From the Program On Cell Adhesion and the Cancer
Research Center, Burnham Institute, La Jolla, California 92037, the
§ Institute for Drug Design and the Department of Chemistry,
Wayne State University, Detroit, Michigan 48202, and the
Department of Pathology, University of Oklahoma Health Sciences
Center, Oklahoma City, Oklahoma 73104
Received for publication, January 30, 2001, and in revised form, March 12, 2001
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ABSTRACT |
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The catalytic clefts of all matrix
metalloproteinases (MMPs) have a similar architecture, raising
questions about the redundancy in substrate recognition across the
protein family. In the present study, an unbiased phage display
strategy was applied to define the substrate recognition profile of
MMP-9. Three groups of substrates were identified, each occupying a
distinct set of subsites within the catalytic pocket. The most
prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P3 through
P2'. This sequence is similar to the MMP cleavage sites
within the collagens and is homologous to substrates the have been
selected for other MMPs. Despite this similarity, most of the
substrates identified here are selective for MMP-9 over MMP-7 and
MMP-13. This observation indicates that substrate selectivity is
conferred by key subsite interactions at positions other than
P3 and P1'. This study shows that MMP-9 has a
unique preference for Arg at both P2 and P1,
and a preference for Ser/Thr at P2'. Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.
Matrix metalloproteinase-9
(MMP-9)1 is a member of the
matrixin family of metallo-endopeptidases (1-3). MMP-9 is historically referred to as gelatinase B because of its ability to cleave gelatin, a
denatured form of collagen, in vitro. Along with MMP-2,
MMP-9 differs from other MMPs because it contains three fibronectin type II repeats that have high binding affinity for collagen. These
repeats are thought to mediate the binding of MMP-2 and -9 to collagen
(1, 2). This binding interaction brings the catalytic pocket of the MMP
in proximity to collagen, thereby enhancing its rate of hydrolysis.
Despite these well characterized biochemical interactions, it is now
clear that MMP-9 is also able to cleave a number of other proteins and
may have a rather wide range of physiologic substrates (4-8).
Much of our understanding of the biological function of MMP-9 comes
from the study of mice lacking this gene. For example, MMP-9-deficient
mice have impaired ossification of the skeletal growth plate, a defect
that has been partially attributed to poor vascularization of
developing bone (9). Studies on these mice also show that MMP-9 is
essential for the recruitment of osteoclasts into developing bones
(10). Other work indicates that MMP-9-deficient mice are resistant to
dermal blistering in a bullous pemphigoid model, an effect that has
been attributed to the inability of these mice to cleave the SERPIN
A first approximation of the substrate recognition specificity of MMP-9
has been gleaned from alignments of its cleavage site within a number
of different proteins (4, 8, 9, 15-17). Nevertheless, a detailed
understanding of subsite preferences for MMP-9 is lacking. Such an
analysis is particularly important because the catalytic cleft of MMP-9
is closely related to other MMPs, raising questions about the
distinction among substrates for these proteases. These issues are
particularly important because many of the current pharmacologic
antagonists of the MMPs have overlapping inhibition profiles.
Furthermore, in the post-genomic era, where the sequences of all
proteins will soon be available, information on substrate recognition
could help identify important physiologic substrates.
With these ideas in mind, we applied an unbiased approach to define the
substrate recognition preference of MMP-9. MMP-9 was used to cleave
substrates within a vastly complex phage display library of random
hexamers. Substrates within this library can be cleaved at any position
within the hexamer, allowing information on the substrate specificity
on both sides of the scissile bond to be obtained. Three families of
substrates were identified. The largest group contains a
Pro-X-X-Hy-(Ser/Thr) motif (X is any
residue, and Hy is a hydrophobic residue) that occupies positions P3 through P2'. This general motif is cleaved
by a number of MMPs and is presumed to represent a collagen-like
substrate (18-23). Nevertheless, substrates within this family that
were selected show considerable selectivity for MMP-9. The second group of substrates are defined by a Gly-Leu-(Lys/Arg) motif at positions P1 through P2'. Members of the third family of
substrates are unique in that they contain Arg residues at both
P1 and P2. Altogether, these findings reveal
multiple modes of substrate recognition by MMP-9 and provide important
insights into the hydrolysis of physiologic substrates that may be
important in biology and pathology.
