From the Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08534
Received for publication, April 29, 2003
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ABSTRACT |
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INTRODUCTION |
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Experiments with mice or fibroblasts lacking MMP-11 also suggest that MMP-11 plays an important role in human cancer. First, mice lacking MMP-11 have a reduced incidence and size of tumors induced by 7,12-dimethylbenzanthracene compared with mice with a functional MMP-11 (9). Second, MMP-11 null fibroblasts, unlike wild type fibroblasts, do not stimulate the implantation rate of MCF7 xenografts in nude mice (9). Third, syngenic tumor cells had a higher rate of apoptosis when injected into MMP-11 null mice compared with wild type mice (10). Hence, the frequent overexpression of MMP-11 in human tumors and the effect of MMP-11 levels on tumor formation in mice both suggest that MMP-11 plays an important role in human cancer.
Relatively little is known about the physiologic substrates for MMP-11. In
particular, human MMP-11, unlike most MMPs, does not readily cleave
extracellular matrix components, such as collagen, laminin, fibronectin, or
elastin (11,
12). In vitro studies
showed, however, that MMP-11 cleaves 1-proteinase inhibitor,
2-macroglobulin, and insulin-like growth factor-binding protein-1,
although the physiologic significance of these reactions is unknown
(11,
13). More importantly, recent
experiments showed that MMP-11 mutants without proteolytic activity did not
stimulate the implantation of MCF7 cells into nude mice
(14). These studies strongly
suggest that MMP-11 has proteolytic activity that is important for its role in
cancer.
In addition to the poor cleavage of extracellular matrix components, human MMP-11 differs from other MMPs in that it contains alanine at residue 235, whereas mouse MMP-11 and other MMPs contain proline at the corresponding residue (12). This alanine residue affects catalytic activity because the human protein MMP-11-A235P, but not MMP-11, readily cleaved laminin and type IV collagen. In fact, human MMP-11-A235P had activity similar to that of mouse MMP-11. It is unclear whether the alanine 235 to proline mutation in human MMP-11 affects catalytic activity, substrate dependence, or both.
In this study, we used phage display libraries to select peptide substrates for human MMP-11. Preferred sequences for MMP-11 cleavage could identify physiologic substrates for MMP-11. Such substrates would be interesting because they are likely to be important for human cancer.
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EXPERIMENTAL PROCEDURES |
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Construction of Control Phage ClonesPositive and negative
control phage clones were constructed to help develop methods for library
screening. A positive control phage was constructed with oligonucleotides that
expressed the hexapeptide PLGLYA because this sequence is known to be a
substrate for MMP-14. Two clones containing the hexapeptides MTQMIS or TALSPQ
were isolated from the library that were not cleaved by MMP-14.
Expression Vectors for MMP-14 and MMP-11Fragments of human MMP-14 and MMP-11 that encoded the catalytic domains were inserted into vectors for expression in E. coli. For MMP-11, a DNA fragment encoding residues 98272 was amplified by PCR from IMAGE clone 2425546 (Research Genetics, Birmingham, AL) with a 5' oligonucleotide that contained NdeI and a 3' oligonucleotide that contained BglII. The amplified DNA fragment was digested with NdeI and BglII and then ligated with pET3b (Novagen, Madison, WI) that was digested with NdeI and BglII. MMP-11 mutants with increased activity, MMP-11-A235P, or decreased activity, MMP-11-E216A, were generated by site-directed mutagenesis. DNA sequencing confirmed the authenticity of the preceding plasmids. For MMP-14, a vector that directed the expression of MMP-11 residues 111298 was created as previously described (15).
Purification of MMP-14 and MMP-11 Catalytic Protein Fragments Plasmids that expressed the catalytic regions of MMP-11 or MMP-14 were transformed into BL21pLysS (Novagen). Recombinant proteins were induced and purified based on published methods (15). Briefly, inclusion bodies were purified from E. coli that were induced to express the desired recombinant protein for 3 h at 37 °C. The purified inclusion bodies were dissolved in buffer with 8 M urea, diluted, and dialyzed against buffer with decreasing amounts of urea to facilitate refolding. Insoluble protein was then removed by centrifugation, and the resulting proteins were stored at 80 °C. The identity of the MMP-11 was confirmed by Western blotting with antibody SL305 (Oncogene Sci.). The concentration of the soluble protein was determined by comparison with bovine serum albumin following Coomassie Blue staining of SDS-poly-acrylamide gels.
