From the Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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A new member of the membrane-type matrix
metalloproteinase (MT-MMP) subfamily tentatively named MT5-MMP was
isolated from mouse brain cDNA library. It is predicted to contain
(i) a candidate signal sequence, (ii) a propeptide region with the
highly conserved PRCGVPD sequence, (iii) a potential furin recognition
motif RRRRNKR, (iv) a zinc-binding catalytic domain, (v) a
hemopexin-like domain, (vi) a 24-residue hydrophobic domain as a
potential transmembrane domain, and (vii) a short cytosolic domain.
Reverse transcriptase-polymerase chain reaction analysis of its
transcripts indicates that MT5-MMP is expressed in a brain-specific
manner consistent with the origin of its EST clone from cerebellum. It
is also highly expressed during embryonic development at stages day 11 and 15. Like other MT-MMPs, MT5-MMP specifically activates
progelatinase A when co-expressed in Madin-Darby canine kidney
cells. Its ability to activate progelatinase A is dependent on its
proteolytic activity since a mutation converting Glu to Ala in the zinc
binding motif HE255LGH renders MT5-MMP inactive
against progelatinase A. In contrast to other MT-MMPs, MT5-MMP tends to
shed from cell surface as soluble proteinases, thus offering
flexibility as both a cell bound and soluble proteinase for
extracellular matrix remodeling processes. Taken together, these
properties serve to distinguish MT5-MMP as a versatile MT-MMP playing
an important role in extracellular matrix remodeling events in the
brain and during embryonic development.
Members of the matrix metalloproteinase
(MMP)1 family have been well
documented as critical players in the breakdown of extracellular matrix
(ECM) under both physiological as well as diseased conditions ranging
from embryo implantation to cancer progression (1-4). Highly modular
in design, all MMPs share three basic functional domains found in the
smallest MMP matrilysin: (i) a signal peptide for extracellular
targeting, (ii) a prodomain with the cysteine-switch for latency, and
(iii) a conserved catalytic domain built around a zinc-binding site
HEXXH as the catalytic core (5, 6). The addition of a
hemopexin-like domain to this basic design eventually led to the
evolution of the rest of the MMP family and confers them with
specificity in substrate as well as inhibitor bindings (5, 6). While
the majority of the MMPs are secreted as soluble enzymes into
extracellular milieu, a subset of MMPs have been identified in recent
years to contain additional sequences downstream of the hemopexin-like
domain capable of anchoring the MMPs on plasma membrane (7-10). Named
after the putative transmembrane domains as membrane-type matrix
metalloproteinase 1 to 4 (MT1-, MT2-, MT3-, and MT4-MMPs), these
enzymes have been proposed to be the master switches of ECM turnover
based on the purported ability of MT-MMPs to activate other MMPs such
as progelatinase A and collagenase 3: two degradative enzymes widely
implicated in tumor invasion and metastasis (7, 11, 12). However, MT-MMPs themselves are synthesized in latent forms and activation is
required for them to exert any proteolytic function (13-16). The
mechanism responsible for MT-MMP activation appears to be mediated by
members of the proprotein convertase family which can specifically
cleave off the prodomain at the carboxyl side of the conserved
RXRXKR motif sandwiched between the pro- and catalytic domains of all MT-MMPs, a mechanism first demonstrated in
stromelysin-3 (13-18). Thus, a proprotein convertase/MT-MMP/MMP cascade could be potentially responsible for the regulation of ECM
turnover at the level of zymogen activation.
Despite the extensive sequence homology and functional overlap among
MT-MMPs, little is known about any functional cooperation among
themselves in executing ECM remodeling. Their patterns of expression
suggest a complex picture with overlapping expression in both normal
and tumor tissues (7-10, 19). For example, breast cancer tissues are
known to express MT1-, MT2-, MT3-, and MT4-MMPs individually or
together as detected by Northern blotting and in situ
hybridization (19-21). In addition, MT1-MMP has been investigated extensively and found to be expressed in other malignant tumors such as
those from human brain, colon, pancreas, liver, gastrointestinal organs, ovary, and cervix (18, 22-27). Among tissues and cell examined, expression of MT1-, MT2-, and MT3-MMPs seems to correlate well with the activation of progelatinase A, suggesting that MT-MMPs may act cooperatively toward progelatinase A in vivo (7,
22-27). With the expansion of the MT-MMP family, it becomes apparent
that the function of MT-MMPs may not be only restricted to
progelatinase A and collagenase 3 activation. In fact, purified MT1-MMP
and MT2-MMP can degrade fibronectin, laminin, type I and III collagens, nidogen, tenascin, aggrecan, and perlecan (13, 14, 16). MT3-MMP appears
to be able to degrade denatured type I collagen (gelatin), native type
III collagen, and fibronectin based on limited studies (28, 29). Taken
together, MT-MMPs are a subgroup of versatile proteinases involved in
ECM remodeling by both activating other MMPs as well as directly
degrading ECM components.