Purified forms of full-length MMP-7, MMP-13, and TIMP-2 were
purchased from Chemicon (Temecula, CA). Ilomastat (GM6001) was purchased from AMS Scientific (Concord, CA). Restriction enzymes were
from Roche Biosciences or New England Biolabs. Oligonucleotides were
synthesized by Integrated DNA Technologies, Inc. Tissue culture media
and reagents were from Irvine Scientific (Irvine, CA). All other
reagents, chemicals, and plasticware were from Sigma or Fisher.
Construction of Substrate Phage Display
Library--
Substrate phage libraries were generated using a modified
version of the fUSE5 phagemid (24-26). A FLAG epitope was engineered at the NH2 terminus of the geneIII protein by annealing
oligonucleotides 5'-CCGGGTTTGTCGTCGTCGTCTTTGTAGTCGGTAC-3' and
5'-CGACTACAAAGACGACGACGACAAAC-3' and ligating them into fUSE5 at the
KpnI and XbaI restriction sites. The random
hexamers were generated by PCR extension of the template
oligonucleotide
5'-GGGGAGGCCGACGTGGCCGTCATCAGGCGGCTCAGGC(NNK)6ACGG CCTCTGGGGCCGAAAC-3', where N is any nucleotide and K is either G or T. The template oligonucleotide also encodes an SGGSG linker positioned in
between the FLAG epitope and the random hexamer. A primer
oligonucleotide 5'-AATTTCTAGTTTCGGCCCCAGAGGC-3' and the template
oligonucleotide were mixed and heated at 65 °C for 2 min. The
heating block was switched off and allowed to cool passively to
40 °C to allow annealing of the extension oligonucleotide to the
template oligonucleotide. Elongation of the template oligonucleotide was performed using Sequenase (United States Biochemical Corp.) (27).
The final cDNA product was precipitated with ethanol, re-suspended
in water, and digested with SfiI. The DNA insert and fUSE5
were mixed and ligated at a 5:1 molar ratio and electroporated into
Escherichia coli MC1061(F Expression and Purification of MMP-9--
The cDNA encoding
the catalytic domain of MMP-9 (28, 29) was generated by PCR and cloned
into pCDNA3 (Invitrogen). The expression vector was used to
transfect HEK 293 cells by electroporation, and individual
antibiotic-resistant colonies were isolated with cloning rings,
expanded, and then screened by reverse transcription-PCR for MMP-9
mRNA. Expression of the proteinaceous catalytic domain was
determined by zymography using conditioned medium from the transfected
cells. Cells expressing the MMP-9 catalytic domain were grown in 150-mm
dishes and conditioned in Dulbecco's modified Eagle's medium with
10% fetal bovine serum supplemented with G418 (200 µg/ml) for 5 days. The catalytic domain of MMP-9 was purified from the conditioned
medium by gelatin-Sepharose chromatography as described previously (28,
29). Fractions containing MMP-9 were pooled and dialyzed against 50 mM Tris, pH 7.8, 10 mM NaCl. The dialyzed
material was then purified further using a 1-ml Hi-Trap Source Q ion
exchange column (Amersham Pharmacia Biotech). Fractions containing
MMP-9 were pooled and dialyzed against 50 mM Tris, pH 7.5, 100 mM NaCl, 10 mM CaCl2 and then
concentrated in a dialysis bag using Aquacide II (Calbiochem). The
final concentration of purified material was typically 1.5-3
mg/ml.
Activation and Active Site Titration of Proteases--
The
purified catalytic domain of MMP-9 was activated by incubation with 2 mM APMA for 16 h at room temperature (29). Activation was monitored by altered migration of the protein on SDS-polyacrylamide gel electrophoresis and zymography. Pro-MMP-13 was activated with APMA
for 3 h at room temperature, and MMP-7 required no activation.