Screening the Phage Display Library with MMP-14, MMP-11, or
MMP-11-A235P0.1 ml of phosphate-buffered saline containing
109 phage was added to each well of a 96-well Reacti-Bind
Metal Chelate Plate (Pierce), and the resulting plate was shaken gently
overnight at room temperature. Unbound phage were removed by washing six times
with 0.2 ml of phosphate-buffered saline containing 0.05% Tween 20. This
procedure yielded
25% of the input phage bound to the plate based
on titering phage that were eluted from the plate with 0.1 ml of 0.02
M EDTA for 10 min. To enrich for MMP-14 substrates, the bound phage
were digested in 0.1 ml of 50 mM Hepes, 10 mM
CaCl2, 100 mM NaCl, 0.1% Brij 30, 10 nM
MMP-14 (pH 7.5) for 6 h at 37 °C with gentle shaking. The resulting
supernatants were then collected and amplified, and the enrichment cycle was
repeated as above. Individual phage were cloned and analyzed as described
under "Results." Likewise, bound phage were digested in 0.1 ml of
50 mM Tris-HCl, 10 mM CaCl2, 100
mM NaCl (pH 7.5) with either 500 nM MMP-11 or 180
nM MMP-11-A235P for 6 h at 37 °C with gentle shaking to enrich
for MMP-11 and MMP-11-A355P substrates. The resulting supernatants were then
collected and amplified, and the enrichment cycle was repeated three more
times. Individual phage were cloned, amplified, and analyzed as described
under "Results." In some cases, the amount of phage released from
the nickel plate was quantified by titering the reaction supernatant and/or
phage eluted from the plate after a 10-min treatment with 0.1 ml of 0.02
M EDTA.
Enzyme Assays with Peptides2 ml of recombinant enzyme were
dialyzed twice against 1 liter of 50 mM Hepes, 100 mM
NaCl, 5 mM CaCl2, 1 µM ZnCl2, 5
mM -mercaptoethanol (pH 7.6) (reaction buffer plus
-mercaptoethanol) to remove amines that would react with fluorescamine.
Peptides (Research Genetics, Huntsville, AL) were dissolved in
Me2SO at 10 mM. The peptide at concentrations ranging
from 0.016 to 1 mM was diluted into reaction buffer for a final
volume of 0.1 ml and equilibrated to 37 °C. Enzyme at concentrations from
1 to 65 nM was then added, and the reaction was continued for
120 h. To quantify the extent of hydrolysis, 0.05 ml of 200
mM potassium borate (pH 10.0) was added followed by 0.05 ml of 1
mg/ml fluorescamine in acetonitrile and gentle mixing. The fluorescent signal
generated by the reaction of the fluorescamine with the primary amines created
by enzymatic digestion of the peptides was measured using a plate reader with
excitation and emission wavelengths of 390 and 490 nm, respectively. The molar
amount of fluorescamine-peptide conjugate was determined using standards
prepared from fluorescamine. These values were then used to calculate the
amount of product formed (16).
kcat/Km was calculated from
nonlinear regression analysis of Michaelis-Menten plots using Enzyme Kinetics
software (Trinity Software).
As indicated under "Results," the digestion of some peptides was also monitored using reversed-phase HPLC separation. In these cases, the reaction was stopped with 10 mM EDTA, and the reaction products were separated using a C18 reversed-phase HPLC column and acetonitrile gradient. The peptide fragments were detected by their absorbance at 210 nM. The results obtained by the HPLC method were in excellent agreement with the results obtained with the fluorescamine method. Finally, analysis of the digestion of peptides MA15 and MA18 with either the wild type or the A235P mutant MMP11 was performed using a Waters Micromass ZQ mass spectrometer interfaced with a Shimadzu HPLC. Analysis was performed using either acetonitrile, water, 0.1 mM ammonium acetate or methanol, water, 0.1% trifluoroacetic acid gradients as mobile phase on a C18 reversed-phase HPLC column using electrospray ionization(+/) ionization.
