From the Departments of Pediatrics (Allergy and
Pulmonary Medicine) and Cell Biology and Physiology, Washington
University School of Medicine, St. Louis, Missouri 63110 and the
§ Departments of Virology and Pathology, Haartman Institute,
University of Helsinki, FIN-00290 Helsinki, Finland
Received for publication, February 28, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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We have cloned a new human matrix
metalloproteinase (MMP-28, epilysin) from human keratinocyte and testis
cDNA libraries. Like most MMPs, epilysin contains a signal
sequence, a prodomain with a PRCGVTD sequence, a zinc-binding catalytic
domain with an HEIGHTLGLTH sequence, and a hemopexin-like domain. In
addition, epilysin has a furin activation sequence (RRKKR) but has no
transmembrane sequence. The exon-intron organization and splicing
pattern of epilysin differ from that of other MMP genes. It has only 8 exons, and 5 exons are spliced at sites not used by other MMPs. Another novel feature of epilysin is that exon 4 is alternatively spliced to a
transcript that does not encode the N-terminal half of the catalytic
domain. Northern hybridization of tissue RNA indicated that epilysin is
expressed at high levels in testis and at lower levels in lungs, heart,
colon, intestine, and brain. RNase protection assay with various cell
lines indicated that epilysin was selectively expressed in
keratinocytes. Recombinant epilysin degraded casein in a zymography
assay, and its proteolytic activity was inhibited by EDTA and by
batimastat, a selective MMP inhibitor. Immunohistochemical staining
showed expression of epilysin protein in the basal and suprabasal
epidermis of intact skin. In injured skin, prominent staining for
epilysin was seen in basal keratinocytes both at and some distance from
the wound edge, a pattern that is quite distinct from that of other
MMPs expressed during tissue repair. These findings suggest that this
new MMP functions in several tissues both in tissue homeostasis and in repair.
The matrix metalloproteinases
(MMPs)1 compose a family of
enzymes that share several common structural features and that function both in the turnover and degradation of extracellular matrix proteins and in the processing, activation, or deactivation of a variety of
soluble factors (1). MMPs, or matrixins, are a subgroup of the much
larger metalloproteinase superfamily, which also includes astacin and
ADAM proteinases, among others. To date 23 different MMPs have been
cloned, and additional members continue to be identified (2).
To be classified as a matrix metalloproteinase, a protein must have
conserved features of two domains, namely the prodomain and the
catalytic domain. The prodomain of a typical MMP is about 80 amino
acids, and all MMPs, except MMP-23 (3), contain the consensus sequence
PRCXXPD. As for all metalloproteinases, the catalytic domain
contains an active site Zn2+ that binds three conserved
histidines in the sequence
HEXXHXXGXXH(S/T)XXXXXXM, which also contains a conserved methionine to the carboxyl side of the
zinc-binding site (metzincins) (4). In an inactive state, the conserved
cysteine residue in the prodomain provides the fourth coordination site
for the catalytic zinc ion. In addition, with the exception of
matrilysin (MMP-7), endometase/matrilysin-2 (MMP-26), and MMP-23, MMPs
have a hinge region, which is often proline-rich, and a so-called
hemopexin-like C-terminal domain (3, 5, 6). Other domains found in MMPs
are specialized to subgroups of enzymes. For example, four
membrane-type MMPs (MMP-14, -15, -16, and -24) have transmembrane and
cytosolic domains, whereas MT4-MMP and MT6-MMP (MMP-17 and -25, respectively) have C-terminal hydrophobic extensions that act as a
glycosylphosphatidylinositol-anchoring signal (7-9). The two
gelatinases (MMP-2 and MMP-9) have gelatin-binding domains. MMP-23
lacks the hemopexin domain and has a novel cysteine array motif and an
immunoglobulin-like C2-type fold domain (3, 10). In addition to a
common domain structure, MMPs share a similar gene arrangement
suggesting that they were generated by duplications of an ancestor
gene. At least eight of the known human MMP genes (MMPs 1, 3, 7, 8, 10, 12, 13, and 20) are clustered on chromosome 11 at 11q21-23. Other
known MMP genes are scattered along chromosomes 1, 8, 12, 14, 16, 20, and 22 (3, 11, 12).
MMPs are secreted or bound or anchored to the cell membrane, and all
function extracellularly or within the secretion pathway. As
demonstrated in defined in vitro studies, almost all MMPs
can cleave or degrade some protein components of the extracellular matrix, and many are able to act on a wide variety of proteins (13).
Notable exceptions to this rule are stromelysin-3 (MMP-11) and MMP-23,
which have no known extracellular matrix substrates (3, 14). In
addition and quite importantly, MMPs can process or degrade nonmatrix
proteins. For example, matrilysin is responsible for activation of the
pro-form of Many of the secreted MMPs, including MMPs 1, 3, 9, 10, 11, and 13, are
not expressed in normal, healthy resting tissues, and with some
exceptions, their production and activity are maintained at nearly
undetectable levels. In contrast, some level of MMP expression is seen
in any repair or remodeling process, in any diseased or inflamed
tissue, and in essentially any cell type grown in culture (25, 26).
Although the qualitative pattern and quantitative levels of MMPs vary
among tissues, diseases, tumors, inflammatory conditions, and cell
lines, a reasonably safe generalization is that activated cells express
MMPs. Some MMPs, including MMPs 7, 19, 24, 25, and 26, are expressed in
healthy tissues (27-31).
In the present study, we report on the cloning and initial
characterization of a novel human MMP, MMP-28, which we call epilysin. We isolated the cDNA for this protein from keratinocyte and testis libraries, and we show that it has the essential domains of a prototypic MMP, as well as several unique features. Because of its
ability to degrade a protein substrate was fully inhibited by EDTA and
a hydroxamate MMP inhibitor, epilysin, is indeed a metalloenzyme. Our
data suggest that epilysin is expressed in intact tissues and
up-regulated in response to injury. Thus, this new MMP may function in
both tissue homeostasis and tissue repair.
Cloning of Human Epilysin cDNA, Exon/Intron Mapping--
A
search of the GenBankTM data base with the peptide string
FDGXXXXLAHAXXPGXXXXGDXHFDXXEXW,
which is conserved among MMPs, returned a homologous sequence within an
82-kb human genomic DNA clone (GenBankTM accession number
AC006237). Nested primers were designed to amplify a 161-bp cDNA
fragment by RT-PCR using HT-1080 human fibrosarcoma line RNA as a
template. The amplified product corresponds to bases 726-886 (see Fig.