The level of active protease was always quantified by active site
titration prior to kinetic studies. The active site of MMP-9 was
titrated using the hydroxamate inhibitor Ilomastat (30). Briefly, 25 nM MMP-9 was incubated with a range of Ilomastat. Inhibition of MMP-9 by Ilomastat was then measured by determining the
rate of cleavage of three concentrations of the peptide, m1A11, using
the fluorescamine incorporation assay (21, 31). The active sites of
MMP-13 and MMP-7 were titrated with human TIMP-2. Briefly, 5-15
nM of each protease were pre-incubated with a range of
TIMP-2 for 5 h at room temperature. Residual MMP-13 activity was
measured by cleavage of MCA-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 (Calbiochem) as substrate, and residual MMP-7 activity was measured by
cleavage of MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2
(Calbiochem) with monitoring at Phage Selection of MMP-9 Substrates--
The substrate phage
library (2 × 1010 phage) was incubated with 2.5 µg/ml MMP-9 in 50 mM Tris, pH 7.4, 100 mM
NaCl, 10 mM CaCl2, 0.05% Brij-35, and 0.05%
BSA for 1 h at 37 °C. A control selection was performed without
protease. The cleaved phage were separated from the non-cleaved phage
by immunodepletion. 100 µg of anti-FLAG monoclonal antibody (Sigma)
was added to the phage samples and then incubated for 18 h with
rocking at 4 °C. The phage-antibody complexes were precipitated by
the addition of 100 µl of Pansorbin (Calbiochem). The cleaved phage
remaining in the supernatant were amplified using K91 E. coli as described previously (32-34) and were then used for
subsequent rounds of substrate selection.
Substrate Phage ELISA--
Hydrolysis of individual phage
substrates was measured using a modified ELISA. 96-well microtiter
plates were coated with anti-M13 antibody (Amersham Pharmacia Biotech,
2.5 µg/ml), in phosphate-buffered saline, overnight at 4 °C. After
coating, the wells were blocked for 1 h at room temperature in
TBS-T (50 mM Tris, pH 7.8, 150 mM NaCl, 0.2%
Tween 20) containing 10 mg/ml BSA. After blocking, 150 µl of
supernatant from an overnight phage culture was added to each well and
incubated for 2 h at 4 °C to allow for phage capture. Unbound
phage were removed with three washes of ice-cold TBS-T. To asses
hydrolysis, MMP-9 (2.5 µg/ml) was added to the appropriate wells in
Incubation Buffer (50 mM Tris, pH 7.5, 100 mM
NaCl, 10 mM CaCl2, 0.05% BSA, 0.05% Brij-35, 50 µM ZnCl2) for 2 h at 37 °C.
Control wells lacked protease. The protease solution was removed, and
the wells were washed four times with ice-cold TBS-T. To measure
hydrolysis of the peptides on phage by MMP-9, anti-FLAG polyclonal
antibody (1.8 µg/ml in TBS-T with 1 mg/ml BSA) was added to each well
and the plates were incubated at 4 °C for 1 h. Binding of
anti-FLAG antibody to FLAG epitope was measured with a horseradish
peroxidase-conjugated goat anti-rabbit IgG antibody (Bio-Rad) followed
by detection at 490 nm. The extent of hydrolysis, taken as a measure of
substrate hydrolysis, was calculated by the ratio of the optical
density at 490 nm of the protease-treated samples versus
samples lacking protease.
Determination of Scissile Bonds--
The cleavage site for MMP-9
within peptide substrates was determined using MALDI-TOF mass
spectrometry. MMP-9 (25 nM) was incubated with 100 µM amounts of each peptide, independently, in 50 mM Tris, pH 7.5, 100 mM NaCl, 10 mM
CaCl2 for 2 h at 37 °C. The mass of the cleavage
products was determined using a Voyager DE-RP MALDI-TOF mass
spectrometer (PerSeptive Biosystems, Framingham, MA). Following
hydrolysis, the peptide samples were prepared according to methods
described previously (35-38). In all cases, no other peaks that
corresponded to other potential cleavage products were observed.