Gel-based Activity Assay for MMP-11 Cleavage of
1-Proteinase InhibitorMMP-11
1-antitrypsin
inhibitor cleavage activity was measured by incubating 5 µgof
1-proteinase inhibitor (Calbiochem 178251) in 50 mM Tris-HCl
(pH 7.5), 100 mM NaCl, 5 mM CaCl2, 1
µM ZnCl2, 26 mM MMP-11 or MMP-11-A235P in
a total volume of 0.05 ml at 37 °C for the indicated times. The reactions
were stopped by adding 10 µl of 3 M
-mercaptoethanol, 10%
SDS, 50 mM Tris-HCl (pH 7.6), 25% glycerol. The reaction products
(15 µl) were separated using an 18% Tris-HCl gel (Bio-Rad) and visualized
with Coomassie Blue.
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RESULTS |
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A screen for MMP-14 substrates was used to test this phage display system.
A phage clone that contained a hexapeptide that was cleaved by MMP-14,
PLGLYA, and two clones with hexapeptides that were not cleaved by
MMP-14, MTQMIS and TALSPQ, were first used to develop methods for library
screening. Based on these studies, conditions were identified where
75%
of the positive control phage were released from the nickel plate, whereas
less than 2% of the negative control phage were released from the nickel plate
(see "Experimental Procedures" for details.) Using these
conditions, two rounds of enrichment were performed with MMP-14. 52 random
phage clones from this enriched pool were then individually digested with
MMP-14 or buffer controls, and the fraction of the phage released by MMP-14
was determined. This analysis showed that 31 of 52 clones were released from
nickel plates at levels equal to or greater than the level for the positive
control phage. Some of these release rates are quantified in
Table I. DNA from these 31
phage were sequenced, and the predicted hexapeptides were aligned
(Table I). Based on this
alignment, a consensus sequence of PL(G/P/A)
L(R/M) was obtained
(Table II). The individual
peptide sequences and the consensus sequence were in excellent agreement with
preceding identification of MMP-14 substrates using phage display and peptide
methods
(1719).
Hence, our studies substantiate the conclusions of other investigators and
validate our phage display method for identifying peptide substrates.
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Identification of MMP-11 Substrates Using Phage DisplayA
fragment of human MMP-11 containing residues 98272 was expressed in
E. coli. Two MMP-11 mutants were also expressed in E. coli
to aid in the identification of MMP-11 substrates. One mutant, MMP-11-E216A,
altered a residue required for catalytic function
(14), whereas a second mutant,
MMP-11-A235P, had increased activity for cleavage of 1-proteinase
inhibitor (12)
(Fig. 1A). This
analysis showed that both MMP-11 and MMP-11-A235P cleaved
1-proteinase
inhibitor, yielding the expected products. Furthermore, MMP-11-A235P was about
10-fold more active than MMP-11, consistent with previous results
(12). Similar results were
obtained using either
-casein,
2-macroglobulin, or insulin-like
growth factor-binding protein-1 as a substrate (data not shown). The cleavage
of
1-proteinase inhibitor was inhibited by AG3340, a broad spectrum MMP
inhibitor (20), with an
IC50 of
6 nM
(Fig. 1B). As
previously described (14), the
active site mutant MMP-11-E216P was inactive in all of the assays tested (data
not shown). Taken together, these findings are consistent with earlier
findings and validate our MMP-11 preparations.