1). By screening a human foreskin keratinocyte cDNA library
(HL1110b, CLONTECH, Palo Alto, CA) by plaque
hybridization with the 32P-labeled 161-bp cDNA
fragment, we obtained three positive clones, which were then sequenced.
The longest clone of 1.5 kb contained exons 3-8 of epilysin (see below
for exon numbering). This cDNA clone was then used to screen a
pooled human testis cDNA library (HL5033t,
CLONTECH). Among the more than 20 positive clones
was a clone that contained the coding regions of exons 1 and 2. Exon-intron boundaries were determined by comparing the cDNA
sequences with the genomic sequence in data base.
Computer Analyses--
Screening of GenBankTM data
base was performed using the TBLASTN program and the NCBI server
(32). Epilysin signal peptide was identified, and its cleavage site was
predicted using the SignalP server (33). The amino acid sequences of
human MMPs 1, 3, 11, 14, and 19 were aligned with epilysin using
ClustalW (34). A phylogenetic tree was drawn based on a ClustalW
alignment of the amino acid sequences of the catalytic domains of all
known human MMPs.
Cell Culture--
Human foreskin fibroblasts, immortalized human
keratinocytes (HaCaT) (35), and human fibrosarcoma HT-1080 cells
(CCL-121, American Type Culture Collection, Manassas, VA) were grown to confluence in Eagle's minimal essential medium containing 10% heat-inactivated fetal calf serum (Life Technologies, Inc.), 100 IU/ml
penicillin, and 50 µg/ml streptomycin. Human colon adenocarcinoma HT-29 cells (HTB38, ATCC) were maintained in RPMI medium. MMP expression was stimulated by treatment with 16 nM phorbol
ester (PMA, Sigma) for 24 h. Primary human keratinocytes were
isolated from normal full thickness adult skin and cultured on
collagen-coated dishes as described (36). U937 cells (ATCC, CRL 1593),
a human monocyte-like cell line, were cultured and differentiated to
stimulated macrophage-like cells by a 24-h treatment with 4 nM PMA and 5 µg/ml lipopolysaccharide (Sigma) as
described (37). Total RNA was isolated using RNAzol B (Tel-Test,
Friendswood, TX).
Northern Blot Analysis--
Nylon filters containing 2 µg of
poly(A)+ RNA from various human tissues
(CLONTECH) were prehybridized with ExpressHyb®
hybridization solution (CLONTECH) and then
hybridized in the same solution with 32P-labeled epilysin
probe, generated by random priming using a 1.5-kb cDNA fragment as
a template. Loading was normalized by hybridization with a RNase Protection Analysis and Determination of Alternative
Splicing--
Expression of epilysin mRNA in cultured cells was
analyzed by ribonuclease protection technique using Direct Protect kit
(Ambion Inc., Austin, TX) and two different RNA probes. A PCR fragment of 161 bp (bases 726-886 in epilysin cDNA) and another fragment of
539 bp (bases 370-908) were cloned to pGEM-T-Easy (Promega), and
plasmid DNA was linearized with SalI. Antisense RNAs of 257 and 635 nt, respectively, were transcribed with T7 RNA polymerase in
the presence of [ Baculoviral Recombinant Epilysin--
For generation of
recombinant protein, nucleotides 536-1732 of epilysin cDNA, coding
for amino acids 123-520 of epilysin protein were amplified
using primers
5'-CGGGATCCGACGATGACGATAAGTTTGCAAAGCAAGGTAACAAATGGTACAAGC-3' (forward, epilysin sequence underlined) and
5'-CGGAATTCTCAGAACAGGGCGCTCCCCGAGTTG-3' (reverse).
The amplification product was digested with BamHI and EcoRI and cloned into the pAcSecG2T baculovirus transfer
vector (PharMingen, San Diego, CA). The resulting expression construct codes for a fusion protein of Schistosoma japonicum
glutathione S-transferase (GST) and amino acid residues
123-520 of epilysin corresponding to the putative furin-activated
enzyme. This vector also provides the signal peptide from the
baculovirus protein gp67 to direct secretion of the fusion protein.
Sf9 cells were transfected with the construct and BaculoGold DNA
(Phar- Mingen) to produce recombinant baculovirus. Following two
rounds of virus amplification, High Five insect cells (Stratagene) were
infected and harvested along with conditioned medium 5 days later. Cell pellets were lysed in 10 mM Tris-HCl buffer, pH 7.5, containing 130 mM NaCl, 1% Triton X-100, 10 mM
NaF, 10 mM NaPi, and 10 mM NaPPi. GST-epilysin fusion protein was purified from cell
lysates and conditioned medium by glutathione-Sepharose affinity
chromatography according to manufacturer's protocol (PharMingen).
Protein eluting from the affinity resin was analyzed by SDS-PAGE and
Western blotting with a rabbit polyclonal anti-GST antibody (Upstate
Biotechnology, Inc.). Unexpectedly, most of the GST-epilysin fusion
protein was found in cell pellets and not in the conditioned medium.
Recombinant Epilysin Produced in E. coli and Casein
Zymography--
To express a fusion protein consisting of S. japonicum glutathione S-transferase (GST) and the pro-
and catalytic domains of epilysin (amino acid residues 23-284, see
Fig. 1) in Escherichia coli, a 802-bp fragment of the MMP-28
cDNA was amplified by PCR using primers
5'-CGGGATCCCAGCCCGCGGAGCGCGGA-3' (forward, epilysin sequence underlined) and
5'-GGAATTCTCACCCATACAGGCTCTGCACGGCCAGC-3' (reverse),
and the PCR product was digested with BamHI and
EcoRI and cloned into the pGEX-6P-2 vector (Amersham
Pharmacia Biotech). The resulting expression vector was then
transformed into BL21(DE3)pLys strain of E. coli. Overnight
bacterial culture derived from a single bacterial colony was diluted
1:10 and incubated at 37 °C for 2 h. Expression of the fusion
protein was then induced by adding
isopropyl-1-thio- Preparation of Antibodies and Immunoblotting Assay for
Epilysin--
An 8-chain branching multiple antigenic peptide of 16 amino acids, DQDERWSLSRRRGRNL, corresponding to the middle of the
catalytic domain of epilysin (amino acid residues 219-234, see Fig.