In contrast to secreted MMPs, MT-MMPs may express their proteolytic
activities more efficiently by anchoring on cell membrane and enjoying
two distinct advantageous properties, which are highly focused on ECM
substrates and more resistant to proteinase inhibitors present in the
extracellular milieu (11, 30). Recently, Nakahara and colleagues (31)
demonstrated that MT1-MMP is localized in the invadopodia of malignant
melanoma cells via the transmembrane/cytoplasmic domain, responsible
for the efficient degradation of subjacent substrates and invasion into
ECM in vitro. MT1-MMP can also confer mouse lung carcinoma
cells metastatic phenotype upon transfection when analyzed in a
tail-vein injection assay in vivo (32). Therefore, recent
attention has been shifting toward the characterization of
membrane-bound MMPs and their biochemical properties (11). In this
report, the identification and characterization of MT5-MMP, the fifth
member of the MT-MMP subfamily, is described.
Cell Lines and Reagents--
MDCK cells and COS 7 were obtained
and maintained as described previously (17, 33). DNA restriction and
modification enzymes were purchased from Promega (Madison, WI).
Oligonucleotide primers were made by the University of Minnesota
microchemical core facility. COS cells are used for transient gene
expression because the pCR3.1 expression vector system is very
efficient in COS cells due to the presence of SV40 T antigen, while
moderately efficient in cells lacking T antigen such as MDCK cells. For
progelatinase A activation, MDCK is preferred because it expresses
higher levels of furin, a putative MT5-MMP activator, than COS
(17).
cDNA Cloning and Sequence Analysis--
The original EST
clone EST27028 was obtained from American Type Cell Culture (ATCC, MD).
The rest of EST27028 was sequenced by primer-walking using an ABI371
automatic sequencer. The resulting sequence was blasted against Genbank
data base and aligned to 321GNFDT of MT1-MMP. The rest of
EST27028 contains basically the entire hemopexin-like domain, followed
by a putative transmembrane domain and cytosolic domain. Since this
gene is homologous, but not identical to known MT-MMPs, it was named
MT5-MMP as the fifth member of the MT-MMP subgroup. An EcoRI
fragment from this clone was then isolated and used as a probe to
isolate the missing part of MT5-MMP from both human and mouse brain
cDNA libraries (Stratagene, CLONTECH, CA). From
the human cDNA library, at least 10 clones have been isolated and
sequenced to find the longest cDNA starting immediately upstream of
the PRCGVPD sequence, but missing the 5' end approximately 100-amino
acid residue. Fortunately, out of 7 clones isolated from the mouse
brain cDNA library, two longest clones 3 and 17 overlap to give
rise to a complete open reading frame. Clone number 17 covers from the
5'-untranslated region to the seventh residue upstream of the end of
MT5-MMP, thus missing the last 6 residues. Clone 3 covers the 3'
portion of the MT5-MMP cDNA. Both clones were sequenced by a
combination of shotgun strategy and primer-walking from both strands to
give rise to the full-length sequence of mouse MT5-MMP. Sequence
alignment was performed via Internet using program Multalin version
5.3.3 at http://www.expasy.ch/www/tools.html using blosum62 with Gap
weight: 12; Gap length weight: 2. The dendrogram was constructed using
ClustalW program.
Tissue Distribution of MT5-MMP as Determined by
RT-PCR--
Premade cDNA panels from mice was purchased from
CLONTECH (Palo Alto, CA) and amplified with two
primers located at the 3' portion of MT5-MMP cDNA
(5'-GTGCATGCACTGGGCCATG-3', 5'-TAGCCTTCCTGCACCCG-3'; 2 min at 94 °C
for denaturation, 33 cycles of 10 s at 94 °C, 30 s at
50 °C for annealing and 30 s at 72 °C for extension,
followed by 10 min extension at 72 °C). To control for the amount of
cDNA used in each reaction, a parallel amplification using primers designed from the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was performed under the same experimental conditions.
Construction of MT5-MMP Expression Vectors--
The cDNA
clone number 17 (missing the last 6 residues) was engineered by high
fidelity PCR to give rise to MT5-MMP Analysis of MT5-MMP Products and Progelatinase A
Activation--
The antibody against human MT5-MMP was raised in
rabbit using GST-hMT5-MMP (Tyr125 to Cys538) as
described previously (34). DNA constructs (1.5 µg each) pCR3.1uniMT5-MMP Generation of Stable MT5-MMP Transfectants--
Expression
vector for MT5-MMP was transfected into MDCK cells and stable clones
were selected and characterized as described previously (13, 17). The
expressed MT5-MMP products were analyzed by immunoprecipitations for
both cell and secreted forms as described above. Synthetic
metalloproteinase inhibitor BB-94 (5 µM, British Biotech,
United Kingdom) was added to the serum-free Dulbecco's modified
Eagle's medium and allowed to incubate with MT5-MMP cells. The
conditioned media were analyzed for MT5-MMP activity by zymography directly, by Western blot after ~10-fold concentration using
Millipore YM10 membrane filtration as described (13, 34).