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MMP-11 and MMP-11-A235P were then used to enrich the phage display library
for substrates. Unfortunately, neither the positive control phage clone for
MMP-14 (PLGLYA) nor any of the phage clones selected with MMP-14 were
good substrates for MMP-11 or MMP-11-A235P. In the absence of phage substrates
to establish conditions for screening, enrichment conditions were chosen based
on the published activity of MMP-11 and MMP-11-A235P (see "Experimental
Procedures" for details). Using these conditions,
25-fold more
phage were released from the pool of phage that had undergone four rounds of
enrichment than from the nascent phage library. 60 phage from both the MMP-11
and MMP-11-A235P enrichments were then individually cloned and tested for
release from nickel plates by MMP-11, MMP11-A235P, or buffer controls. This
analysis showed that 7 of 60 phage clones from the MMP-11 enrichment and 34 of
60 phage clones from the MMP-11-A235P enrichment were released from nickel
plates at 100-fold or higher levels than the negative control clones. This
analysis also showed that all phage released by MMP-11 could be released by
MMP-11-A235P and vice versa. An additional test was performed to ensure that
phage clones were released by MMP-11 instead of potentially contaminating
E. coli proteases. Putative MMP-11 or MMP-11-A235 substrate phage
were first digested with MMP-11-A235P either in the presence or absence of 0.1
µM AG3340, a broad spectrum MMP inhibitor. This analysis showed
that the release of 32 of 41 phage clones selected with either MMP-11 or
MMP-11-A235P was inhibited 95% or more by AG3340. DNA from these 32 phage
clones were then sequenced, and the predicted hexapeptides were aligned
(Table III). Three groups of
peptide substrates for MMP11 were readily identifiable. Based on this
alignment, the four clones from Group B had hexapeptides that shared the
sequence G(G/A)E
LR, whereas two clones had hexapeptide sequences that
were represented by neither Group A nor Group B. The majority of peptides
belong to Group A. Separate analysis of these 26 peptides belonging to Group A
indicated that the hexapeptides were well represented by the consensus
sequence A(A/Q)(N/A)
(L/Y)(T/V/M/R)(R/K)
(Table IV). As a final test
that these phage were released by MMP-11, 13 phage clones were treated with
either MMP-11 or MMP-11-E216A. This analysis showed that at least 100-fold
more phage were released with MMP-11 than with MMP-11-E216A. Hence, this phage
screen isolated two classes of hexapeptides and two unique hexapeptides
outside these classes that were specifically released from nickel plates by
both MMP-11 and MMP-11-A235P but not by MMP-11-E216A.
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Determination of Kinetic Parameters for MMP-11 Substrate
PeptidesSeveral peptides were synthesized based on the hexapeptide
sequences identified by phage display so that kinetic parameters could be
determined and compared with known substrates. In particular, decapeptides
were synthesized where the hexapeptide sequences identified by phage display
were flanked by a pair of glycine residues at both the N and C termini as in
the phage. The N terminus was blocked with an acetyl group to eliminate
primary amines in the peptides. The peptides were then incubated with either
MMP-14, MMP-11, MMP-11-A235P, or MMP11-E216A, and the extent of cleavage was
determined using fluorescamine, which reacted with the primary amines created
by peptide cleavage to generate fluorescent conjugates. Nonlinear regression
analysis of Michaelis-Menten plots was used to determine
Km and Vmax that were then
used to calculate kcat/Km
values (Table V). Although
comparisons between the columns of Table
V are subject to the vagaries of the percentages of active enzyme
after refolding, certain conclusions are evident. For instance, peptide MA13,
which contained the hexapeptide PLGLYA used for the positive control
phage in the MMP-14 screen, was a good substrate for MMP-14 but was not
cleaved at a detectable rate by MMP-11 or MMP-11 mutant proteins, as expected
from the phage assays (Table
V). MA15 and MA18 contained the hexapeptides GAN
LVR and
YAE
LRM that represented the two major classes of sequences identified
from the MMP-11 phage screens. Consistent with the phage assays, both MA15 and
MA18 were good substrates for MMP-11, better substrates for MMP-11-A235P, and
not cleaved at detectable rates by either MMP11-E216A or MMP-14
(Table V). MA16 and MA17
contained hexapeptides QPRGVW and TDAWLS from the two phage clones isolated in
the MMP-11 screen that were not represented by either of the major classes.
Surprisingly, neither MA16 nor MA17 were cleaved at detectable rates by MMP-11
or MMP11235A. Possible reasons for the isolation of these sequences in
the phage display screen will be considered under "Discussion." As
expected from the sequence, MA16 was a good substrate for MMP-14. The results
using the fluorescamine assay with MA16 and MA17 were confirmed by HPLC
analysis (data not shown). MA20 contained the hexapeptide PLA
LWA that
was previously found to be a good MMP14 substrate and a weak substrate for
mouse MMP-11 (21). Consistent
with these results, MA20 was a good substrate for MMP-14, a weak substrate for
MMP-11-A235P, and a poor substrate for MMP-11
(Table V). Finally, MA21
contained the decapeptide GAAGA
MFLEA that spanned the natural MMP-11
cleavage site in
1-proteinase inhibitor. As expected, MA21 was a good
substrate for MMP-11, a better substrate for MMP-11-A235P, and not cleaved at
detectable rates by MMP-14. Based on these results, we conclude that MMP-11
and MMP-11-A235P cleaved hexapeptides from the two classes identified using
MMP-11 at rates that were similar to those for other MMPs with their preferred
peptide substrates. Furthermore, MMP-11-A235P cleaved these substrates at
rates 25-fold faster than MMP-11, with Km
values
2-fold lower than wild type MMP-11
(Table VI). The site of
cleavage indicated in Tables V
and VI was determined using a
combination of HPLC separation of the reaction products after the digestion of
peptides MA15 or MA18 with wild type MMP11 or the mutant MMP-11-A235P with
subsequent determination of the molecular weight of the product peaks by mass
spectroscopy. Fragments consistent with the proposed cleavage sites were
observed for both peptides.