1), was used as an antigen (Research Genetics, Huntsville, AL). Rabbits were first immunized with 0.5 mg of the peptide in complete Freund's adjuvant, and three booster injections with 0.5 mg of the peptide in
incomplete Freund's adjuvant were given 2, 6, and 8 weeks later by a
commercial operation (Research Genetics). Antibodies were purified from
whole serum, harvested at 10 weeks after primary injection, by affinity
chromatography with the peptide coupled to
N-hydroxysuccinimide-Sepharose 4B according to the
manufacturer's instructions (Amersham Pharmacia Biotech). For
immunoblotting, confluent cultures of HaCaT cells were washed with
serum-free medium and were incubated under serum-free conditions for an
additional 48 h. The medium was then collected and concentrated
70-fold using a Centricon microconcentrator (Amicon, Beverly, MA). 10 µl of concentrated conditioned medium was mixed with an equal amount of Laemmli sample buffer containing 10% Expression of Epilysin in CHO Cells, Immunofluorescence
Staining--
Chinese hamster ovary (CHO) cells were transfected with
a cDNA construct coding for epilysin with a C-terminal 10-aa
influenza virus hemagglutinin tag under transcriptional control by
cytomegalovirus promoter in pcDNA3 vector (Invitrogen, San Diego,
CA) using FuGENE 6 transfection reagent according to the
manufacturer's instructions (Roche Molecular Biochemicals). 24 h
after transfection, transfected cell clones were selected for neomycin
resistance as described (40). For immunofluorescence staining, a pool
of transfected cells was plated on glass coverslips, and 3 days later,
the cells were fixed with 3% paraformaldehyde in phosphate-buffered
saline (PBS, 0.14 M NaCl in 10 mM phosphate
buffer, pH 7.4). After fixing the coverslips were washed three times
with PBS and blocked with 5% bovine serum albumin in PBS for 30 min.
Affinity-purified epilysin antibody was then added (1:100 dilution) in
0.5% bovine serum albumin in PBS and incubated 1 h at room
temperature with mild shaking. After three washes with the same buffer,
fluorescein isothiocyanate-conjugated anti-rabbit IgGs (Jackson
ImmunoResearch Laboratories, West Grove, PA) were added and incubated
for 1 h. Coverslips were then washed 5 times with PBS and mounted
on glass slides using Vectashield anti-fading agent (Vector
Laboratories, Inc., Burlingame, CA).
Immunohistochemistry--
Individual, 4-mm-wide, full thickness
biopsies of human skin used for keratinocyte culture were placed into
the wells of 6-well cluster dishes and covered with Dulbecco's
modified Eagle's medium containing antibiotics. 24 h later,
tissues were fixed in 10% buffered formalin and processed for paraffin
embedding. Deparaffinized 5-µm sections were processed for
immunohistochemistry using alkaline phosphatase as described (41).
Endogenous peroxidase activity was blocked by incubation in 0.3%
H2O2 for 30 min at room temperature. Affinity-purified anti-human epilysin antibody was diluted 1:1000. Bound antibody was detected using a Vectastain ABC Elite kit (Vector Laboratories) following the manufacturer's instructions. Peroxidase activity was detected using 3,3'-diaminobenzidine tetrahydrochloride as
chromogenic substrate. Sections were counterstained with Harris hematoxylin. For negative controls, sections were processed with preimmune serum.
Cloning and Sequencing of a cDNA Encoding Human Epilysin,
Comparison with Other MMPs--
To identify undiscovered MMPs, we
searched the GenBankTM data base using the TBLASTN program
and a peptide query sequence
FDGXXXXLAHAXXPGXXXXGDXHFDXXEXW, which defines a partial consensus sequence of a metalloproteinase catalytic domain. Among the more than 100 hits was a human genomic DNA
clone (GenBankTM accession number AC006237) that was
submitted by Whitehead Institute/MIT Center for Genome Research and had
been sequenced as a part of the Human Genome Project sequencing
chromosome 17. There was no annotation that the sequence would code for
proteins. After translation of the genomic DNA in three forward reading frames, several peptide sequences typical of MMPs, including a propeptide sequence PRCGVTD and a catalytic domain sequence HEIGH, were
identified, and these sequences were separated by putative intronic sequences.
To assess if this genomic region was transcribed to an mRNA, two
sets of primers were designed; the forward and reverse primers were
directed to different suspected exons. As a source of RNA, we used the
human fibrosarcoma cell line HT-1080, as these cells are known to
express a wide variety of MMPs (40, 42). cDNA was synthesized using
random hexamer primers and was amplified by PCR. Two-stage PCR with
nested primers produced an amplified DNA fragment of expected size (161 bp), and the nucleotide sequence of this fragment was identical to that
of presumed exonic portions of the genomic sequence (data not shown).
To obtain the full-length cDNA for this novel MMP, we screened a
human keratinocyte cDNA library using the PCR product as a probe.
(RNase protection analysis of various cell lines revealed that epilysin
is expressed in cultured human keratinocytes; see Fig. 6.) Among the
three positive clones, we isolated and characterized a 1.5-kb cDNA
that contained sequence coding for part of the prodomain, the entire
catalytic and hemopexin-like domains, a stop codon TGA, and 85 bp of
3'-untranslated region. To determine the 5' end of this MMP transcript,
we screened a testis cDNA library with a probe corresponding to the
5' end of the 1.5-kb keratinocyte cDNA insert. (Hybridization of
tissue-RNA blots revealed that epilysin is expressed in human testis at
high levels; see Fig. 5.) Of the positive clones, we identified one
containing a cDNA insert that coded for the missing portion of the
prodomain and for about 170 bases of 5'-untranslated region. The open
reading frame, starting from the first ATG codon, contains 1560 nucleotides and codes for a 520-aa protein with a calculated
molecular mass of 59 kDa (Fig. 1).
We named this new MMP epilysin. By using the accepted consecutive
number nomenclature, epilysin would be assigned MMP-28.
The domain structure and organization of epilysin is predictable for an
MMP (Figs. 1 and 2). By using an analysis program available at the
SignalP server (33), we identified a typical hydrophobic signal
sequence of 22 amino acids at the N terminus of epilysin (Fig. 1). The
signal sequence is followed by a prototypic MMP prodomain with the
conserved cysteine-switch sequence PRCGVTD (Figs. 1 and 2). In this
sequence, a proline that is present in all other human MMPs except
MMP-19 (43) is replaced by a threonine (Fig.
