characterized. DNA was isolated from both
positive clones, and their nucleotide sequence was determined by
standard procedures. This sequence analysis revealed that one of these clones (1.2) had an insert of 750 bp, which was entirely contained in
the 810-bp sequence determined for clone 1.1. A detailed comparative analysis of the sequence obtained for the largest clone with those corresponding to other MMPs suggested that it was incomplete at the
5'-end. To extend the partial cDNA sequence toward the 5'-end, we
performed 5'-RACE experiments using a specific oligonucleotide deduced
from the end of the 1.1 clone and RNA from human ovary as a template.
Successive 5'-RACE experiments performed in similar conditions led us
finally to obtain a fragment long enough to contain the entire coding
information for the identified MMP. Computer analysis of the obtained
sequence (Fig. 1) revealed an open
reading frame coding for a protein of 390 amino acids with a predicted
molecular mass of 43.9 kDa. This sequence contains four potential sites
of N-glycosylation (N-L-T, N-H-T, N-A-T, and N-V-T, at
positions 92, 148, 232, and 316, respectively). However, computer
analysis using the algorithm developed by Nielsen et al.
(34) revealed that the deduced amino acid sequence lacks a recognizable
signal sequence at its N-terminal end, which is in contrast to all the
remaining MMPs. Further analysis of the identified amino acid sequence
revealed a significant similarity with other human MMPs, the maximum
percentage of identities (35%) being with stromelysin-3. Most of these
identities are concentrated in the putative catalytic domain of the
novel sequence (Figs. 1 and 2). This
domain contains 176 residues including the consensus sequence
HEXGHXXXXXHS (at positions 211-222) involved in
the coordination of the zinc atom at the active site of MMPs. The
catalytic domain also shows a methionine residue, located seven amino
acids C-terminal to the zinc-binding site, that is absolutely conserved
in all MMPs (Fig. 2). This residue has been proposed to play an
essential role in the structure of the active site of these enzymes
(35). The predicted protein sequence also contains a putative prodomain with a single cysteine residue, which can be part of the activation locus characteristic of MMPs. However, it is noteworthy that this residue is located in a sequence context (A-L-C-L-L-P-A) that does not resemble the consensus P-R-C-G-V-P-D motif involved in maintaining the latency of MMPs (Fig. 2). Similarly, the
C-terminal domain of the identified sequence is shorter than those of
other MMPs, with the exception of matrilysin, and does not exhibit
significant sequence similarity to hemopexin. Finally, the putative
prodomain and catalytic domains of the identified sequence are
separated by a R-X-R-R furin activation consensus sequence.
This sequence has been shown to mediate the intracellular activation of
a number of MMPs, including MT-MMPs and stromelysin-3. Taking together
all these structural comparisons, we suggest that the isolated cDNA
codes for a novel human MMP that we propose to call MMP-23; MMP-22
(also known as C-MMP) corresponds to the last family member recently
identified in chicken embryos (14). Finally, it must be mentioned that
during revision of this manuscript, we have been aware of the release
by GenBank of a series of nucleotide sequences related to that reported
herein for human MMP-23. Two of these sequences (accession numbers
AB010961 and AF056200; see also Ref. 36) deposited by Ohnishi et
al. and Gururajan et al., and cloned from uterus and
testis cDNA libraries, are essentially identical to that reported
here for MMP-23 isolated from an ovarian cDNA library. Gururajan
et al. (36) have also provided evidence that the MMP-23 gene
is duplicated, but the gene product resulting of this duplication would
be virtually identical in amino acid sequence to that of MMP-23 gene.
Furthermore, the sequences for the putative rat and mouse homologs of
human MMP-23 have also been recently released by GenBank (accession numbers AB010960 and AF085742, deposited by Ohnishi et al. and D. Pei, respectively). These sequences are about 84% identical to
the human enzyme and maintain all specific features of this MMP,
including the lack of a recognizable signal sequence at the N-terminal
end, a short prodomain with a cysteine residue located in a sequence
unrelated to the consensus P-R-C-G-V-P-D activation locus of MMPs, as
well as a short C-terminal domain with no sequence similarity to
hemopexin. It is also of interest that both rat and mouse sequences
have a conserved methionine residue at exactly the same position than
that proposed in the present work as the starting residue for the human
enzyme. Furthermore, both cDNA sequences have an in-frame stop
codon closely upstream of the ATG codon encoding this methionine
residue. Taken together, these data strongly suggest that the first
methionine residue shown in Fig. 1 is the true translation start site
of human MMP-23.

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Fig. 1.
