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Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo, Spain
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Introduction |
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The association of MMPs with malignant tumors is well documented both in vitro and in vivo (7) . In general, MMP levels, as compared with nonmetastatic counterparts, are abnormally elevated in metastatic tumor cell lines. On the other hand, high MMP levels are associated with invasive lesions and poor clinical prognosis in cancer patients (8 , 9) . However, the finding of new members of the MMP family and their subsequent functional characterization at both genetic and biochemical levels has opened new views on the role of these enzymes in tumor progression (10) . Thus, evidence is accumulating that MMPs are not exclusively involved in the proteolytic breakdown of the tissue barriers for metastatic spread. In fact, MMPs have been reported to play direct roles in other critical events in tumor evolution that occur at earlier stages, events such as tumor promotion, modulation of the growth of the primary tumor, and angiogenesis (11, 12, 13) .
The finding that MMPs may be involved in such diverse biological processes during tumor progression has stimulated the search for new family members that could participate in some of the events associated with malignant tumors. In this work, we describe the molecular cloning and complete nucleotide sequence of a cDNA coding for matrilysin-2, a new MMP that shares a number of structural and functional properties with matrilysin (MMP-7). We also report the expression of the gene in Escherichia coli and perform an analysis of the enzymatic activity of the recombinant enzyme with the finding that matrilysin-2 is a potent enzyme with a wide substrate specificity on extracellular matrix and basement membrane proteins. Finally, we establish the chromosomal location of the matrilysin-2 gene in the human genome and analyze its expression in human tumors from different sources.
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Materials and Methods |
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Screening of EST Database.
A search of the GenBank database of human ESTs for sequences with
homology to MMPs, allowed us to identify a sequence (AI 743415;
deposited by National Cancer Institute, Cancer Genome Anatomy Project)
that was derived from a pool of cDNA libraries and that showed
similarity with sequences of known human MMPs. To obtain this DNA
fragment, we performed PCR amplification of a human cDNA from placenta
(Clontech, Palo Alto, CA) with two specific primers
5'-CCATTTTGACAAGAATGAACA (mat2–1) and 5'-CATTAAGGT ATGTCAGATGAA
(mat2–2) derived from the AI 743415 sequence. The PCR reaction was
carried out in a GeneAmp 2400 PCR system (Perkin-Elmer/Cetus) for 40
cycles of denaturation (94°C, 15 s), annealing (54°C, 20 s), and extension (72°C, 20 s). The 232-bp PCR product that was
amplified from human placenta cDNA was cloned, and its identity was
verified by nucleotide sequencing.
5'-Extension of Isolated cDNAs.
The 5'-ends of cloned cDNAs were extended by successive cycles of RACE
using RNA from human placenta and the Marathon cDNA amplification kit
(Clontech), essentially as described by the manufacturer. 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'-ATGCAGCTCGTCATCTTAAGAGTT (mat2–14) and 5'-CAGGTTGAATAAGTCCTCTGTGCT
(mat2–15). After gel purification, the amplification product was
cloned and sequenced by standard procedures.
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'-AAATTCTGGAAATCCTGGAGTTGTCC (mat2–4) and 5'-GTGGGGTA
CATTATGGAGCTCTG (mat2–5). Amplification conditions were as follows: 35
cycles of denaturation (94°C, 15 s), annealing (66°C, 15 s), and extension (68°C, 2 min) using the Expand Long PCR kit.
Radiation hybrid mapping was carried out using the Genebridge 4 panel
(14)
. DNA samples from this panel (25 ng) were
PCR-screened for the presence of the genomic sequences flanked by the
primers mat2–4 and mat2–5 Amplification conditions were the same as
above. PCR results were converted to a vector of 93 0’s (no
amplification), 1’s (amplification), and 2’s (blanks and
uncertainities) and submitted to the mapping server of the Whitehead
Institute/Massachusetts Institute of Technology Center for
Genome Research.
Expression Analysis.