Molecular Cloning of MT5-MMP--
A search of the public EST data
base maintained in the National Center for Biotechnology Information
produced a few MMP candidate genes. One such clone, EST27028 from human
cerebellum with homology to stromelysin-3, actually resembles closely
the hemopexin-like domain of MT1-MMP in a BLAST search of sequence data
bases. To prove that this EST clone is part of a novel MT-MMP gene, the remaining portion of the open reading frame was sequenced to reveal the
presence of a putative transmembrane domain and cytosolic domain. The
resulting sequence shows strong homology to human MT3-MMP, thus named
MT5-MMP according to the current terminology of this subgroup (7-10).
A 1.5-kilobase fragment from this EST clone was isolated and used as a
probe to screen for full-length clones from both human and mouse brain
cDNA library (from CLONTECH and Stratagene). So
far, a cumulative sequence for the human MT5-MMP covers the entire
predicted open reading frame except the pro-domain upstream of the
conserved PRCGVPD cysteine switch. A 5'-rapid amplification of cDNA
ends strategy is currently underway to recover the missing 5' end.
However, the screening for mouse MT5-MMP yielded the full-length open
reading frame with both 5'- and 3'-untranslated region (Fig.
1). The mouse and human MT5-MMP are over
95% homologous, thus, representing indeed a novel gene of the MT-MMP
subgroup.
Structural Features of MT5-MMP and Its Relationship to Other
MMPs--
As shown in Figs. 1 and 2,
MT5-MMP possesses similar structural feature as MT1-, MT2-, and
MT3-MMPs with the characteristic Pro, catalytic, hemopexin-like,
stem/transmembrane/cytosolic domains, maintaining overall sequence
identities of 53, 52.1, and 64.4%, respectively. The homology between
MT5-MMP and MT1-, MT2- and MT3-MMPs is more significant among
subdomains as displayed in Fig. 2B, with identity scores
71.1, 68.8, and 86% for catalytic domains, 57.9, 56.2, and 67.4% for
the hemopexin domain, as well as 23.4, 25.5, and 48.7% for the
stem/transmembrane/cytosolic domain, respectively. However, MT4-MMP
appears to be as distant to MT5-MMP as the other non-membrane type
MMPs, e.g. stromelysin-3 and interstitial collagenase (Fig.
2B). To further establish the relatedness between MT5-MMP
and other MMPs, a phylogenetic tree was constructed using alignment
from the catalytic domains of 18 MMPs (not shown). As expected, both
human and mouse MT5-MMP are grouped together with MT3-MMP as a
sub-branch of the MT-MMP subfamily. Consistent with the homology
scores, MT4-MMP is positioned between the MT-MMP subgroup and the main
branch of collagenases/stromelysins/gelatinases.
Tissue Distribution of MT5-MMP--
To gain insight into the
possible role of MT5-MMP in physiological processes, normal tissues
were screened for MT5-MMP expression by Northern blotting. Both human
and mouse MT5-MMP appears to be expressed in minute quantity in tissues
examined (data not shown). Mouse MT5-MMP appears to migrate slightly
below the 28 S rRNA with an estimated size of 4.5 kilobases. A more
sensitive technique, namely RT-PCR, was then employed to analyze the
distribution of MT5-MMP RNA in mouse tissues. As shown in Fig.
3, adult brain appears to be the major
site of MT5-MMP expression, whereas testis is weakly positive. The rest
of adult tissues are virtually negative for MT5-MMP RNA transcript,
consistent with the Northern blot analysis (data not shown). Since MMPs
have been consistently implicated during the development of embryo,
MT5-MMP expression profile was also obtained among developing mouse
embryos. MT5-MMP expression has an onset of day 11 and persists to day
15 before dropping around day 17 before birth (Fig. 3).
Characterization of MT5-MMP Protein Products and Their
Activities--
Since MT5-MMP is highly homologous to other known
MT-MMPs capable of activating progelatinase A (7-9), MT5-MMP is
hypothesized to be a cell membrane-associated activator of
progelatinase A. To test this possibility, MT5-MMP Catalytically Inactive MT5-MMP Fails to Activate Progelatinase
A--
Full-length MT5-MMP was constructed by adding back the missing
6 residues to the carboxyl terminus of clone 17. This full-length MT5-MMP construct is functionally indistinguishable from MT5-MMP Secretion of MT5-MMP Ectodomain into Conditioned
Media--
Full-length MT5-MMP as well as MT5-MMP The MT-MMPs have been recognized as key regulators for ECM
remodeling under both physiological and pathological conditions (11).