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Structural Explanations for MMP-11 Enzymatic Properties
X-ray crystallography has shown that the catalytic domains of MMPs have a very
similar architecture consisting of a five-stranded sheet and three
helices (22,
23). Differences between these
structures occur mainly at the S1' "specificity loop" and
the loop just above it connecting
strand 5 with helix 2. The recent
structure of mouse MMP-11 shows that it has most of the common structural
features of the MMPs (Fig. 2)
(24).
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These structural comparisons form the basis for an explanation of the increased activity of mouse MMP-11 and human MMP-11-A235P relative to human MMP-11. In particular, human residue Ala235 corresponds to mouse Pro239 based on sequence alignments (data not shown). Pro239 of mouse MMP-11 overlays Pro217 of MMP-8 (25), part of the conserved MXP motif that forms a turn directly below the catalytic site (Fig. 2) and is likely to interact with the substrate backbone. Hence, replacement of a turn-inducing proline by alanine is likely to disrupt this loop and, consequently, the interaction of the substrate with the active site.
These structural comparisons also form the basis for an explanation of the
poor cleavage by MMP-11 of substrates having proline in position 3.
Fig. 2 indicates one major
difference between the conformation of mouse MMP-11 and other MMPs of which
MMP-8 is typical. In MMP-11, the loop connecting strands 4 and 5
(upper left) is curved upwards, resulting in distances of up to 9
Angstroms between backbone atoms of equivalent residues of the two structures.
Because the sequences of human and mouse MMP-11 catalytic domains are almost
identical and are totally conserved in this region, the conformation of this
loop in human MMP-11 is likely to be the same as mouse MMP-11. Inhibitor
residue proline at position 3 in the MMP-8/PLG complex is nestled into a
pocket formed by the rings of His162 and Phe164.
His183 of MMP-11 is oriented similarly to His162 of
MMP-8 (residues not shown). In contrast, Phe185 of MMP-11 is turned
in the opposite direction from Phe164 of MMP-8, and thus no cavity
is formed in MMP-11. Hence, the absence of this cavity in human MMP-11 caused
by the altered conformation of this loop could explain why MMP-11, in contrast
to MMP-8, does not prefer proline in position 3. Consistent with this
hypothesis, other MMPs of known structure have the corresponding phenylalanine
or tyrosine positioned like MMP-8 and prefer proline in position 3.
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DISCUSSION |
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In vitro reactions with peptides confirmed that peptides from both
classes were hydrolyzed by MMP-11 and MMP-11-A235P. These in vitro
reactions also showed that the
kcat/Km values for MMP-11
cleavage of these substrates was modest relative to the values for other MMPs
with peptide substrates. In particular, optimal peptide substrates for MMP-1,
MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14 from combinatorial peptide libraries
had kcat/Km values with their
respective MMPs of 1.6, 82, 6.9, 120, 49, and 6.9
mM1 s1
(19). Hence, the
kcat/Km values of 0.7 and 2.0
mM1 s1
for the digestion of MA15 and MA18, respectively, by MMP-11 were on the low
end of the range for known MMP peptide substrates. The ST3 (MMP-11)
kcat/Km values reported
previously using dansyl-heptapeptides ranged from 1300 to 16,700
M1 s1
(see Table I in Ref. 21). The
range of these values is similar to values reported here for wild type MMP-11
using peptide substrates MA15 and MA18 (710 and 2030
M1 s1)
and for the mutant MMP-11-A235P (2900 and 20,800
M1 s1)
(Table V). However, although
the individual Km values reported for ST3
(MMP-11) for two dansyl-heptapeptides are 10100-fold lower than those
reported here (Table VI), our
kcat values are 10100-fold higher (see Table II in
Ref. 21). There are two
reasonable explanations for these differences. First, the earlier report used
mouse ST3 (MMP-11) instead of the recombinant human MMP-11 and the mutant
MMP-11 (A235P) used in these studies. Second, the earlier report used
dansylated peptides, whereas this study used un-modified peptides. Thus, the
10100-fold lower Km values reported using
the dansyl-heptapeptides could have resulted from solvation entropy effects
caused by the addition of the dansyl group to the peptide substrates
(26). Interestingly, although
the addition of the dansyl group could have increased the apparent affinity
(lower Km) because of a +S from
solvation entropic effects, the dansyl group could also have resulted in a
lower catalytic activity of the enzyme. The higher turnover number could also
be attributed to the longer length of the substrate used in these studies.