Analysis of matrilysin-2 expression in human tissues was first
performed by PCR amplification of a panel of cDNAs from commercially
available libraries (Clontech). To do that, total 
-phage DNA from
the diverse human cDNA libraries was amplified with two primers
(mat2–1, and mat2–2) specific for matrilysin-2, and the PCR reaction
was performed as described above. To study matrilysin-2 expression in
human tumors, we performed RT-PCR amplification with RNA from tumor
biopsies using the RNA PCR kit from Perkin-Elmer. After RT using 1 µg
of total RNA and random hexamers as primer according to the
instructions of the manufacturer, the whole mixture was used for PCR
with primers mat2–6 (5'-ACCTACTTGACATGCAGATGC) and mat2–9
(5'-GAGGTCCTAAAGGTCTTAAA CC). For ß-actin amplification, 2-µl
aliquots were amplified in the same way with the oligonucleotides
5'-GTGGGGCCGCTCTAGGCAC and 5'-TTTGATGTCACGCACG ATTT. Negative controls
were performed using all of the reagents with the exception of random
hexamers. Amplification products were analyzed by agarose gel
electrophoresis.
Expression, Refolding, and Purification of Human Matrilysin-2.
A 734-bp fragment of the matrilysin-2 cDNA containing the pro- and
catalytic domains, was generated by PCR amplification with primers
5'-GAAGATCTAGTTCCAGTGCCC CCTGCT (BglII-promat2), and
5'-CGGAATTCCGTTAAGGTATGTCAGATGA (EcoRI-promat2) using the
full-length matrilysin-2 cDNA in pUC18. The PCR amplification was
performed for 30 cycles of denaturation (95°C, 10 s), annealing
(46°C, 10 s), and extension (68°C, 1 min), using the Expand
High Fidelity PCR system. Because of the design of the
oligonucleotides, the amplified fragment could be cleaved at both ends
with EcoRI and BglII and ligated in frame into
the pRSETB E. coli expression vector (Invitrogen), thereby
adding a NH2-terminal His6
tag to the protein. The resulting pRSET-proM2 vector was transformed
into BL21(DE3)pLysS competent E. coli cells and expression
was induced by the addition of IPTG (0.5
mM final concentration) followed by further
incubation for 3–20 h at 30°C. Recombinant protein obtained in
inclusion bodies was solubilized using 20 mM Tris
buffer (pH 7.6) containing 6 M GdnHCl, and 5
mM DTT, and purified in a Superdex-75 column
(Amersham-Pharmacia, Uppsala, Sweden) equilibrated with 20
mM Tris buffer (pH 7.6) containing 3
M GdnHCl, and 5 mM DTT.
After SDS-PAGE analysis, peak fractions with the recombinant protein
were pooled and the GdnHCl concentration adjusted to 6
.M Refolding was achieved by dilution (1:50) into
a 50 mM Tris buffer (pH 7.6) containing 5
mM CaCl2, 200
mM NaCl, 50 µM
ZnSO4, 0.05% Brij 35, 2.5
mM oxidized glutathione, 2.5
mM reduced glutathione, 20% glycerol, and
2.3 M urea, and stirring overnight at 4°C.
The refolding mixture was applied to a Ni-chelated nitriloacetic
acid agarose column (Qiagen) equilibrated with the above buffer.
The protein bound via the NH2-terminal
His6 tag was eluted with a 50
mM Tris buffer (pH 7.6) containing 5
mM CaCl2, 200
mM NaCl, 0.05% Brij 35, and 100
mM imidazole. Fractions containing purified
protein were combined and dialyzed against 50 mM
Tris/HCl (pH 7.6), 5 mM
CaCl2, 200 mM NaCl, and
0.05% Brij 35.
Enzymatic Assays.
Enzymatic activity of purified recombinant matrilysin-2 was detected
using the synthetic fluorescent substrates
Mca-ProLeu-Gly-Leu-Dpa-Ala-Arg-NH2 (QF-24),
Mca-Pro-Leu-Ala-Nva-Dpa-AlaArg-NH2 (QF-35),
and Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 (QF-41;
provided by C. G. Knight, University of Cambridge, United Kingdom).
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 DMSO of
1%. The fluorometric measurements were made in a MPF-44A
Perkin-Elmer spectrofluorometer
(ex = 328 nm and
em = 393 nm, where ex stands for
excitation and em stands for emission.). For inhibition assays,
matrilysin-2 (20 nM) and inhibitors were preincubated for
30 min at 20°C, with BB-94 (British Biotech Pharmaceuticals, Oxford,
United Kingdom) at concentrations ranging from 0 to 100 nM.