2). In addition, after the cysteine
switch sequence, there is an 11-aa insertion, which is not present in
other known MMPs, followed by an RRKKR furin recognition sequence
(Figs. 1 and 2). The catalytic domain is highly conserved relative to
other MMPs, and as for secreted MMPs, an 8-aa insertion present only in
MT-MMPs (e.g. MMP-14) is lacking from epilysin (Fig. 2). The
catalytic center with three histidine residues, HEIGHTLGLTH, is unique
in that no other MMP has threonine within this sequence. A 39-aa hinge
region is followed by a typical hemopexin-like domain. There is no
hydrophobic transmembrane sequence typical of membrane-inserted MMPs or
a hydrophobic extension typical of
glycosylphosphatidylinositol-anchored proteins. In addition, epilysin
has two putative N-glycosylation sites as follows: one in
the N-terminal part of the catalytic domain and another in the second
pexin-like repeat of the hemopexin domain (Fig. 1). The calculated
molecular mass of the proenzyme without the signal sequence is 56 kDa;
the active, furin-processed enzyme is estimated to be 45 kDa. These
weights do not include any contribution in mass by glycosylation.
Comparison of the epilysin amino acid sequence with other MMPs by
ClustalW program (Fig. 2) and construction of a phylogenetic tree on
the basis of the catalytic domain sequences (Fig.
3) indicate that epilysin is most closely
related to some other recently cloned MMPs, including MMP-19, MMP-23,
MT4-MMP (MMP-17), MT6-MMP (MMP-25), and stromelysin-3 (MMP-11) (3, 8,
43-45). The number of identical and similar residues with MMP-19
catalytic domain is 46 and 60%, respectively.
Structural Organization of the Human Epilysin Gene--
We mapped
the exon/intron junctions and determined an exon-intron map of the gene
by comparing the cDNA and genomic sequences (Fig.
4A). Exon-intron boundaries
and the sizes of exons and introns are summarized in Table
I. All exons were contained within the genomic BAC clone except exon 1, and hence, we do not yet know the size
of intron 1 (Table I). The exon-intron structure of epilysin is unique
compared with other MMP genes. Whereas most MMP genes have 10 exons,
the epilysin gene has only 8 exons, similar to that of stromelysin-3
(MMP-11) (Fig. 4B) (46). Furthermore, only three of the
seven splice sites (splice sites between exons 1 and 2, 5 and 6, and 6 and 7) are at positions conserved among most MMP genes. None of the
unique splice sites are similar to those other "nontraditional" MMP
genes characterized to date, such as MMP-7, MMP-11, or MMP-14. Overall,
the organization of the epilysin gene is similar to that of MMP-19,
with one overt difference being that exon 8 of epilysin corresponds to
exons 8 and 9 of MMP-19 (Fig. 4B) (47). The exon/intron
boundaries conform to the GT/AG rule for splice sites (48) (Table
I).
Analysis of Epilysin Expression in Human Tissues and Cell
Lines--
To analyze the expression of epilysin in different human
tissues, we hybridized a Northern blot containing mRNA from various human tissues with a 1.5-kb epilysin cDNA probe. At least three different transcripts of 2.6, 2.0, and 1.2 kb were detected in many
tissues (Fig. 5), and this heterogeneity
is likely due to alternative splicing (see below). The 2.6-kb
transcript was most abundant in all tissues, and among tissues, the
relative levels of three transcripts were about the same. Epilysin
mRNA is highly expressed in testis and at lower levels in lungs,
heart, colon, intestine, and brain (Fig. 5).
To determine which cell types express epilysin, we screened RNA from
several cell lines known to actively express a variety of MMPs. To
obtain maximal specificity and sensitivity, we developed an RNA
protection assay for epilysin mRNA. The human cells we used
included HT-29 colon carcinoma cells (49), U937 monocytic-like cells
(50), HT-1080 fibrosarcoma cells (42), neonatal foreskin fibroblasts
(51, 52), immortalized HaCaT keratinocytes (53), and normal primary
keratinocytes (53). Because the transcription of many MMP genes is
strongly up-regulated by PMA, some cells were treated with this agent
for 24 h before RNA was isolated. U937 cells were treated with a
combination of PMA and lipopolysaccharide, which mediates
differentiation to a macrophage-like phenotype accompanied by a potent
induction of several MMPs (37, 50).
Although epilysin is expressed in several tissues, of the cell types
tested, epilysin mRNA was detected only in keratinocytes (Fig.
6). HaCaT keratinocytes showed the
highest expression of epilysin mRNA, and primary keratinocytes,
which were grown on collagen to induce MMP expression (54), had a
somewhat lower level of expression (Fig. 6). All other cell lines were
negative for epilysin expression. Because we could amplify a portion of epilysin cDNA from HT-1080 RNA by RT-PCR, these fibrosarcoma cells express epilysin at low levels.
To determine whether epilysin mRNA is translated into a protein, we
generated rabbit polyclonal antibodies against a 16-amino acid multiple
antigenic peptide in the middle of the catalytic domain. Anti-epilysin
antibodies were affinity-purified using the same peptide. The
specificity of the antibody was tested by immunofluorescence staining
of transfected CHO cells and by immunoblotting of conditioned medium
(Fig. 7). CHO cells transfected with an epilysin expression construct showed a predictable range of recombinant protein production. Whereas some clones showed prominent fluorescence for epilysin, other selected clones had no staining (Fig.
7A). In addition, we used immunoblotting to assess if
epilysin protein is released by HaCaT keratinocytes. As a positive
control, we used a fusion protein of S. japonicum
glutathione S-transferase and amino acid residues 123-520
of epilysin, corresponding to the putative furin-activated enzyme. A
strongly immunoreactive band of about 58 kDa was detected in
HaCaT-conditioned medium, and the predicted 75-kDa band was seen in the
fusion protein preparation (Fig. 7B). In addition to the
58-kDa band, we detected in HaCaT-conditioned medium a slightly smaller
band of about 55 kDa of much lower intensity (Fig. 7B). As
discussed below, this smaller band could be a product of alternative
splicing or alternative glycosylation. Together, these findings
indicate the epilysin mRNA codes a secreted protein that is
produced and released by keratinocytes.
Detection of Alternative Splicing--
During the cloning of
epilysin, we generated a probe by RT-PCR using primers complementary to
sequences in exons 3 and 5 (see "Experimental Procedures"). In
addition to the expected PCR product, we obtained a shorter cDNA of
nearly equal intensity and a weak band of intermediate size (Fig.