Much of the attention has been focused on the ability of MT1-, MT2-,
and MT3-MMPs to activate progelatinase A or procollagenase 3 on cell
surface (7-10, 12). Synthesized as zymogens themselves, activation of
proMT-MMPs is a pre-requisite for their proteolytic functions. The
mechanism responsible for MT-MMP activation may rely on a basic motif
RXKR found in all MT-MMPs, which is recognized and cleaved
at the carboxyl end by members of the proprotein convertase family such
as furin localized intracellularly in the trans-Golgi network (13, 17).
Thus, MT-MMPs could potentially be the cell surface anchor of a
proteolytic cascade from PC to the soluble MMPs dedicated to the
proteolysis of ECM components. The central role of MT-MMPs as key
regulators in ECM degradation is further strengthened by the discovery
that MT1-, MT2-, and MT3-MMPs can cleave ECM components directly,
making MT-MMPs the most versatile MMPs discovered so far (13, 14, 16,
28, 29). The identification of MT5-MMP enlarges the MT-MMP subgroup to
include 5 distinct members. This addition ensures that the MT-MMPs
overtake the collagenases as the largest subgroup of the MMP
superfamily. This finding is likely to reinforce the notion that
membrane-associated proteolysis plays a critical role in ECM
degradation necessary for many important biological and pathological
processes (11, 36, 37).
Sequence alignment indicates that MT5-MMP is closely related to
MT3-MMP, followed by MT1- and MT2-MMPs. MT4-MMP appears to be only
marginally related to MT5-MMP. The domain structure of MT5-MMP follows
the general design of other MT-MMPs with signal peptide, prodomain,
RXKR activation motif, catalytic domain, hinge region
hemopexin-like domain, and the stem/transmembrane/cytosolic domains.
Despite a high degree of homology, MT5-MMP contains two short segments
with divergent sequences: the hinge and stem regions with unique
dibasic motifs potentially recognizable by proprotein convertases
(Figs. 1 and 2, Ref. 18). These two segments may confer unique function
to MT5-MMP.
Unlike other MT-MMPs, MT5-MMP's expression is highly restricted. In
general, the level of expression for MT5-MMP tends to be much lower
than the other MT-MMPs since conventional Northern blot fails to detect
any meaningful transcript. RT-PCR analysis of various mouse tissues and
developing embryos identified brain as the primary site for MT5-MMP
expression and embryos also express this gene at day 11-15 stage. This
pattern of expression suggests an unique role for MT5-MMP in mediating
ECM turnover during normal biological process. More detailed studies
are under way to define the precise location of MT5-MMP expression in
mouse brain and developing embryos, especially in comparison with the
other MT-MMP. The expression profile of MT5-MMP in human normal or
cancer tissues is also under investigation.
The biochemical properties of MT5-MMP mirror those of MT1-, MT2-, and
MT3-MMPs closely (7-9). MT5-MMP is able to activate progelatinase A
when co-expressed or added exogenously, albeit slightly less efficient
than MT1- and MT3-MMPs. However, the ECM degrading activity of MT5-MMP
remains to be defined. In an effort to produce and purify active enzyme
for substrate studies, stable clones of MT5-MMP have been generated to
express full-length MT5-MMP. The 63-kDa species is a major product of
MT5-MMP expressed both transiently and stably (Figs. 4 and 6). It is
not clear whether this species represents the fully activated form of
MT5-MMP, or simply the proform of MT5-MMP. Protein species with similar
molecular weight have also been identified for at least MT1- and
MT3-MMPs (7, 9). The fact that progelatinase A is activated by MT5-MMP in a proteolysis- dependent pathway suggests that at least a portion of
MT5-MMP must have been activated by the cells, presumably in the
trans-Golgi network by furin (13).
The most striking feature of MT5-MMP may be its tendency to be shed
from the cell surface efficiently (see Fig. 6). A summary model is
presented in Fig. 7. The MT5-MMP protein
is synthesized, packaged, and delivered to cell surface where
proteolytic cleavage on the stem region releases soluble MT5-MMP
species into the extracellular space (Fig. 7). For example, MDCK cells
stably expressing wild type MT5-MMP secrete and accumulate a pair of
immunoreactive proteins at 44-46 kDa in 3-8 h in culture media, a
molecular mass range consistent with the predicted molecular mass of
mature MT5-MMP without the transmembrane/cytosolic domain (Fig.
6A). On zymography, media conditioned for 48 h contain
only a 28-kDa gelatinolytic species in the absence of synthetic MMP
inhibitor BB94 (Fig. 6B). The inclusion of BB94 in the
conditioned media actually enhanced the accumulation of the 28-kDa
species as well as additional gelatinolytic species including the
putative 48-50-kDa mature transmembraneless MT5-MMP species (Fig.