Inhibition assays with TIMPs (kindly provided by Drs. V. Knäuper
and G. Murphy) were performed at the same conditions with
20-nM concentration of the different inhibitors. Cleavage
of type-I, type-II, and type-IV collagens, type I gelatin, type I
laminin, fibronectin, and fibrinogen (purchased from Sigma) by
recombinant matrilysin-2 was followed by SDS-PAGE. All of the assays
were performed in the above-described assay buffer for 16 h at
37°C. The enzyme:substrate ratio (w:w) used in these experiments was
1:10. Finally, to examine the activation of other MMPs by matrilysin-2,
the proenzymes of human gelatinase A (pro-MMP2), stromelysin-1
(pro-MMP3), gelatinase B (pro-MMP9), and collagenase-3 (pro-MMP13) were
incubated with active matrilysin-2 at a 1:1 w:w enzyme:substrate ratio.
Incubations were performed in assay buffer for 16 h at 37°C. The
processing of proMMPs was assessed by SDS-PAGE.
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Results |
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-phage DNA prepared from a human placenta cDNA library. A detailed comparative analysis of the sequence, obtained for the identified EST with the sequence of MMPs, clearly indicated that it was not long enough to encode a functional MMP. To extend the partial cDNA sequence toward the 5' end, we performed 5'-RACE experiments using RNA from human placenta as a template. Successive 5'-RACE experiments 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 (European Molecular Biology Laboratory accession number AJ251531) showed the presence of an open reading frame coding for a protein of 261 amino acids with a predicted molecular mass of 29.7 kDa.
This sequence contains two potential sites of
N-glycosylation (N-G-T and N-V-T, at positions 64
and 133, respectively) and exhibits overall sequence similarity with
other MMPs, the maximum percentage of identities being with MMP-12
(about 50%). The identified amino acid sequence also shows the
presence of a series of structural features characteristic of MMPs
(Fig. 1)
. Thus, the finding of a NH2-terminal hydrophobic
sequence adjacent to the initiator methionine suggests the presence of
the signal peptide that targets these enzymes to the secretory pathway.
This sequence is followed by a prodomain region with the activation
locus containing the essential cysteine residue (at position 82) for
maintaining enzyme latency. Finally, the identified sequence contains a
catalytic domain of about 170 residues including the consensus sequence
HEXGHXXGXXHS (at positions 208–219)
involved in the coordination of the catalytic zinc atom at the active
site of MMPs. Similarly, the structural zinc-ligands (His-158, Asp-160,
His-173, and His-186), and the structural calcium-ligands (Asp-165,
Asp-188, and Glu-191) strictly conserved in MMPs, are also present in
this domain of the identified protein. This catalytic domain also shows
a methionine residue located seven amino acids COOH-terminal to the
zinc-binding site, that is absolutely conserved in all of the MMPs. The
presence of a Thr residue immediately before the first His of the
zinc-binding site (Fig. 1)
is also remarkable. This residue is
exclusively found in human, mouse, rat, and cat matrilysin among all
vertebrate MMPs and has been defined as a specific structural signature
of this enzyme (15
, 16)
. More interestingly, the predicted
amino acid sequence lacks the hinge sequence and the COOH-terminal
hemopexin-like domain characteristic of most MMPs. In fact, these
domains are present in all human MMPs characterized to date with the
exception of matrilysin (MMP-7) and MMP-23 (17
, 18)
.
However, MMP-23 does exhibit a unique COOH-terminal domain rich in
cysteine and proline residues and lacks the cleavable signal sequence
and cysteine-switch region generally present in all MMPs, thus
being very distinct from the novel MMP described herein.
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Chromosomal Location of the Human Matrilysin-2 Gene.