Nucleotide sequence of MMP-23 cDNA
isolated from human ovary. The deduced amino acid sequence is
shown below the nucleotide sequence. The single cysteine residue
present in the prodomain sequence, the furin-like cleavage site, and
the potential sites for N-glycosylation are
underlined. The sequence corresponding to the zinc-binding
site is boxed.
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Fig. 2.
Partial comparison of the amino acid sequence
of MMP-23 with other human MMPs. The amino acid sequences of human
MMPs were extracted from the SwissProt data base and the multiple
alignment was performed with the PILEUP program of the GCG package
(30). Conserved residues around the cysteine switch region and the
zinc-binding site characteristic of MMPs are shown in bold.
For comparison purposes, numbering in each protein starts in the
initiator methionine.
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Chromosomal Mapping of the Human MMP-23 Gene--
To determine the
chromosomal location of the human MMP-23 gene, we first used a
PCR-based strategy to screen a panel of somatic cell hybrid lines
containing a single human chromosome in a rodent background. The
sequence-tagged site specific for the MMP-23 gene was generated by
using two specific oligonucleotides whose sequence was derived from a
noncoding sequence flanking the second exon of the gene, and from a
coding sequence of this same exon. As can be seen in Fig.
3, positive amplification results were
only obtained in the hybrid containing the autosome number 1. Since no
amplification products were observed in the hybrids containing the
remaining human chromosomes, we can conclude that the MMP-23 gene maps
to chromosome 1. To localize more precisely the MMP-23 gene within
chromosome 1, we first isolated MMP-23 genomic clones from a human PAC
genomic library and used them for FISH experiments on human metaphase
spreads. As shown in Fig. 4, and in
complete agreement with the human-rodent somatic hybrid studies,
fluorescent signals corresponding to biotinylated MMP-23 clones were
located on chromosome 1 and no other chromosome site was labeled above background. After DAPI banding of 60 metaphases showing hybridization in both chromosomes 1, the MMP-23 fluorescent signal was assigned to
the telomeric region of the short arm of chromosome 1, in the p36
region. A number of genes have been already mapped to this region,
including that encoding p73, a recently identified p53-related protein
(37). However, no other MMP genes have been previously found to map at
this chromosome site (38-44).

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Fig. 3.
Chromosomal mapping of the human MMP-23
gene. 100 ng of total DNA from the 24 monochromosomal somatic cell
lines was PCR-amplified with primers 5'-CGAAACCACAGGCAGAC and
5'-CTGTAGGTGAGGTTG AAGTGGTC as described under "Experimental
Procedures." pBR322 digested with HaeIII (Marker V,
Boehringer Mannheim) was used as a size marker.
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Fig. 4.
FISH mapping of genomic DNA clones for
MMP-23. The hybridization spots corresponding to the MMP-23 PAC
clone are detected in the telomeric region of chromosome 1. Metaphase
cells were counterstained with DAPI.
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Production of Recombinant MMP-23 in Bacterial Cells and Analysis of
Its Enzymatic Activity--
According to the above structural data,
MMP-23 has a number of structural features characteristic of previously
identified MMP family members. However, its deduced amino acid sequence
also shows some unique features that could affect its putative role as
a proteolytic enzyme. As a preliminary step to elucidate whether the
isolated MMP-23 cDNA codes for a biologically active proteinase, we
expressed the cloned cDNA in E. coli. A partial cDNA
coding for the catalytic and C-terminal domains of human MMP-23 was
subcloned into the expression vector pRSETB, and the resulting plasmid
was transformed into E. coli BL21(DE3)pLysS. Transformed
bacteria were induced with IPTG, and the resulting recombinant protein was purified and refolded as described under "Experimental
Procedures." However, all attempts to detect any proteolytic activity
of this recombinant protein against substrates commonly used for
analyzing MMPs were unsuccessful. Similar results were obtained when
other constructs containing the prodomain of MMP-23 or lacking the
C-terminal domain of this enzyme were used (data not shown). A
possibility to explain these negative results could be based on the
inappropriate folding of the recombinant enzymes. We then decided to
prepare an expression vector for a chimeric enzyme consisting of the
propeptide domain of MMP-19 and the catalytic domain of MMP-23. This
chimeric construct was chosen because it would allow autoactivation to occur, thus facilitating the subsequent analysis of the enzymatic activity of the recombinant protein. Transformed bacteria with this
construct were induced with IPTG and protein extracts analyzed by
SDS-PAGE. According to the obtained results, insoluble fraction of the
bacteria transformed with the recombinant plasmid contained a protein
of the expected size (31 kDa), which was not present in the control
extracts (Fig. 5). This recombinant
protein was purified and refolded as described under "Experimental
Procedures," and its degrading activity against specific substrates
for MMPs was examined. The chimeric MMP-23 only displayed a low
proteolytic activity on the synthetic peptide
McaPLGLDpaARNH2, insufficient to perform accurate kinetic
studies. We were, however, able to demonstrate that autoproteolytic
degradation during incubation of the proenzyme preparation at 37 °C
was abolished in the presence of TIMP-1, confirming that the activity
seen with our enzyme preparation was due to a matrix metalloproteinase
(Fig. 5, and data not shown). Since the MMP-23 catalytic domain
contains two cysteine residues, which are unique among MMPs, we
speculate that either the refolding is not very efficient for this
particular enzyme, or it might have very different enzymatic properties
when compared with the classical MMPs. Consistent with this, no
apparent activity of the recombinant protein was detected against other
synthetic quenched fluorescent peptide substrates such as
McaPLANvaDpaARNH2 and McaPChaGNvaHADpaNH2, which are good stromelysin and collagenase substrates, respectively (45, 46). Similarly, no apparent activity was detected against gelatin.