8A). Similar amplified
products were generated using a different forward primer in exon 3, and all PCR products were positive in Southern blotting (Fig.
8A). We gel-purified and sequenced the amplified DNA, and we
found that the longer amplification product contained sequences for exons 3-5, whereas the shorter amplification product represented an
mRNA species that contained exons 3 and 5 but lacked exon 4. To
assess the relative abundance of the different splice forms, another
PCR product was cloned and used to generate an RNA probe for RNase
protection analysis. This probe covers 179 nt of exon 3, the entire
exon 4 (225 nt), and 135 nt of exon 5. In addition to epilysin-specific
sequences, the RNA probe contains 96 nt of vector-derived sequence
giving the total length of 635 nt. HaCaT RNA was then hybridized with
this probe, and nonhybridized probe was degraded by RNase treatment.
The protected fragments included two strong bands of ~550 and 180 nt
and a weaker band of 130 nt. The ~550-nt fragment could be protected
by a mRNA transcript that contains exons 3-5 (expected size 539 nt), and the ~180-nt fragment would represent probe protected by exon
3 (expected size 179 nt). The weaker ~130-nt band could be derived
from probe protected by an mRNA species containing exon 5 but not
exon 4 (expected size 135 nt). The 135-nt band was much weaker than the
179-nt band suggesting that another species of mRNA transcripts
containing exon 3 but lacking both exons 4 and 5 is expressed in testis
and by HaCaT cells. RNase protection using RNA from testis tissue and
RNA from HaCaT cells gave identical results (Fig. 8B).
Production of Recombinant Epilysin (Pro- and Catalytic Domains) in
Bacterial Cells and Analysis of Its Enzymatic Activity--
To
determine whether epilysin cDNA codes for an enzyme with
proteolytic activity, we expressed recombinant epilysin in E. coli as a fusion protein consisting of GST and the pro- and
catalytic domains of epilysin. Because the prodomain may be necessary
for the correct re-folding of the recombinant protein, this region was
included in the construct. SDS-PAGE analysis and Coomassie Blue
staining of proteins bound to glutathione-Sepharose revealed a single
major protein of the expected size (56 kDa) and a minor 33-kDa band
(Fig. 9, lane1). After
treatment with PreScission protease, the 56-kDa protein was cleaved
into two proteins with apparent molecular masses of 34 and 32 kDa
(lane 2), which were identified by immunoblotting as
epilysin and GST, respectively (data not shown). After PreScission
cleavage, recombinant epilysin was eluted from the column matrix with
high salt concentration (500 mM NaCl) (lane 3).
Zymogram analysis with 4-16% SDS-PAGE impregnated with blue casein
indicated that both the 56-kDa fusion protein and the 34-kDa free
epilysin had caseinolytic activity. This proteolytic activity was
completely inhibited by exclusion of calcium and zinc and addition of
10 mM EDTA in the incubation buffer, indicating dependence
on divalent cations, calcium, and zinc (Fig. 9). Incubation of the
casein zymogram gel in the presence of 10 µM batimastat,
a specific MMP inhibitor, also completely inhibited the caseinolytic
activity (data not shown). This compound is a substrate-based inhibitor
containing a hydroxamic acid moiety that chelates the active site zinc
cation and renders all MMPs catalytically inactive. PreScission
protease (46 kDa) did not have any detectable caseinolytic activity
(Fig. 9, lane 2).
Expression in Human Skin--
Because epilysin mRNA and
protein were detected in cultured human keratinocytes, we assessed if
this MMP is expressed in human skin. For this study, we incubated
small, uniformly sized pieces of normal adult human skin for 24 h
in culture medium and then fixed and processed the samples for
immunohistochemistry using affinity-purified antibody. During the
incubation, epidermal cells migrated down the edge of the cut surface
of the biopsy in an attempt to heal the "wounded" tissue. In other
studies, we have demonstrated that the expression of MMPs in this
ex vivo model mirrors that seen in vivo. In
the center of the tissue specimens, at some distance (about 2 mm) from
the wound edge, staining for epilysin protein was seen in the intact
epidermis (Fig. 10A). The staining intensity was strongest in basal keratinocytes and
progressively weaker in suprabasal cells. A different pattern of
staining was seen at the wound edge (Fig. 10C). Here,
intense staining for epilysin was seen in migrating keratinocytes at
the wound edge and in stationary basal keratinocytes several cells
behind the wound front. In contrast to intact epidermis, epilysin was
not detected in suprabasal keratinocytes near the wound front (Fig.
10C). No dermal cells were stained for epilysin protein
(Fig. 10, A and C), and no reactivity was
detected in samples processed with preimmune serum (Fig. 10,
B and D).
Here we report the identification, gene, domain organization, and
tissue expression of a new member of the matrix metalloproteinase gene
family. Based on the sequential numerical nomenclature, this new
protein would be designated MMP-28. Because of its prominent expression
in the epidermis and its catalytic activity as an endopeptidase, we
call this new MMP "epilysin." Epilysin has all the key domains of a
typical MMP as follows: a signal peptide, a conserved
cysteine-containing prodomain, a conserved histidine-containing
catalytic domain, a hinge, and a hemopexin domain. It degrades casein,
and its proteolytic activity requires divalent cations and is inhibited
by a synthetic MMP inhibitor. In contrast, epilysin does not include
domains characteristic of other metalloproteinases subfamilies, such as the disintegrin and thrombospondin-like domains found in ADAMs and
tsADAMs, respectively, or a transmembrane domain as is found in most
membrane-type MMPs (55).
The unique exon-intron structure suggests that epilysin diverged early
from other MMPs. However, the splicing pattern is very close to that of
MMP-19, to which epilysin is also most closely related at the amino
acid sequence level. A notable difference between MMP-19 and epilysin
is that MMP-19 has no furin recognition site between its pro- and
catalytic domains. Unlike many MMP genes, which are clustered on the
long arm of chromosome 11, the locus for epilysin is present on
chromosome 17. In addition, we have recently isolated the cDNA for
mouse epilysin (data not shown). Comparison of the amino acid sequences
of the catalytic domains indicates that the coding regions of the mouse
and human epilysin genes are highly conserved (97% identical
residues), suggesting an important function of this region for enzyme activity.