Clearly, further experiments will be needed to test these hypotheses.
A comparison of human MMP-11 with other MMPs showed that alanine 235 is unusual in that most MMPs contain proline at the corresponding residue. Previous work (12) and results from this study showed that MMP11-A235P was more active than MMP-11. The recent mouse MMP-11 crystal structure offers an explanation for the effect of this mutation on catalytic activity. In particular, this proline forms a turn below the active site that is likely to interact with the substrate. Hence, it is not surprising that mutations that could disturb this turn, such as alanine, decrease catalytic activity. Interestingly, whereas MMP11-A235P was catalytically more active than MMP-11, the substrate preference appeared unchanged.
The identification of two classes of MMP-11 substrates suggests that subsite preferences for MMP-11 cleavage are dependent upon other subsite residues. A similar conclusion was obtained when studying peptide cleavage by MMP-1 and MMP-9 (27) and MMP-3 (28). These investigators found that combining optimal residues for different subsites identified in one context frequently yielded peptides that were not better substrates than either single substitution. Like MMP-11, multiple classes of substrates were found for MMP-9 and MMP-14 using phage display (17, 18, 29). Taken together, these studies suggest that the context dependence of subsite preferences are a common feature of MMP cleavage sites.
The likelihood that optimal subsite residues depend upon other subsite residues has implications for identifying optimal peptide substrates. In particular, this finding suggests that an unbiased approach to substrate identification, such as phage display, should be used to identify the major classes of peptide substrates. The peptides identified using phage display could then be used as templates for further peptide optimization using combinatorial peptide libraries where non-natural amino acids can be incorporated. Such a two-tiered strategy overcomes the inability of phage display libraries to test non-natural residues and the difficulty of sampling a large number of sequences with combinatorial peptide libraries.
Many proteins are reported to be substrates for different MMPs (reviewed in
Ref. 1). Based on this
compilation, none of the substrates for MMP-1, MMP-2, MMP-3, MMP-7, MMP-8,
MMP-10, MMP-12, MMP-13, or MMP-14 contain cleavage sites that match the MMP-11
consensus sites identified in this study. There were, however, a few proteins
in the grouping with cleavage sites similar to the MMP-11 consensus sequences
including proTNF (AQA
VRS), decorin (AAS
LKG), laminin 5
(AAA
LTS), and proMMP-9 (VAE
MRG). Although strict adherence to the
MMP-11 consensus sequences may prevent identification of physiologic MMP-11
substrates, this analysis suggests that relatively few substrates for other
MMPs are likely to be MMP-11 substrates. The hypothesis that MMP-11 does not
cleave the known substrates for most MMPs is further supported by the
inability of human MMP-11 to cleave extracellular matrix components, including
collagen, laminin, fibronectin, or elastin
(11,
12). The apparent substrate
differences between MMP-11 and other MMPs suggest that MMP-11 is likely to
have unique substrates. Identification of these putative MMP-11 specific
substrates will be interesting given the likely role of MMP-11 in human
cancer. Although these substrates should be found by searching the protein
data bases, this analysis is complicated by the large number of proteins that
contain sequences that are similar to the consensus sequences from phage
display methods. Determining which of these potential substrates are
physiologic substrates then becomes a difficult task.