Taken together, these preliminary functional analyses suggest that the
cloned cDNA encodes for a MMP whose substrate is likely to be
distinct to those corresponding to well defined family members such as
collagenases, stromelysins or gelatinases. Nevertheless, the
possibility that the majority of the recombinant protein is not
correctly folded cannot be definitively ruled out.

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Fig. 5.
SDS-PAGE analysis of chimeric MMP-19/MMP-23
produced in E. coli. Aliquots (5 µl) of bacterial
extracts (lane 1, control; lane 2, transfected
with MMP19-MMP23 pRSETB), purified MMP-19/MMP-23 (lane 3),
and autoproteolyzed MMP-19/MMP-23 (lane 4) were analyzed by
SDS-PAGE. The recombinant protein is indicated by an arrow.
Molecular mass markers are indicated on the left.
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Analysis of MMP-23 Expression in Human Tissues--
To investigate
the presence of MMP-23 mRNA transcripts in human tissues, Northern
blots containing poly(A)+ RNAs prepared from a variety of
tissues (leukocytes, colon, small intestine, ovary, testis, prostate,
thymus, spleen, pancreas, kidney, skeletal muscle, liver, lung,
placenta, brain, and heart) were hybridized with the full-length
cDNA isolated for MMP-23. As shown in Fig.
6, a transcript of about 1.35 kilobase
pairs was predominantly detected in ovary, testis, prostate, and heart. A transcript of the same size was also weakly detected in intestine, colon, placenta, lung, and pancreas. Furthermore, a second transcript of about 2.4 kilobase pairs was exclusively observed in ovary. The
predominant expression of MMP-23 in human reproductive tissues such as
ovary, testis, and prostate suggests that this novel MMP could
participate in some of the tissue remodeling processes taking place in
these tissues during physiological conditions. Nevertheless, the
finding of MMP-23 transcripts in other tissues, such as heart, strongly
suggests that the function of this enzyme is not restricted to
reproductive processes.

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Fig. 6.
Northern blot analysis of MMP-23 expression
in human tissues. About 2 µg of polyadenylated RNA from the
indicated tissues were analyzed by hybridization with the full-length
cDNA isolated for human MMP-23. The positions of RNA size markers
are shown. Filters were subsequently hybridized with a human actin
probe to ascertain the differences in RNA loading among the different
samples.
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DISCUSSION |
In this work, we describe the finding of a new human proteinase
belonging to the MMP family, which we have tentatively called MMP-23.