The predicted molecular mass of secreted pro-epilysin is 56 kDa, and if
it is cleaved in the secretion pathway at its furin-recognition site,
the released protein would be about 45 kDa. These sizes, however, do not account for additional mass contributed by
glycosylation. Indeed, by immunoblotting analysis of HaCaT
keratinocyte-conditioned medium, we detected a protein of about 58 kDa
and a less prominent band of about 55 kDa. These two bands may be due
to differential glycosylation, such as is characteristic of
collagenase-1 (56), or they may reflect two distinct isoforms. By using
RT-PCR and RNase protection assays, we determined that epilysin is
transcribed into at least two different mRNAs, one of which lacks
exon 4. Because exons 3-5, among others, end with split codons for
glycine, splicing exon 4 would not affect the amino acid sequence coded by exon 5 (Table I). Omission of exon 4 would reduce the predicted molecular mass of pro-epilysin by about 8 kDa, within the range of the
size difference we detected by immunoblotting. Because our antibody was
raised against sequences coded by exon 5, the epitope would be present
in the putative smaller isoform. Although we do not yet know if the
smaller transcript is translated, splicing exon 4 would place the
cysteine switch of the prodomain much closer to the zinc coordination
site (Fig. 1). This reorganization may have significance for the
structure of the catalytic pocket and for the activation state of the
pro-enzyme.
In our Northern hybridization analyses we could detect at least three
different MMP-28 transcripts of about 2.6, 2.0, and 1.2 kb. Whereas the
longest transcript of 2.6 kb most probably corresponds to the sequence
presented in Fig. 1 with some more 5'-untranslated region and poly(A)
tail added, the other transcripts remain uncharacterized. They could be
either products of alternative splicing or different utilization of
polyadenylation sites. At least the 1.2-kb transcript is too short to
code for a full-length enzyme. During the screening of the libraries we
isolated clones containing intronic sequences (data not shown). Whether
they are cloning artifacts or real functional transcripts remains to be shown.
Another unusual feature of epilysin is that it is expressed in normal,
intact tissues, such as testis, intestine, lung, and skin, and this
pattern of expression suggests that this MMP may serve a role in tissue
homeostasis. Similarly, matrilysin (MMP-7) is expressed by the
epithelium of intact mucosal tissues (31, 49, 57), and we recently
reported that matrilysin functions in innate immunity, a homeostatic
function, by activating prodefensin peptides (15). Although matrilysin
is expressed by mucosal epithelium, including that of small intestine,
injured colon, airways, and exocrine glands, it is not expressed in the
epidermis (31, 49). It is tempting to speculate that epilysin
participates in host defense in intact epidermis by processing
antimicrobial proteins. Indeed, because it is expressed by basal and
suprabasal keratinocytes, released epilysin may not encounter a matrix
substrate in intact skin. Thus, although epilysin is a member of the
matrix metalloproteinases gene family, we cannot yet conclude that
matrix components are physiologic substrates for this enzyme.
In addition to tissue homeostasis, epilysin may serve a distinct and
additional role in repair of cutaneous wounds. In response to injury,
several MMPs are produced by the epidermis in functionally distinct
subpopulations of keratinocytes (58). For example, collagenase-1
(MMP-1), stromelysin-2 (MMP-10), and gelatinase-B (MMP-9) are produced
by basal keratinocytes at the migrating front, whereas stromelysin-1
(MMP-3) is expressed by the hyperproliferative cells just behind these
migrating cells (59-63). Distinct from the localization of these MMPs,
prominent staining for epilysin was seen in basal keratinocytes at the
migratory front and in many cells behind the wound edge. Again, this
pattern is similar to that for matrilysin in wounded epithelium.
Although it is not found in cutaneous wounds (58), matrilysin is
expressed by migrating and stationary epithelial cells in wounds and
ulcerations of mucosal tissues, such as lung and intestine (64, 65).
Demonstrating an essential role for matrilysin in mucosal repair,
airway epithelial wounds do not repair in MMP-7-null mice (65). Thus,
matrilysin serves at least two distinct roles in mucosal tissues as
follows: one in innate defense and the other in epithelial repair and
migration. Epilysin may have equally critical roles in skin. Our future
studies will be directed at determining the function of this new
MMP.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-defensins (15), a class of secreted antimicrobial
peptides, and several MMPs can cleave and inactive the serpin
1-proteinase inhibitor (16, 17), which is an in
vivo substrate for gelatinase-B (MMP-9) (18). In addition, several
MMPs, such as MMP-1, -2, -3, -7, and -11, among others, directly
modulate the activity of several growth factors, such as tumor necrosis
factor-
, insulin-like growth factor-1, epidermal growth factors, and
fibroblast growth factors (19-24). Thus, matrix degradation is neither
a sole nor a common functional feature of MMPs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin
cDNA probe. Hybridization and washes were performed according to
manufacturer's instructions.
-32P]UTP and hybridized with 10 µg
of total cellular RNA. After an overnight hybridization, unpaired RNA
was degraded by treatment with RNase A and RNase T1 (10 and 400 units/ml, respectively) at 37 °C for 30 min followed by
isopropyl alcohol precipitation. Protected RNA fragments were
fractionated by 5% SDS-PAGE containing 6 M urea and
visualized by autoradiography. Alternative splicing was first detected
when RT-PCRs to amplify regions containing bases 370-960 and
440-960 (forward primers 5'-GTGGGTGTCCCAGCTACCTGTC-3' and
5'-TGCGGGGTTACAGATACCAACAG-3', reverse primer
5'-CTCTTGTAGTAGGGCGCCATGAG-3') using HaCaT cDNA as a template gave
three specific products of different size. Specificity of the
amplification products was determined by Southern blotting using a
[
-32P]ATP-labeled internal oligonucleotide probe
5'-GCGGCGAAGCGCACTTCGACCAAGATGAGC-3', and the amplification
products were purified from gel and sequenced. The presence of
alternatively spliced mRNA in HaCaT cells was verified by RNase
protection analysis using the 635-nt RNA probe described above.