Kinetic Measurements of Peptide Hydrolysis--
The kinetic
parameters of substrate hydrolysis were measured using a fluorescamine
incorporation assay that has been described previously (21, 31, 34,
39). Briefly, MMP-9, MMP-7, or MMP-13 were incubated with individual
peptide substrates at concentrations ranging from 100 to 800 µM in 50 mM Tris, pH 7.5, 100 mM
NaCl, 10 mM CaCl2, 50 µM
ZnCl2. At selected time points, the reactions were stopped
by the addition of 1,10-phenanthroline. Peptide hydrolysis was
determined by the addition of fluorescamine followed by detection at
Computational Molecular Modeling--
The terminology
established by Schechter and Berger is used to describe the subsites in
the protease active site and the correlating positions in substrates
(40). This method describes the former as a set of subsites designated
as S (S1, S2, etc.) and the latter as P
(P1, P2, etc.). The primed sites are to the
carboxyl-terminal and unprimed sites are to the amino-terminal side of
the scissile bond. The x-ray structure of MMP-9 has not been reported
to date. We have previously reported a computational three-dimensional model for the catalytic domain of MMP-9, which was generated by homology modeling taking advantage of sequence alignment and
conservation of secondary structural elements (41-43). This model
shows high sequence and three-dimensional homology to the structure of
MMP-2 (42), and it indicates an extended cleft for binding of substrate that can accommodate at least six amino acids from the substrate. We
considered molecular models for representative members of each class of
substrates (peptide A10 and peptide C11), but we concluded that there
is not sufficient sequence information for the group II substrates to
draw meaningful conclusions. Therefore, the modeling work was
concentrated on groups I and III. The molecular models of the sequences
flanking the sites of MMP-9 cleavage in clones A10 and C11
(Ac-SGPLFYSVTA-NH2 and Ac-SGRRLIHHTA-NH2,
respectively) were constructed using the SYBYL molecular modeling
program and were docked into the active site of MMP-9 (42). Each
enzyme-substrate complex contained the hydrolytic water molecule
positioned in the active site near the Glu-402 side chain, the general
base that promotes it for the hydrolytic reaction. The enzyme-substrate complexes were energy-minimized using the AMBER 5.0 software package (44, 45). The protocol for the energy minimization was described previously (42).
Construction of the Phage Library--
We created a system for
displaying random hexamer substrates on the surface of phage. The fUSE5
polyvalent phage display vector (24-26) was modified to express random
hexamers at the amino terminus of the geneIII protein, and an
octapeptide FLAG epitope at the amino-terminal end of the random
hexamers. The library comprises 2.4 × 108 independent
transformants, giving a 75% confidence that each of the 6.4 × 107 possible random hexamer sequences are represented in
the library. Sequencing of phage confirmed the randomness of the
hexamer insert. Under the selection conditions, greater than 95% of
phage could be immunodepleted using an anti-FLAG antibody (data not shown).
Selection of Peptide Substrates for MMP-9--
Optimal substrates
were selected by exposing the phage library to a recombinant form of
the catalytic domain of MMP-9 expressed in HEK 293 cells (29). The
recombinant catalytic domain of MMP-9 was purified on
gelatin-Sepharose, followed by ion exchange chromatography (29). The
protease was activated with 2 mM APMA for 16 h at room
temperature, and the active site was titrated with the hydroxamate inhibitor Ilomastat (29, 30). Phage selections were performed with 2.5 µg/ml (56 nM) active MMP-9. Following three rounds of exposure to MMP-9, individual phage clones were selected for
sequencing. An alignment of the motifs revealed three groups of
structurally distinct substrates (Table
I). Substrates in group I contain the
motif with sequence Pro-X-X-Hy-(Ser/Thr). Further
analysis shows that, within this larger motif, Arg is favored at
P2 and Ser/Thr is favored at P1 (consensus
motif noted in Table I). Substrates in group II contain a
Gly-Leu-(Lys/Arg) motif. Interestingly, both groups I and II are
related to substrates that have been described previously for other
MMPs and are somewhat related to sites of cleavage in collagen
(21-23). Group III substrates, however, appear to represent a novel
recognition sequence as it contains the Arg-Arg-Hy-Leu (group IIIA) and
Arg-X-Leu (group IIIB) motifs. The two subclasses that comprise group
III have not been described previously as a substrate motif for
MMP-9.