To establish the chromosomal location of the human matrilysin-2 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 matrilysin-2 gene
was generated by using two specific oligonucleotides presumably
corresponding to different exons of the gene. As can be seen in Fig. 2
, positive amplification results were obtained only in the hybrid
containing the autosome number 11. Because no amplification products
were observed in the hybrids containing the remaining human
chromosomes, we can conclude that the matrilysin-2 gene maps to
chromosome 11. To determine more precisely the chromosomal location of
the gene, we next performed a PCR-based screening of a panel of
radiation hybrids containing human chromosome fragments in a rodent
background. Computer analysis of positive amplification results
indicated that the matrilysin-2 gene is located at 3.2 cR from
top of chromosome 11 linkage group. This location at the short arm of
chromosome 11, around 11p15, differs from that reported for other MMP
genes that have been previously found to map at the long arm of this
chromosome and clustered at 11q22 (19)
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. This recombinant protein was purified using
size-exclusion and Ni-NTA-agarose chromatography, and then was
subjected to a refolding procedure as described previously
(18)
. However, and similarly to other MMPs, all of the
proenzyme was autoactivated after performing the last step of dialysis,
which resulted in the generation of a putative active enzyme with a
molecular mass of about 19 kDa (Fig. 3A)
. This size is
identical to that obtained after SDS-PAGE analysis of the recombinant
protein produced by using a cDNA construct containing the catalytic
domain of matrilysin-2 exclusively (data not shown). To assess the
substrate specificity of the recombinant matrilysin-2, a series of
synthetic quenched fluorescent peptides that is commonly used for
assaying MMPs were used. As shown in Fig. 3B
, the general
MMP substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2
(QF-24), the collagenase/gelatinase substrate
Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 (QF-41), and
the stromelysin substrate
Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2 (QF-35) were
hydrolyzed by matrilysin-2. These hydrolytic activities were completely
abolished in the presence of EDTA (50 mM) or the
hydroxamic-acid-based inhibitor BB-94 (Fig. 3B)
. Finally, we
examined the potential inhibition of active matrilysin-2 by different
available TIMPs. To this purpose, we used a constant enzyme
concentration of 20 nM in the quenched
fluorescent assay and used
Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 (QF-41) as
substrate. As shown in Fig. 3C
, TIMP-1 and TIMP-4 blocked
the hydrolyzing activity of matrilysin-2 against the synthetic peptide.
By contrast, the inhibitory potential of TIMP-2 was apparently lower.
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, the active matrilysin-2 was able to degrade at a variable extent a
number of protein substrates like type IV collagen, fibronectin,
fibrinogen, and type I gelatin. Type IV collagen and gelatin were
extensively degraded, whereas the digestion of fibronectin and
fibrinogen generated both small and large protein fragments. In the
latter case, the A
and Bß chains were completely degraded, but the
chain remained virtually intact. In all of the cases, the degrading
activity was completely blocked by MMP inhibitors including EDTA,
synthetic hydroxamic-acid-based compounds like BB-94, and TIMPs (data
not shown). By contrast, type I and type II collagens were resistant to
hydrolysis, which is consistent with the fact that matrilysin-2 lacks
all of the structural determinants to act as a triple-helical
collagenase.
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, matrilysin-2 was able to process progelatinase B
generating the final active form of Mr
82,000. This activating ability was time-dependent, reaching the
maximal activation rate at 24 h (data not shown). However,
matrilysin-2 was not able to process to a significant extent any of the
other tested proMMPs, which suggests that both matrilysin-2 and
progelatinase B could act coordinately as part of a proteolytic
cascade. Taken together, these results provide evidence that
matrilysin-2 is a potent enzyme with a wide range of substrates and
with the inhibitory profile characteristic of members of the MMP family
of endopeptidases.
Analysis of Matrilysin-2 Expression in Normal and Tumor Tissues.