-D-galactopyranoside (0.5 mM final concentration) followed by further incubation at
25 °C for 4 h. Recombinant protein obtained in inclusion bodies
was solubilized by sonication in the presence of
N-lauroylsarcosine and affinity-purified with glutathione-Sepharose as described (38). Recombinant fusion protein
bound to glutathione-Sepharose was then digested with PreScission
protease according to manufacturer's instructions (Amersham Pharmacia
Biotech) to remove the GST tag. Between the GST domain and epilysin,
the fusion protein has the recognition sequence (LEVLFQGP) for
PreScission Protease. Recombinant epilysin (pro- and catalytic domains)
was then eluted with 50 mM Tris-HCl buffer, pH 7.5, containing 500 mM NaCl, 5 mM dithiothreitol,
and 0.1% Brij-35. Caseinolytic activity was measured by zymography using a 4-16% SDS-PAGE blue casein zymogram gel (NOVEX, San Diego, CA) according to manufacturer's protocol. After electrophoresis, the
zymogram gel was washed and incubated in 50 mM Tris-HCl
buffer, pH 7.5, containing 1-2.5% Triton X-100 and either 1) 10 mM EDTA, or 2) both 5 mM CaCl2 and
1 µM ZnCl2, or 3) 10 µM
batimastat in the presence of 5 mM CaCl2 and 1 µM ZnCl2.
-mercaptoethanol and resolved by SDS-PAGE through a 4-15% gradient gel. Recombinant baculoviral GST-epilysin fusion protein was used as a positive control.
Proteins were then electrophoretically transferred to nitrocellulose
(Schleicher & Schuell) using a semi-dry blotting apparatus at 2.5 mA/cm2 for 30 min. Membranes were blocked with 5% milk in
PBS/Triton X-100 (0.5%) and incubated with 0.15 µg/ml of
affinity-purified antibodies in 50 mM Tris-HCl buffer
containing 500 mM NaCl, 0.1% Tween 20, and 0.1% bovine
serum albumin, pH 8.5. After five washes in the same buffer, the bound
antibodies were detected using biotinylated anti-rabbit IgG antibodies
and peroxidase-conjugated streptavidin (Dakopatts, Copenhagen, Denmark)
and enhanced chemiluminescence Western blotting detection system
(Amersham Pharmacia Biotech) as described (39).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence of the human epilysin
(MMP-28) cDNA and its deduced amino acid sequence. The deduced
amino acid sequence is shown below the DNA sequence. The
first ATG and the termination codon TGA are in bold. Numbers
on the right and left refer to the positions of
nucleic acids and amino acid residues, respectively. Pro-sequence
PRCGVTD and zinc-binding site HEIGHTLGLTH are inverted, and the furin
recognition sequence RRKKR is boxed. Predicted signal
peptide cleavage site is indicated with an arrow, and the
furin cleavage site is marked with an arrowhead. Two
potential N-glycosylation sites are underlined.
Vertical bars indicate the exon limits, and exons are
numbered as indicated in Table I. Our cDNA clones cover the
sequence between bases 1 and 1817. First consensus polyadenylation
signal AATAAA is found about 500 bp downstream in the genomic sequence
(GenBankTM accession number AC006237), and the sequence
between bases 1818-2332 is derived from this deposited sequence and
represents the most probable 3' end of epilysin mRNA. The
nucleotide sequence data are in the GenBankTM nucleotide
sequence data base with the GenBankTM accession number
AF219624.
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Fig. 2.
Comparison of the amino acid sequence of
epilysin with other human MMPs. Peptide sequences for human MMPs
including collagenase-1 (MMP-1), stromelysin-1 (MMP-3), stromelysin-3
(MMP-11), membrane-type-1 matrix metalloproteinase (MT1-MMP and
MMP-14), and MMP-19 were retrieved from GenBankTM and
aligned with epilysin (MMP-28) peptide sequence using ClustalW program.
Identical amino acid residues in all six MMPs are indicated
below the sequences. Epilysin domains are indicated
above the sequence.
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Fig. 3.
Dendogram of the catalytic domains of human
MMPs. The amino acid sequences of the catalytic domains of human
MMPs were retrieved from GenBankTM and aligned with
ClustalW to generate a phylogenetic tree. Epilysin is most closely
related to MMPs 19, 23, 11, and 17. Other clusters of MMPs were formed
by MT-MMPs excluding MT4-MMP (MMPs 14, 15, 16, and 24), gelatinases
(MMPs 2 and 9), stromelysin-1 and -2 (MMPs 3 and 10), and
collagenases-1 and -2 (MMPs 1 and 8).
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Fig. 4.
Organization of the human epilysin gene.
Comparison with other human MMP genes. A, organization of
the epilysin gene was drawn based on the comparison of the cDNA
sequence with the sequence of the genomic BAC clone hRPC.161_P_9. Exons
are numbered from the 5' end of the gene and depicted by black
boxes. The noncoding regions of the first and last exons are
depicted by open boxes. The size of the first intron is
unknown; it was not present in the genomic BAC clone. The positions of
the transcription start site (ATG), stop codon (TGA), pro-sequence
(PRCGVTD), furin cleavage site (RRKKR), and the catalytic zinc-binding
site (HEIGHTLGLTH) are indicated below the gene graph. Base
positions in the BAC clone are indicated above the gene
graph. B, comparison of exon and domain structures of
members of MMP family. The exons in human epilysin (MMP-28), gelatinase
A (MMP-2), collagenase-1 (MMP-1), stromelysin-1 (MMP-3), matrilysin
(MMP-7), stromelysin-3 (MMP-11), membrane type-1 matrix
metalloproteinase (MT1-MMP, MMP-14), and MMP-19 are shown as
boxes, with their sizes in nucleotides below. Open
boxes indicate untranslated sequences. Filled boxes
indicate different domains of the matrix metalloproteinases as follows:
signal peptide, prodomain, catalytic domain, hinge region,
hemopexin-like domain, transmembrane domain, and intracellular domain.
FN, fibronectin-like domain of gelatinase A. The locations
of the exon-intron splicing sites in the epilysin gene differ markedly
from other MMPs. Only the splice sites between exons 1 and 2, 5 and 6, and 6 and 7 are at conserved positions among most MMP genes, whereas
all the splice sites of epilysin gene are utilized also in
theMMP-19 gene.
The exon-intron junctions in the human MMP-27 gene
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Fig. 5.
Northern blot analysis of epilysin expression
in a variety of human tissues. 2 µg of poly(A)+ RNA
from the indicated tissues were analyzed by hybridization with the
cDNA for human epilysin. Migration of RNA size markers is shown on
the left. Filters were subsequently hybridized to a human
-actin probe to control the loading of RNA. At least three different
transcripts of 2.6, 2.0, and 1.2 kb were detected.
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Fig. 6.