The ability of MMP-9 to hydrolyze each of the phage clones was assessed
in a semiquantitative manner using a modified ELISA. Individual phage
clones were captured into microtiter plates using anti-M13 antibody.
Captured clones were exposed to MMP-9 (2.5 µg/ml). A polyclonal
anti-FLAG antibody was used to measure the liberation of the FLAG
epitope by hydrolysis of the substrate with MMP-9. Results are
expressed as the extent of hydrolysis relative to untreated phage
clones (Table I). In general, the group I substrates were hydrolyzed
most efficiently. However, a number of the substrates within groups II
and III were cleaved to the same degree as group I substrates.
Identification of Scissile Bonds within MMP-9 Substrates--
To
identify the position of the scissile bonds, 10 peptides, representing
each of the three groups of substrates, were designed and synthesized.
Following exposure to MMP-9, the scissile bond was determined by
analyzing the cleavage products by MALDI-TOF analysis (Table
II). This analysis revealed that each
substrate contains a hydrophobic residue at the P1'
position. The substrates in group I contain Pro at the P3
position. The single peptide from group II contained a Gly at
P1, Leu at P1', and a Lys at P2'.
The motifs of groups I and II, and locations of their scissile bonds,
are similar to MMP cleavage sites in collagen and gelatin (21-23).
Interestingly, however, the peptides from group III were all cleaved
after an Arg residue, and in several cases an Arg-Arg occupied the
P2 and P1 positions.
Kinetic Characterization of Substrate Hydrolysis by MMP-9--
The
Michaelis constant (Km) and first-order rate
constant of substrate peptide turnover (kcat)
were measured by incubating a range of each peptide with MMP-9. Peptide
hydrolysis was measured by incorporation of fluorescamine onto newly
formed amino termini as previously described (31, 33, 34). From these
measurements, kcat and Km
were derived for each peptide using double-reciprocal plots of 1/[S]
versus 1/vi. Results from this
analysis are shown in Table II. Among the peptides, the
kcat values ranged from 9 s
Interestingly, the three peptides cleaved most efficiently by MMP-9
were from group I and contained Arg at the P2 position. This led us to hypothesize that group I and III substrates might represent a larger set of substrates whose relationship to one another
is not entirely evident from the sequences of only 100 clones. To test
this idea, we synthesized a mutant peptide (A11m1) that contains a Thr
In almost every case, the MMP-9 substrates selected from the phage
library contain a hydrophobic residue at P1', a finding that is entirely consistent with the fact that MMPs are known to have a
deep hydrophobic S1' pocket (1, 2, 46). To assess the
contribution of this hydrophobic residues to substrate binding and to
substrate turnover, we synthesized another mutant peptide (A11m2) based
on the sequence of peptide A11, but containing Ala, rather than Leu, at
P1' (Ac-SGKIPRT Kinetic Characterization of Substrate Hydrolysis by MMP-7 and
MMP-13--
Since many of the selected substrates contained a motif
similar to that described for other MMPs
(P-X-X-Hy), we measured the degree to which MMP-7
and MMP-13 could cleave these MMP-9 substrates. These two MMPs were
used for comparison because substrates for both proteases have been
selected using a similar phage display approach. Interestingly, most of
the substrates we tested were cleaved more efficiently by MMP-9. The
kcat/Km ratios ranged from
2.6- to 47-fold higher for MMP-9 than for either MMP-7 or MMP-13 (Table
II). Only peptide A7 deviated from this trend. It is also worth noting
that, although the substrates in group IIIA (Arg-Arg) were not cleaved
rapidly by MMP-9, we were unable to detect any hydrolysis of these
peptides by either MMP-7 or -13.