To investigate the presence of matrilysin-2 transcripts in human
tissues, Northern blots containing poly(A)+ RNAs
prepared from a variety of normal 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 this enzyme. However,
matrilysin-2 transcripts were virtually undetectable in all of the
analyzed tissues (data not shown). Nevertheless, PCR amplification
experiments with cDNAs from different human tissues allowed us to
confirm that matrilysin-2 is expressed in placenta, the tissue
originally used for cloning this enzyme (Fig. 5A)
. In addition, these PCR amplification experiments allowed
us to detect the expression of matrilysin-2 in a sample from a uterus
(Fig. 5A)
. Additional studies confirmed that expression in
human uterus is only circumscribed to very specific phases of the
menstrual
cycle.4
According to these data, we can conclude that matrilysin-2 exhibits a
very restricted expression pattern in normal tissues. To evaluate the
possibility that this enzyme was produced by human tumors, we performed
a preliminary survey of the expression of this gene in a series of
tumor cell lines and malignant tumors of diverse sources. These
analyses revealed that matrilysin-2 expression could be detected in
tumor cells of different origin such as promyelocytic leukemia (HL-60),
colorectal adenocarcinoma (SW480), and lung carcinoma (A549), as well
as in a variety of tumors, including endometrial, lung, and prostate
adenocarcinomas (Fig. 5B
and data not shown). These results,
together with the above data showing that matrilysin-2 is active
against a variety of extracellular matrix and basement membrane
proteins, indicate that this novel enzyme may be added to the growing
list of proteases potentially implicated in tumor progression.
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Discussion |
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The approach used to identify matrilysin-2 involved a search of the EST databases, followed by the screening of a human placenta cDNA library using as probe a EST fragment amplified from this tissue and showing sequence similarity to the catalytic domain of MMPs. After identification of positive clones, and successive RACE-5' experiments, we could isolate and clone a full-length cDNA coding for matrilysin-2, a protein composed of 261 amino acid residues. Structural analysis of the amino acid sequence of this protein showed that it exhibits several protein domains characteristic of MMPs: the signal peptide, the prodomain, and the catalytic domain. However, similarly to matrilysin, matrilysin-2 lacks the hinge region and the COOH-terminal hemopexin-like domain present in other MMPs. An additional structural feature exclusively shared by matrilysin and matrilysin-2 among all vertebrate MMPs is the presence of a Thr residue immediately adjacent to the Zn-binding site. This residue is present in matrilysin from different species and has been defined as a specific structural signature of this enzyme, although its functional relevance has not been clarified (15 , 16) . The identification and cloning of a second human MMP with the same structural design as matrilysin provides additional support to the proposal by MacDougall and Matrisian (20) that matrilysins would be classified within a specific subfamily of MMPs. In fact, the MMPs were originally separated into three groups according to substrate specificity: collagenases, stromelysins (including matrilysin), and gelatinases. More recently, two additional subfamilies were created to accommodate the membrane-anchored MT-MMPs as well as other MMPs, such as stromelysin-3 and MMP-23, whose structural and/or functional properties do not fit well in any of the remaining subgroups. As an alternative to this traditional scheme, it has been proposed that MMPs could be grouped on the basis of the presence or absence of specific domains in their structure (1 , 20) . In this alternative classification, matrilysin would be the prototype of the minimal-domain MMP category composed of proteases containing exclusively the three domains necessary for secretion, latency, and catalytic activity. It is remarkable that, in addition to matrilysin and matrilysin-2, this subfamily would contain at least one other MMP, found in soybean leaves, that also possesses the same minimal-structural design as vertebrate matrilysins (21) .
In addition to all of these structural features, matrilysin-2 also
shares with matrilysin a wide degrading activity against proteins
present in extracellular matrix and basement membranes. Thus, the
refolded catalytic domain that is produced in E. coli
exhibits strong proteolytic activity against synthetic peptides used
for analyzing the enzymatic properties of MMPs. Furthermore, it
efficiently degrades gelatin, fibronectin, fibrinogen, and type IV
collagen. By contrast, and consistent with a series of structural
properties of matrilysin-2 including lack of both hemopexin-domain and
specific residues present in the catalytic domain of collagenases, this
enzyme does not cleave triple-helical collagens such as type I and type
II collagens. It is remarkable that matrilysin-2 may digest fibrinogen
in a very similar manner to that recently reported for MT1-MMP
(22)
. In fact, both are able to degrade the A and Bß
chains of this protein whereas the chain remains essentially
intact. MT1-MMP fibrinolytic activity has been proposed to be essential
in neovascularization processes. Similarly, the observation that
matrilysin-2 may act as a fibrinolysin opens the possibility that this
protease may participate in processes involving fibrin formation and
degradation, such as angiogenesis, that are necessary for tumor
progression (13)
. It is also of interest that matrilysin-2
was able to activate progelatinase B, one of the MMPs more frequently
associated with malignant tumors. This observation suggests that both
enzymes could form part of an activation cascade similar to those
operating for activating other MMPs (23)
. Additional
studies will be required to provide more definitive evidence of the
putative collaborative role of both enzymes in some of the
tissue-remodeling processes in which these enzymes are produced,
including tumor progression.