6B). Since BB94 fails to inhibit the shedding process, it is
unlikely that MT5-MMP is secreted autocatalytically (Fig. 7). The fact
that both MCF7 and T47D breast cancer cells can also shed MT5-MMP from
cell surface suggests that the shedding of MT5-MMP can be
generalized.2 It is noteworthy that the two most divergent
segments in MT5-MMP, i.e. the hinge region immediately
downstream of the catalytic domain and the stem region just
NH2-terminal to the transmembrane domain, contain multiple
dibasic motifs which could be targets for the members of the proprotein
convertase family (see Figs. 1 and 7, Ref. 18). It is of interest to
note that MT5-MMP is processed into smaller fragments, including the
44-46-kDa species within the cells as described in Fig. 6A.
It is possible that the 44-46-kDa species observed in the conditioned
media may be generated inside the cell prior to secretion via
proprotein convertase-mediated cleavage in a similar fashion as the
proposed activation process at the RXRXKR motif
(13, 17, 18). Further investigation will be needed to clarify the
location and mechanisms responsible for the efficient shedding of
MT5-MMP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6 which includes a FLAG tag at
its COOH terminus for detection purposes using M2 monoclonal antibody
(33). A chimeric primer,
5'-GTCACTTGTCATCGTCGTCCTTGTAGTCCCGCTTATAGTAGGTGAC-3', was designed to
cover part of the MT5-MMP sequence at the carboxyl end as well as the
FLAG sequence. This primer was paired with T3 primer from vector to
amplify MT5-MMP
6 from template clone number 17. The resulting
fragment was cloned into pCR3.1uni and confirmed by sequencing as
described (17). Full-length MT5-MMP was constructed by using another
primer, 5'-CTCATACCCACTCCTGGACTGGCCGCTTATAGTAGGTGAC-3', containing the
missing 6 residues at the COOH terminus to amplify the entire open
reading frame with T3 primer from clone 17. The PCR fragment was cloned
and characterized as described above. MT5-MMP(E252A) was made by
sequential PCR as described previously using the following primers to
convert Glu252 to Ala:
5'-GCCGTGCATGCACTGGGCCAT-3' and
5'-ATGGCCCAGTGCATGCACGGC-3'. The mutant was cloned into the
same pCR3.1uni vector and confirmed by DNA sequencing as described
(17).
6, pCR3.1uniMT5-MMP, and pCR3.1uniMT5-MMP(E252A) were transfected into COS 7 cells and their protein products were analyzed by immunoprecipitation as described previously (13, 17). To
isolate cytosolic as well as membrane fractions, cells were disrupted
initially by repeated freeze-thaw cycles and fractionated by
centrifugation to partition into supernatants as cytosolic fraction and
pellets as membrane fractions. The pellets were washed extensively and
extracted with Triton X-100 (1%) in Tris-buffered saline as described
(8). Both cytosolic as well as membrane fractions were analyzed by
Western blot as described (13). For progelatinase A activation,
pCR3.1GelA (0.1 µg) was transfected either alone or with
pCR3.1uniMT5-MMP
6, pCR3.1uniMT5-MMP, or pCR3.1uniMT5-MMP(E252A) into
MDCK cells and the conditioned media were analyzed for gelatinase
activity by zymography as described previously (13).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence of the mouse MT5-MMP
cDNA and its deduced protein sequence. The amino acid sequence
is shown in uppercase and the DNA sequence in
lowercase. The numbering on the right is for the
amino acid sequence. The predicted signal peptide cleavage is indicated
by a downward arrow. The cysteine switch,
RXRXKR motif, are bold. The
zinc-binding site of the catalytic domain is bold and
underlined. The dibasic motifs are bold. Vertical
lines separate distinct subdomains of MT5-MMP into pre-, prodomain
(Pro), catalytic (Cat), hinge (H), hemopexin-like
(Pex), stem (S), transmembrane (T),
and cytosolic (C) domains as depicted in the lower
portion.
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Fig. 2.
Homology analysis of MT-MMPs.
A, sequence alignment of MT5-MMP with the other MT-MMPs. The
name of the MT-MMPs are on the left column. The number of
amino acid residues were on the right. The cysteine
switches, proprotein convertase recognition sites, and the catalytic
and transmembrane domains are boxed. The dibasic motifs are
bold. The downward arrow indicates the putative
processing sites for the maturation of these enzymes. The
vertical lines separate neighboring subdomains. The
consensus for catalytic zinc-binding sites is shown in bold type.
B, percentage of identity among distinct subdomains between
MT5-MMP and the rest of MT-MMPs. The gene names are on the
left. Percentage in each box represents the identity score
using MT5-MMP as 100. MT1-, MT2-, MT3-, MT4-, and MT5, MT1, -2, -3, -4, -5-MMPs; Pro, prodomain; CAT, catalytic domain;
H/Pexin, hinge and hemopexin-like domains;
S/TM/C, stem/transmembrane/cytosolic domains.
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Fig. 3.
Tissue distribution of mouse MT5-MMP.