None of the MMP-11 cleavages sites from 1-proteinase inhibitor,
2-macroglobulin, and insulin-like growth factor-binding protein-1 agree
with the consensus sequences from the phage display screen. In particular,
MMP-11 cleaved
1-proteinase inhibitor,
2-macroglobulin, and
insulin-like growth factor-binding protein-1 at sites spanning AGA
MFL,
VGF
YES, and ALH
VTN, respectively
(11,
13). Consistent with the poor
agreement between these sequences and the phage consensus sequences, a peptide
from
1-proteinase inhibitor (MA21) had a
kcat/Km value that was 10-
and 29-fold lower than those for MA15 and MA18, peptides based on the phage
sequences. Perhaps, additional constraints imposed by the protein structure or
additional sites within
1-proteinase inhibitor contribute to cleavage
by MMP-11. Alternatively,
1-proteinase inhibitor may simply be a poor
substrate for MMP-11. Additional work is needed to address this issue.
Two hexapeptides (PRGVWG and TDAWLS) identified in phage clones that were released from nickel plates by MMP-11 and MMP-11-A235P were not substrates for these enzymes when contained in decapeptides. Although the explanation for this discrepancy is unknown, we hypothesize that these peptides might bind MMP-11 and MMP-11A235P but not be hydrolyzed by these enzymes. If this occurred, then the binding of MMP-11 or MMP-11-A235P to phage containing these peptides might interfere with the binding of the adjacent hexahistidine tag to the nickel plate and thereby cause the phage to be released. Consistent with this explanation, preliminary data show that these peptides inhibit MMP-11. Regardless of the mechanism, these results highlight the need to substantiate phage display results with peptide experiments.
Peptide sequences derived from the MMP-11 selected phage were used to
search the protein data bases for potential MMP-11 substrates. This analysis
revealed that many proteins contained the tetrapeptide sequences AANL, GANL,
AELR, or GELR. Some proteins identified in these searches were reported
substrates for other MMPs, such as perlecan and cell surface-bound Fas ligand,
and proteins related to reported MMP substrates, such as pro-transforming
growth factor-1, several protocadherins, acid-labile subunit of the
insulin-like growth factor binding protein complex, integrin
1, integrin
5, integrin
m, and integrin
8 (see Ref.
30 for review of potential MMP
substrates). A recent paper
(31) has identified a novel
intracellular isoform of MMP-11 that is translated in its activated form.
These provocative results imply the existence of potential intracellular
targets for MMP-11 in addition to those extracellular proteins that are the
standard fare of MMPs. Consequently, the most interesting protein identified
in the search of the data base was the estrogen receptor
, which
contains the sequence 288AANL291 in the hinge region
separating the DNA-binding domain and the ligand-binding domain. Although the
ligand-binding and the DNA-binding domains of estrogen receptor
and
are highly conserved, the hinge domains are not conserved, and the
estrogen receptor
lacks this sequence
(32). Preliminary in
vitro data indicate that only the estrogen receptor
is cleaved by
MMP-11. Although these results are intriguing, the large number of proteins
identified by this search suggests that only a small fraction are likely to be
physiologically relevant MMP-11 substrates. Identifying these substrates will
require additional studies.
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FOOTNOTES |
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Present address: Epitomics, Inc., 1015 Grandview Dr., South San Francisco,
CA 94080.
Present address: GlaxoSmithKline-Molecular Oncology, 1250 S. Collegeville
Rd., UP1450, Collegeville, PA 19426.
¶ Present address: Rm. H3B-330, DuPont Hospital for Children, 1600 Rockland
Rd., Wilmington, DE 19899.
|| Present address: Bristol-Myers Squibb Pharmaceutical Research Institute,
311 Pennington-Rocky Hill Rd., Pennington, NJ 08534.
** Present address: Bristol-Myers Squibb Pharmaceutical Research Institute,
Route 206 & Province Line Road, Lawrenceville, NJ 08543.
Present address: Incyte Pharmaceuticals, Stine Haskell Research Center,
1090 Elkton Rd., 115-36, Newark, DE 19711.
To whom correspondence should be addressed. Tel.: 302-283-7919; Fax:
302-283-7846; E-mail:
cAlbright{at}incyte.com.
1 The abbreviations used are: MMP, matrix metalloproteinase; HPLC, high
pressure liquid chromatography.
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ACKNOWLEDGMENTS |
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REFERENCES |
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