As in a previous study to define the physiological role of matrilysin-2, in this work we have also examined the expression pattern of this enzyme in human tissues. In agreement with the highly restricted expression of most MMPs in normal adult tissues under quiescent conditions, matrilysin-2 expression was detected at significant levels only in placenta and uterus. The expression in placenta could suggest a role for this enzyme in embryo implantation and/or placental development and function, as previously suggested for other MMPs produced by this tissue (24) . On the other hand, the expression in samples from uterine tissue suggests that matrilysin-2, likely acting in concert with other enzymes, could participate in any of the matrix-remodeling processes occurring in this tissue during the human menstrual cycle (25 , 26) . In marked contrast to the restricted expression pattern of this enzyme in normal tissues, a preliminary survey of tumor tissues and cell lines for matrilysin-2 expression revealed that this gene is widely expressed in a number of malignancies, including endometrial, lung, and prostate adenocarcinomas. Additional studies, now in progress, will be necessary to clarify the clinical significance of the expression of this broad-spectrum protease in human tumors as well as to elucidate its precise functional role in the context of other MMPs with potential involvement in tumor progression (7 , 9 , 10) .
Finally, in this work we have established the chromosomal location of the human matrilysin-2 gene. According to radiation hybrid mapping, this gene localizes to the short arm of chromosome 11, within a region that does not contain any other known MMP gene. These results provide additional evidence that MMP genes have been very dynamic in their evolutionary pathways, and with the exception of several MMP genes clustered in the long arm of chromosome 11, all of the remaining MMP genes are widely dispersed in different human chromosomes. Interestingly, it has been reported that the region containing the matrilysin-2 gene (11p15) is frequently altered in several human tumors (27) . Consequently, it will be of interest to analyze the possibility that matrilysin-2 may be a direct target of these genetic abnormalities.
In conclusion, we have identified and characterized matrilysin-2, the smallest MMP described thus far. This enzyme exhibits significant structural similarities with matrilysin, as well as with a previously described plant enzyme, which suggests that all of them may form part of a specific class of MMPs characterized by exclusively possessing the minimal domains required for the catalytic activity of these endopeptidases. Matrilysin-2 has a wide substrate specificity on extracellular matrix and basement membrane proteins, which is consistent with its potential role in tumor progression. Also in agreement with this possibility, matrilysin-2 has a very restricted expression pattern in human tissues, but is widely found in primary tumors and tumor cell lines from diverse sources. Additional studies will be required to elucidate the precise role of this protease in any of the connective-tissue turnover and remodeling processes occurring during tumor progression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from
Comisión Interministerial de Ciencia y Tecnología-Spain
(SAF97-0258) and by Plan FEDER (Fondos Europeos para el Desarrollo
Regional; 1FD97-0214). The Instituto Universitario de Oncologia is
supported by Obra Social Cajastur-Asturias. 
2 To whom requests for reprints should be
addressed, Departamento de Bioquímica y Biología
Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo,
Spain. Phone: 34-985-104201; Fax: 34-985-103564; E-mail: CLO{at}correo.uniovi.es
3 The abbreviations used are: MMP, matrix
metalloproteinase; MT-MMP, membrane-type MMP; EST, expressed sequence
tag; RT, reverse transcription; TIMP, tissue inhibitor of
metalloproteinases; RACE, rapid amplification of cDNA ends; IPTG,
isopropyl-1-thio-ß-D-galactopyranoside.
Received 4/11/00. Accepted 7/19/00.