Reverse transcribed cDNA (0.5-ng aliquot each) from a multiple
tissue panel (CLONTECH) including heart, brain,
spleen, lung, liver, skeleton muscle, kidney, and testis (lanes
2-9) were amplified to give rise to the 400-base pair fragment of
MT5-MMP. Similar cDNA preparations from whole embryos aged days 7, 11, 15, and 17 (E7 to 17 in lanes 10-13) were analyzed as
the adult tissues. To control for the amount of cDNA in each
reaction, a parallel PCR reaction using the glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was carried out as suggested by the
supplier.
6FLAG, an
expression vector derived from the clone 17 of MT5-MMP (missing the
last 6 residues at the COOH terminus), was transfected into COS cells
and the products were analyzed by immunoprecipitation and
immunoblotting as described (17). Consistent with the presence of a
putative transmembrane domain at its COOH terminus, MT5-MMP is detected by immunoprecipitation as a 63-kDa major species only in the lysates, not the conditioned media, of cells transfected with MT5-MMP
6FLAG (Fig. 4A, lanes 2 and
4), while mock transfected cells are negative (Fig.
4A, lanes 1 and 3). As reported for MT1-MMP (7,
13), this species may represent the proenzyme of MT5-MMP. In addition, there are minor species slightly above the 63-kDa main species which
may represent the minor glycosylated form. Interestingly, a visible
protein species around 130 kDa was also detected from the MT5-MMP
6
transfected cells, which may represent the dimeric form of MT5-MMP
(Fig. 4A, lane 4). To further clarify its physical localization, transfected cells were harvested, disrupted, and separated into two main fractions: cytosolic and membrane. As shown by
immunoblotting in Fig. 4B, MT5-MMP is mainly associated with
the membrane fraction, not the cytosol (Fig. 4B, lanes 4 versus
2), while the mock transfected COS cells are negative in both
fractions (Fig. 4B, lanes 1 and 3). Taken
together, these data suggest that the majority of MT5-MMP are membrane
associated as reported for other MT-MMPs (7, 8, 9). The ability of
MT5-MMP
6FLAG to activate progelatinase A was tested in
co-transfection experiment in MDCK cells. As shown in Fig.
4c, MT5-MMP
6FLAG activated co-expressed progelatinase A
specifically, whereas progelatinase B was not activated (Fig. 4c,
lanes 1-5). In the same experiment, similar amounts of MT1- and
MT3-MMPs were also transfected to serve as positive controls.
MT5-MMP
6FLAG appears to be slightly less efficient than MT1- and
MT3-MMPs in mediating progelatinase A activation.
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Fig. 4.
Mouse MT5-MMP activates progelatinase A. A, MT5-MMP is cell-bound, not secreted. Control (lanes
1 and 3) and mouse MT5-MMP 6 (lanes 2 and
4) expression vector were transfected into COS cells and the
MT5-MMP products were labeled with [35S]Met (100 µCi/ml, Amersham) for 3 h. The supernatants (lanes 1 and 2) and cell lysates (lanes 3 and
4) were harvested and analyzed by immunoprecipitation using
anti-MT5-MMP antisera as described under "Materials and Methods."
The arrowhead indicates the main MT5-MMP species while the
line above the arrowhead marks the putative dimer
of MT5-MMP. B, MT5-MMP is in the membrane fraction, not the
cytosol. COS cells were transfected with control (lanes 1 and 3) or MT5-MMP
6 (lanes 2 and 4)
as in A and processed into cytosolic (lanes 1 and
2) and membrane fractions (lanes 3 and
4) as described under "Materials and Methods." Both
fractions were analyzed by immunoblotting using anti-MT5-MMP antisera.
The MT5-MMP-specific species is indicated by an arrowhead.
C, MT5-MMP activates progelatinase A. MDCK cells (lane
5) were transfected with gelatinase A expression vector (0.1 µg,
lanes 1-4) and control vector (1.5 µg, lane
1), MT1-MMP (lane 2), MT3-MMP (lane 3), or
MT5-MMP
6 (lane 4). The conditioned media were analyzed
by zymography. Gel B, gelatinase B/MMP9; Gel A,
gelatinase A/MMP2; MT1, MT1-MMP; MT3, MT3-MMP;
MT5, MT5-MMP; MT5
6, MT5-MMP with a deletion of
the last 6 residues.
6 in
progelatinase A activation (data not shown), with similar efficiency, thus suggesting the last 6 residues are not required for the gelatinase A activation. In an initial attempt to characterize the mechanism responsible for MT5-MMP-mediated progelatinase A activation, a full-length mutant named MT5-MMP(E252A) was generated by substituting Glu252 with an Ala residue within the catalytic motif
HE252LGH. A similar mutation has been shown to render
progelatinase A catalytically inactive while maintaining overall
structural integrity (33, 35). As shown in Fig.