The strategy followed to identify MMP-23 was first based on a computer
search of the EST data base, looking for sequences with similarity to
previously characterized MMP family members. A single sequence
presumably encoding the C-terminal region of a new MMP was identified,
PCR-amplified from human ovary cDNA, and used to screen an ovarian
cDNA library. After screening of this library and further 5'-RACE
experiments, a full-length cDNA coding for MMP-23 was finally
isolated and characterized. Structural analysis of the identified
sequence for MMP-23 shows that it exhibits a series of protein domains
characteristic of MMPs, including a prodomain, a catalytic domain, a
hinge region, and a C-terminal domain. However, a more detailed
analysis of the sequence deduced for MMP-23 reveals a number of
specific structural features for this novel human proteinase. Thus, the
identified sequence lacks the recognizable signal sequence present at
the N-terminal end of all the remaining MMPs, suggesting that this
novel enzyme could function in an intracellular compartment. However,
the use of alternative secretory mechanisms as proposed for other
proteins such as basic fibroblast growth factor, which are secreted
despite lacking signal peptide, can not be excluded (47). In addition, the prodomain identified for MMP-23 is unusually short although it
contains a single Cys residue that can be part of the activation locus
characteristic of MMPs. In this regard, it is well known that MMPs are
synthesized as inactive precursors with an N-terminal propeptide that
maintains the latency of the enzymes through a Cys-switch mechanism
(48). According to this mechanism, coordination of the unpaired
cysteine residue in the propeptide with the zinc ion in the active site
leads to inactivation of the enzyme. Disruption of the Cys-zinc bond by
limited proteolysis or conformational perturbations leads to opening of
the switch and subsequent autocatalytic cleavages, finally resulting in
the generation of a catalytically competent enzyme. The single Cys
residue in the propeptide region of human MMP-23, which is conserved in
the putative mouse and rat homologs of this protein (GenBank accession
numbers AF085742 and AB010960), could participate in maintaining the
latency of this enzyme. Nevertheless, the absence of conserved residues around this Cys residue in MMP-23, when compared with other MMPs (Fig.
2), could suggest that these consensus residues
(P-R-C-G-V-P-D) are not essential for the appropriate
functioning of the Cys-switch mechanism in all MMPs. Additionally, in
relation with the activation mechanism of proMMP-23, the presence of a
stretch of basic residues linking the pro- and the catalytic domains is
indicative of a furin-mediated activation for this enzyme, in a similar
fashion to that described for stromelysin-3 and MT-MMPs (49, 50).
An additional distinctive feature of the structure determined for
MMP-23 derives from its unique C-terminal domain. All human MMPs
characterized to date, with the exception of matrilysin, contain an
hemopexin-like region of about 200 amino acids organized into four
recognizable repeats (51, 52). In contrast, the C-terminal domain of
MMP-23 contains a domain of only 100 residues that lacks any
significant similarity with hemopexin. Because this domain has been
reported to be important in defining the substrate specificity of MMPs,
and in mediating interactions with inhibitors (53, 54), the occurrence
of a shortened domain in MMP-23 could be relevant in determining the
substrate specificity and catalytic properties of this novel enzyme. In
this regard, it is also worth mentioning that MMP-23 also lacks a
series of structural features distinctive of the diverse MMP
subclasses, including the Asp, Tyr, and Gly residues located close to
the zinc-binding site of collagenases, the fibronectin-like domain of
gelatinases, and the transmembrane domain of MT-MMPs (55-59).
Taking all these structural data collectively, it seems clear that
MMP-23 cannot be classified in any of the previously defined MMP
subfamilies. Consequently, it must be placed into the growing group of
"other MMPs," which includes enzymes such as macrophage metalloelastase, stromelysin-3, MMP-19, or enamelysin, all of them
having distinctive structural and/or functional properties (5-8). In
this work, we have also examined the activity of recombinant MMP-23
produced in E. coli as a fusion with the MMP-19 prodomain, and purified and refolded following the same procedure previously used
for producing other active MMPs (5, 8, 60). This recombinant protein
shows low proteolytic activity against a synthetic peptide commonly
used for analyzing the enzymatic properties of MMPs. Nevertheless, the
fact that the detected proteolytic activity is inhibitable by TIMP-1
suggests that it corresponds to a bona fide MMP. A likely
possibility to explain this low activity can be that the majority of
the recombinant protein is not correctly folded. However, the
possibility that this novel enzyme may have specific substrates whose
nature is currently unknown, cannot be be definitively ruled out. Also
consistent with the above discussed distant relationship between MMP-23
and other human MMPs, chromosomal mapping of the MMP-23 gene has shown
that it is located at chromosome 1, a unique position among all MMP
genes mapped to date (38-44). Furthermore, the pattern of MMP-23
expression in human tissues is also somewhat unusual. Thus, in this
work, we have provided evidence that this gene is abundantly expressed
in a number of normal tissues, which is in marked contrast with the
highly restricted expression of most MMPs in adult tissues under normal
quiescent conditions. It is also of interest that MMP-23 is
predominantly expressed in reproductive tissues, and particularly in
ovary, suggesting that this proteinase could play some specific role in
any of the matrix-remodeling processes occurring in these tissues (61).
The availability of specific reagents for MMP-23 generated in this work
will be very helpful to examine the functional relevance of this enzyme
in physiological processes. Finally, the isolation of genomic clones
for murine MMP-232 opens the
possibility of generating animals deficient in this gene, which will
contribute to clarification of its precise role in the context of other
proteases produced in reproductive processes.