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 H. Yamamoto, A. Vinitketkumnuen, Y. Adachi, H. Taniguchi, T. Hirata, N. Miyamoto, K. Nosho, A. Imsumran, M. Fujita, M. Hosokawa, et al. Association of matrilysin-2 (MMP-26) expression with tumor progression and activation of MMP-9 in esophageal squamous cell carcinoma Carcinogenesis, December 1, 2004; 25(12): 2353 - 2360. [Abstract] [Full Text] [PDF]
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R. Pilka, H. Domanski, S. Hansson, P. Eriksson, and B. Casslen Endometrial TIMP-4 mRNA is high at midcycle and in hyperplasia, but down-regulated in malignant tumours. Coordinated expression with MMP-26 Mol. Hum. Reprod., September 1, 2004; 10(9): 641 - 650. [Abstract] [Full Text] [PDF]
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 V. Quesada, L. M. Sanchez, J. Alvarez, and C. Lopez-Otin Identification and Characterization of Human and Mouse Ovastacin: A NOVEL METALLOPROTEINASE SIMILAR TO HATCHING ENZYMES FROM ARTHROPODS, BIRDS, AMPHIBIANS, AND FISH J. Biol. Chem., June 18, 2004; 279(25): 26627 - 26634. [Abstract] [Full Text] [PDF]
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 X. S. Puente and C. Lopez-Otin A Genomic Analysis of Rat Proteases and Protease Inhibitors Genome Res., April 1, 2004; 14(4): 609 - 622. [Abstract] [Full Text] [PDF]
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Y.-G. Zhao, A.-Z. Xiao, H. I. Park, R. G. Newcomer, M. Yan, Y.-G. Man, S. C. Heffelfinger, and Q.-X. A. Sang Endometase/Matrilysin-2 in Human Breast Ductal Carcinoma in Situ and Its Inhibition by Tissue Inhibitors of Metalloproteinases-2 and -4: A Putative Role in the Initiation of Breast Cancer Invasion Cancer Res., January 15, 2004; 64(2): 590 - 598. [Abstract] [Full Text] [PDF]
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H. I. Park, Y. Jin, D. R. Hurst, C. A. Monroe, S. Lee, M. A. Schwartz, and Q.-X. A. Sang The Intermediate S1' Pocket of the Endometase/Matrilysin-2 Active Site Revealed by Enzyme Inhibition Kinetic Studies, Protein Sequence Analyses, and Homology Modeling J. Biol. Chem., December 19, 2003; 278(51): 51646 - 51653. [Abstract] [Full Text] [PDF]
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F. Goffin, C. Munaut, F. Frankenne, S. Perrier d'Hauterive, A. Beliard, V. Fridman, P. Nervo, A. Colige, and J.-M. Foidart Expression Pattern of Metalloproteinases and Tissue Inhibitors of Matrix-Metalloproteinases in Cycling Human Endometrium Biol Reprod, September 1, 2003; 69(3): 976 - 984. [Abstract] [Full Text] [PDF]
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 R. Visse and H. Nagase Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases: Structure, Function, and Biochemistry Circ. Res., May 2, 2003; 92(8): 827 - 839. [Abstract] [Full Text] [PDF]
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R. Pilka, C. Whatling, H. Domanski, S. Hansson, P. Eriksson, and B. Casslen Epithelial expression of matrix metalloproteinase-26 is elevated at mid-cycle in the human endometrium Mol. Hum. Reprod., May 1, 2003; 9(5): 271 - 277. [Abstract] [Full Text] [PDF]
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Y.-G. Zhao, A.-Z. Xiao, R. G. Newcomer, H. I. Park, T. Kang, L. W. K. Chung, M. G. Swanson, H. E. Zhau, J. Kurhanewicz, and Q.-X. A. Sang Activation of Pro-gelatinase B by Endometase/Matrilysin-2 Promotes Invasion of Human Prostate Cancer Cells J. Biol. Chem., April 18, 2003; 278(17): 15056 - 15064. [Abstract] [Full Text] [PDF]
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Q.-L. Li, S. A. Illman, H.-M. Wang, D.-L. Liu, J. Lohi, and C. Zhu Matrix metalloproteinase-28 transcript and protein are expressed in rhesus monkey placenta during early pregnancy Mol. Hum. Reprod., April 1, 2003; 9(4): 205 - 211. [Abstract] [Full Text] [PDF]