5A, MT5-MMP(E252A) can be
expressed in COS cells as protein species indistinguishable from
wild-type MT5-MMP (Fig. 5, lanes 2 and 3). In
fact, MT5-MMP(E252A) appears to accumulate to a higher level than the
wild type enzyme. In a parallel experiment, MT5-MMP(E252A) fails to
activate co-transfected progelatinase A in MDCK cells, while the
full-length wild type MT5-MMP activated the co-transfected
progelatinase A at similar degree as MT5-MMP
6 (Fig. 5B).
Thus, the proteolytic activity of MT5-MMP is required for its ability
to mediate progelatinase A activation.
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Fig. 5.
Catalytic activity of MT5-MMP is required for
gelatinase A activation. A, expression of full-length
wild type and catalytic mutant of MT5-MMP in COS cells. COS cells were
transfected with control expression vector (lane 1),
full-length MT5-MMP (lane 2), or full-length MT5-MMP(E252A)
(lane 3). MT5-MMP products were analyzed by
immunoprecipitated as described in the legend to Fig. 4. B,
catalytic mutant of MT5-MMP (MT5EA) fails to activate progelatinase A. MDCK cells (lane 1) were transfected with gelatinase A
(Gel A) expression vector (0.1 µg, lane 2) and
full-length wild type MT5-MMP (1.5 µg, lane 3) or
full-length mutant MT5-MMP(E252A) (1.5 µg, lane 4).
Conditioned media (48 h) were analyzed by zymography as described in
the legend to Fig. 4. MT5, MT5-MMP; MT5EA,
MT5-MMP(E252A) mutant.
6 were introduced
into MDCK cells and stable clones were selected in the presence of G418. A panel of positive clones for both constructs were obtained and
shown to be able to activate progelatinase A added exogenously (data
not shown). Surprisingly, in addition to the endogenous progelatinase B
on the zymograms, additional gelatinolytic species were observed in
MT5-MMP positive clones only, suggesting that some MT5-MMP products be
secreted into the conditioned media. To examine this possibility, a
stable clone F591 expressing full-length MT5-MMP was labeled with
[35S]Met under serum-free conditions for 3 and 8 h.
Both cell lysates and conditioned media were analyzed by
immunoprecipitation using the rabbit anti-MT5-MMP antibody. As shown in
Fig. 6A, a 63-kDa species was
identified as the major MT5-MMP associated with the cells, similar to
the protein species produced in transiently transfected COS cells (Fig.
6A, lanes 2 and 3). However, there is a general
shift of MT5-MMP products from the full-length 63-kDa species to
smaller fragments around 29, 40-46 kDa in cell lysates of MT5-MMP
stable transfectants (Fig. 6A, lanes 2 and 3). In
the conditioned media, a major 46-kDa and a minor 44-kDa species were also detected during the course of 3-8-h incubation (Fig. 6A, lanes 5 and 6). To analyze the proteolytic activity of
the shed MT5-MMP species, serum-free media conditioned for 48 h
were collected from F591 and analyzed on gelatin-zymogram. The major
gelatinolytic MT5-MMP species migrates around 28 kDa instead of 44-46
kDa, indicating that the 44-46-kDa MT5-MMP species may be unstable in
the culture media (Fig. 6B, lane 2), while the control
transfected MDCK cells are negative for this activity (Fig. 6B,
lane 1). The discrepancy between the zymogram and
immunoprecipitation may be the result of further processing of the
44-46-kDa species into smaller ones during the 48-h incubation period.
Since many MMPs undergo autocatalytical processing into smaller
fragments, a synthetic MMP inhibitor BB94 was included in the
conditioned media to inhibit further fragmentation (34). As a result,
more gelatinolytic species were observed in the presence of BB94
including a pair of 48-50-kDa species on zymogram (5 µM,
Fig. 6B, lane 3), similar to the 44-46-kDa species
identified by immunoprecipitation (Fig. 6A, lane 6). This apparent migratory difference is probably due to the non-reduced and
reduced conditions employed by these two electrophoresis procedures. Interestingly, BB94 appears to have enhanced the amount of the 28-kDa
species in addition to the 48-50- and 29-33-kDa species (Fig.
6B, lane 3), suggesting the catalytic domain alone (28-kDa species) may decay autocatalytically. To confirm the identity of these
gelatinolytic species in lanes 1-3 of Fig. 6B,
the same conditioned media were concentrated 10-fold, analyzed by
Western blotting using anti-MT5-MMP antisera and shown in lanes
4-6. The gelatinolytic species observed on zymography in
lanes 2 and 3 of Fig. 6B are
immunoreactive on immunoblots (Fig. 6B, lanes 4-6), albeit
with varying degrees of intensity presumably due to the differences in
structural integrity of the catalytic domain. MT5-MMP products which
are immunoreactive, but negative on zymography, may contain the
hemopexin domain only, lacking a functional catalytic domain (Fig.
6B, lane 2 versus 5). A similar pattern of MT5-MMP products
are secreted by MCF7 and T47D cells stably transfected with
MT5-MMP.2 Thus, it is
concluded that MT5-MMP is shed into the culture media efficiently.