Expression of epilysin in cultured
cells. RNase protection analysis. Confluent cultures of colon
adenocarcinoma cells (HT-29), histiocytic lymphoma cells (U937), human
fibrosarcoma cells (HT-1080), human foreskin fibroblasts, immortalized
human keratinocytes (HaCaT), and primary keratinocytes were treated
with PMA (40 nM) and lipopolysaccharide (5 µg/ml) for
24 h where indicated. Total RNA was then extracted and analyzed by
RNase protection for the presence of epilysin mRNA as described
under "Experimental Procedures." Protected RNA fragments were
fractionated by 5% TBE-PAGE containing 6 M urea and
visualized by autoradiography. Undigested probe (257 nt) and protected
fragment (161 nt) are indicated on the left. Migration of
RNA size markers is shown on the left. Specific signal for
epilysin could be detected only in HaCaT and keratinocyte samples.
Full-length probe is also protected to a minor extent because of
residual template DNA. Equal loading of the RNAs was confirmed by
separate RNase protection analysis for cyclophilin mRNA
(cyclo).
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Fig. 7.
Expression of epilysin in cultured
cells. A, phase-contrast microscopy of transfected pool
of CHO cells. CHO cells were transfected with an expression construct
for recombinant epilysin, and neomycin-resistant cells were selected
with G418. This transfected pool was then plated on glass coverslips
and fixed with paraformaldehyde 3 days later. B,
immunofluorescence staining. This field shows one CHO clone expressing
epilysin at high levels (marked with arrows). Other cells
seen in phase contrast (A) have low or no expression.
C, Western blot. Concentrated medium from HaCaT cells and
purified recombinant fusion protein of glutathione S-transferase and
catalytic and hemopexin domains of epilysin (GST-M28) were separated by
electrophoresis in 4-15% SDS-PAGE under reducing conditions. The
proteins were electrophoretically transferred to nitrocellulose and
immunostained for the presence of epilysin protein.
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Fig. 8.
Alternative splicing of epilysin.
A, RT-PCR and Southern blotting. Epilysin cDNA was
amplified by RT-PCR using two different forward primers in the exon 3 and a reverse primer in exon 5. PCRs were then separated by
electrophoresis in a 1.5% agarose gel and stained with ethidium
bromide (lanes 1 and 2). For identification, DNA
was transferred onto a nylon membrane by Southern blotting and probed
with an internal oligonucleotide probe in exon 5 of epilysin
(lanes 3 and 4). Lanes 1 and
3, forward primer 370F; lanes 2 and 4, forward primer 440F. M, 100-bp marker, 500-bp band has
higher intensity of staining. B, RNase protection analysis.
Total RNA from human testis or HaCaT cells was hybridized with an
antisense RNA probe spanning exons 3-5 of epilysin. Unbound probe was
then degraded with RNases, and protected RNA fragments were
fractionated by 5% TBE-PAGE containing 6 M urea and
visualized by autoradiography. The protected fragments included a
539-nt fragment corresponding to mRNA containing all three exons, a
179-bp fragment corresponding to exon 3, and a faint band or 135 nt
corresponding to exon 5, as indicated on the right.
Migration of RNA size markers is shown on the left. To avoid
overexposure of the film, the amount of the probe loaded in the
1st lane corresponds only ~5% of the probe used in
hybridizations.
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Fig. 9.
Epilysin is a metalloproteinase capable of
degrading casein. Recombinant epilysin was expressed in E. coli as a fusion protein that consists of GST, prodomain of
epilysin, and catalytic domain of epilysin. Fusion protein was purified
by affinity chromatography on glutathione-Sepharose, and this fusion
protein was cleaved with PreScission protease to release epilysin from
GST. Washing of the glutathione-Sepharose matrix with 500 mM NaCl selectively released recombinant epilysin, whereas
GST and PreScission protease remained bound to the matrix. Samples of
affinity purified fusion protein (lanes 1), fusion protein
cleaved with PreScission protease (lanes 2), and free
epilysin eluted from glutathione-Sepharose matrix (lanes 3)
were then separated by electrophoresis in 4-15% SDS-PAGE under
reducing conditions, and protein was stained with Coomassie Blue
(left panel) or analyzed by casein zymography in a 4-16
SDS-PAGE impregnated with blue casein in the presence of calcium and
zinc (middle panel) or in the absence of divalent cations
and in the presence of EDTA (10 mM) (right
panel). Migration of molecular mass markers is shown on the
left. Migration of the fusion protein is indicated with an
asterisk, epilysin (pro- and catalytic domains) with an
arrow, and free GST with an arrowhead, all on the
right.
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Fig. 10.
Expression of epilysin in the epidermis of a
healing wound. Four-mm punch biopsies of normal human skin
(n = 4 donors) were cultured on a cell culture dish for
24 h, fixed in buffered formalin, embedded, and stained for
epilysin protein using affinity-purified antibodies. A,
middle of the biopsy. Staining for epilysin was seen in keratinocytes
in all layers of intact epidermis, with more prominent signal in basal
keratinocytes. Staining for epilysin was not seen in any cell type in
the underlying dermis. C, wound (biopsy) edge. An intense
signal for epilysin protein was seen in basal keratinocytes at the
wound edge and in basal keratinocytes some distance from the migratory
front. B and D, serial sections of those shown in
A and C were processed with preimmune
serum.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Prof. Norbert Fusenig of German Cancer Research Center (Heidelberg, Germany) for HaCaT cells.
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Note Added in Proof |
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After our article was published in the "JBC papers in press" on December 19, 2000, we learned that another manuscript, "MMP-28, a New Human Matrix Metalloproteinase with an Unusual Cysteine-Switch Sequence Is Widely Expressed in Tumors" by George N. Marchenko and Alex Y. Strongin, had been accepted for publication in Gene. The gene identified and characterized in the Gene paper is identical to that described in the current publication.
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FOOTNOTES |
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* This work was supported by The Academy of Finland, the Finnish Cultural Foundation, and National Institutes of Health Grant AR45254.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF219624.
¶ William S. Keck fellow at Washington University School of Medicine. To whom correspondence should be addressed: Dept. of Pathology, Haartman Institute, University of Helsinki, P. O. Box 21 (Haartmaninkatu 3), FIN-00014 Helsinki, Finland. Tel.: 358-9-191-26469; Fax: 358-9-191-26475; E-mail: jouko.lohi@helsinki.fi.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M001599200
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ABBREVIATIONS |
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The abbreviations used are: MMP, matrix metalloproteinase; GST, Schistosoma japonicum glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; kb, kilobase pair; bp, base pair; nt, nucleotides; aa, amino acids; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary.
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