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Q. Li, H. Wang, Y. Zhao, H. Lin, Q. A. Sang, and C. Zhu Identification and specific expression of matrix metalloproteinase-26 in rhesus monkey endometrium during early pregnancy Mol. Hum. Reprod., October 1, 2002; 8(10): 934 - 940. [Abstract] [Full Text] [PDF]
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H. I. Park, B. E. Turk, F. E. Gerkema, L. C. Cantley, and Q.-X. A. Sang Peptide Substrate Specificities and Protein Cleavage Sites of Human Endometase/Matrilysin-2/Matrix Metalloproteinase-26 J. Biol. Chem., September 13, 2002; 277(38): 35168 - 35175. [Abstract] [Full Text] [PDF]
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J. Zhang, Y.-J. Cao, Y.-G. Zhao, Q.-X. A. Sang, and E.-K. Duan Expression of matrix metalloproteinase-26 and tissue inhibitor of metalloproteinase-4 in human normal cytotrophoblast cells and a choriocarcinoma cell line, JEG-3 Mol. Hum. Reprod., July 1, 2002; 8(7): 659 - 666. [Abstract] [Full Text] [PDF]
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E. Llano, G. Adam, A. M. Pendas, V. Quesada, L. M. Sanchez, I. Santamaria, S. Noselli, and C. Lopez-Otin Structural and Enzymatic Characterization of Drosophila Dm2-MMP, a Membrane-bound Matrix Metalloproteinase with Tissue-specific Expression J. Biol. Chem., June 21, 2002; 277(26): 23321 - 23329. [Abstract] [Full Text] [PDF]
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N. D. Marchenko, G. N. Marchenko, and A. Y. Strongin Unconventional Activation Mechanisms of MMP-26, a Human Matrix Metalloproteinase with a Unique PHCGXXD Cysteine-switch Motif J. Biol. Chem., May 17, 2002; 277(21): 18967 - 18972. [Abstract] [Full Text] [PDF]
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T. Fujimoto, S. Parry, M. Urbanek, M. Sammel, G. Macones, H. Kuivaniemi, R. Romero, and J. F. Strauss III A Single Nucleotide Polymorphism in the Matrix Metalloproteinase-1 (MMP-1) Promoter Influences Amnion Cell MMP-1 Expression and Risk for Preterm Premature Rupture of the Fetal Membranes J. Biol. Chem., February 15, 2002; 277(8): 6296 - 6302. [Abstract] [Full Text] [PDF]
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B. S. Nielsen, F. Rank, J. M. Lopez, M. Balbin, F. Vizoso, L. R. Lund, K. Dano, and C. Lopez-Otin Collagenase-3 Expression in Breast Myofibroblasts as a Molecular Marker of Transition of Ductal Carcinoma in Situ Lesions to Invasive Ductal Carcinomas Cancer Res., October 1, 2001; 61(19): 7091 - 7100. [Abstract] [Full Text] [PDF]
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E. Llano, A. M. Pendas, P. Aza-Blanc, T. B. Kornberg, and C. Lopez-Otin Dm1-MMP, a Matrix Metalloproteinase from Drosophila with a Potential Role in Extracellular Matrix Remodeling during Neural Development J. Biol. Chem., November 10, 2000; 275(46): 35978 - 35985. [Abstract] [Full Text] [PDF]
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M. Balbin, A. Fueyo, V. Knauper, J. M. Lopez, J. Alvarez, L. M. Sanchez, V. Quesada, J. Bordallo, G. Murphy, and C. Lopez-Otin Identification and Enzymatic Characterization of Two Diverging Murine Counterparts of Human Interstitial Collagenase (MMP-1) Expressed at Sites of Embryo Implantation J. Biol. Chem., March 23, 2001; 276(13): 10253 - 10262. [Abstract] [Full Text] [PDF]
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 M. J.G. Jimenez, M. Balbin, J. Alvarez, T. Komori, P. Bianco, K. Holmbeck, H. Birkedal-Hansen, J. M. Lopez, and C. Lopez-Otin A regulatory cascade involving retinoic acid, Cbfa1, and matrix metalloproteinases is coupled to the development of a process of perichondrial invasion and osteogenic differentiation during bone formation J. Cell Biol., December 24, 2001; 155(7): 1333 - 1344. [Abstract] [Full Text] [PDF]
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