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Fig. 6.
MT5-MMP is shed into culture media.
A, immunoreactive MT5-MMP species is secreted into culture
media. MDCK cells stably transfected with pCR3.1 control vector
(lanes 1 and 4) or wild type full-length MT5-MMP
(lanes 2, 3, 5, and 6) were labeled with
[35S]Met (100 µCi/ml, Amersham) for 3 h
(lanes 1, 2, 4, and 5) or 8 h (lanes
3 and 6). The conditioned media (lanes 4-6)
were collected and cells were washed and lysed with RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS,
50 mM Tris, pH 8.0; lanes 1-3). Both the media
and cell lysates were then immunoprecipitated and analyzed essentially
as described (13, 17). The lysate panel was exposed for 6 h, and
the media for 48 h. The two arrows indicate the
secreted MT5-MMPs. B, zymography and Western blotting of
secreted MT5-MMP. Control MDCK cells (lanes 1 and
4) and MT5-MMP transfectants (lanes 2, 3, 5, and
6) were conditioned with serum-free media for 48 h in
the absence (lanes 1, 2, 4, and 5) or presence of
BB94 (5 µM, lanes 3 and 6). The
cleared conditioned media were analyzed by zymography directly as
described (lanes 1-3) (13). The same conditioned media were
then concentrated 10-fold and analyzed by Western blotting with
anti-MT5-MMP antisera as described in "Materials and Methods"
(lanes 4-6). The two arrows indicate the two
high molecular weight gelatinolytic bands, the bracket and
arrowhead for the lower species of MT5-MMP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Summary model for MT5-MMP shedding. The
proMT5-MMP is processed to active form either in the trans-Golgi
network or on the cell surface, then shed as mature species around
44-46-kDa species or the catalytic domain at 28-kDa species. As
described in the text, the hinge (H) and stem (S)
regions contains multiple RR or KK dibasic motifs (arrows)
which could be cleaved by proprotein convertase family (18). The basic
motif in the stem region (underlined) resembles a classic
furin recognition site RXR/KXK/RR (18).
C, control; MT5, MT5-MMP. H, hinge
region; Pexin, hemopexin-like; Pro, prodomain and
proenzyme; CAT, catalytic domain; S, stem;
N, nucleus. Arrowheads indicate potential
processing site for shedding.
It has been reported that alternative splicing of MT3-MMP in both human
and rat can generate soluble-type MT3-MMP (29, 38). This may represent
one strategy for cells to secrete soluble MT-MMPs into the
extracellular space. However, this mechanism does not seem to apply to
MT5-MMP. First, no splicing variants were uncovered during the library
screening process with over 20 clones analyzed. Second, a single 63-kDa
species was detected from COS cells transfected with MT5-MMP cDNA
constructs, consistent with that of the full-length protein. The
culture media from the COS-transfected cells do not contain the
predicted 55-60-kDa splicing variant (Figs. 4 and 5). Thus, MT5-MMP is
likely to be generated post-translationally as soluble enzymes from the
cell surface. Although it has been suggested that MT1-MMP may undergo
intercellular transfer from stromal cells to the invading cell via
soluble intermediates (13, 19), the secretion of MT1-MMP has been
demonstrated recently (20, 39). Thus, shedding may be a general
mechanism for MT-MMPs to increase their versatility. Further research
into the shedding mechanisms of MT-MMPs could potentially enhance our
understanding of MT-MMPs' role in ECM remodeling and cancer
progression and MT5-MMP may offer a good model system to uncover the
mechanism of shedding.
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ACKNOWLEDGEMENTS |
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I thank Dr. Jianxun Lei for expressing human GST-MT5-MMP fusion protein and the production of anti-MT5-MMP antisera; Stephen J. Weiss, Hideaki Nagase, and Fred Woessner for thoughtful inputs and comments for this study.
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FOOTNOTES |
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* This work was supported in part by National Cancer Institute Grant CA76308, American Heart Association Grant-in-Aid 9750197N, the Elsa Pardee Foundation, a University of Minnesota grant-in-aid, the Minnesota Medical Foundation, and the Arthritis Foundation (MN chapter).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) AJ010262.
To whom correspondence should be addressed: 3-249 Millard Hall,
435 Delaware St. S.E., University of Minnesota, Minneapolis, MN 55455. Tel.: 612-626-1468; Fax: 612-625-8408; E-mail:
peixx003{at}tc.umn.edu.
2 D. Pei, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MMP, matrix metalloproteinase; MT1-, MT2-, MT3-, MT4-, and MT5-MMP, membrane-type matrix metalloproteinase-1, -2, -3, -4, -5; ECM, extracellular matrix; RT-PCR, reverse transcription-polymerase chain reaction; MDCK, Madin-Darby Canine Kidney; EST, expressed sequence tag; GST, glutathione S-transferase.
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REFERENCES |
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