Modeling Substrate Interactions with MMP-9--
Molecular modeling
studies were conducted to help visualize how substrates might dock into
the enzymatic cleft of MMP-9. Energy-minimized models of MMP-9 were
constructed according to procedures described under "Experimental
Procedures," and then docked with peptides A10
(Ac-SGPLFYSVTA-NH2) representing group I and C11
(Ac-SGRRLIHHTA-NH2) representing group III.
A primary feature of the group I substrates is a Pro residue at the
P3 position. The unique conformational features of Pro introduce the appropriate "bend" needed for the optimal substrate binding at this site. This Pro residue, occupying the S3
subsite of MMP-9, is illustrated in Fig.
1A (white
arrow). The group I substrates also contain an invariant
hydrophobic residue at the P1' position. This residue
protrudes into the deep S1' pocket of the protease, as
depicted by the orange arrows.
Amino acids with long basic side chains at P2 and
P1, such as Arg, are the defining features of the
substrates in group III. Although the presence of these residues at
P2 and P1 is somewhat surprising, the
energy-minimized models support the observation that these residues
bind favorably. An Arg at P2 is likely to interact with the
backbone carbonyl moieties of His-405, Gly-408, and the side chain of
Asp-410, all of which contribute to the S2 subsite within
MMP-9 (white arrow in Fig. 1B). These
electrostatic interactions are predicted to contribute to favorable
binding of this class of substrates. Many of the group III substrates also contain an Arg residue at P1. The favorable
interaction of Arg into the S1 subsite can be explained by
the somewhat unusual nature of this subsite. It is essentially a
hydrophobic binding surface that would be predicted to accommodate the
hydrophobic side chains of amino acids such as Ala, Phe, and Tyr.
However, the docking studies of peptides with Arg at P2
show that the hydrophobic surface of the S2 subsite could
also bind to the extended methylene group in the side chain of Arg. In
addition, the hydrophobic channel of the S2 subsite
contains the backbone carbonyl of Pro-180, which is likely to engage in
electrostatic interaction with the basic side chain of Arg, stabilizing
the interaction.
Because of their association with a number of diseases, the MMPs
have received considerable attention as drug targets (15-17). Much of
the effort in this area has focused on the design of small molecule
antagonists with two primary features; 1) a hydroxamate moiety that
binds to the proteases catalytic zinc, and 2) a rather large
hydrophobic moiety that fits into the deep S1' pocket present in all MMPs (30). This synthetic strategy has focused structure-activity studies to essentially two positions within the
catalytic pocket. An understanding of the interactions between the
substrate and other key subsites within the catalytic pocket is lacking.
We have identified three families of peptide substrates for MMP-9 that
each appear to interact differently with the catalytic cleft of the
protease. Substrates in group I are cleaved most efficiently by MMP-9.
These peptides all contain a Pro at P3 and a large
hydrophobic residue at P1'. In this respect, the group I
substrates are similar to collagen-like sequences that are known to be
cleaved by the MMPs (19, 20). A prior analysis of a small series of
synthetic peptides based on collagen, and containing a similar
Pro-X-X-Hy core, showed
kcat/Km ratios ranging from
340 to 1000 mM Apparently, individual MMPs do exhibit a great deal of selectivity for
peptides containing the P-X-X-Hy core sequence.
For example, most of the MMP-9 substrates within group I are selective for MMP-9 over MMP-7 and MMP-13. Some of the
kcat/Km ratios for these
peptides are up to 47-fold higher for MMP-9 than for the other MMPs
tested. These findings suggest that substrate selectivity can be
conferred by subsite interactions outside of the dominant
P3 and P1' subsites that are common among MMP substrates.
Since phage substrate selections have been performed for MMP-3, -7, -9, and -13, an analysis of the frequency by which individual residues
occupy distinct subsites can be used as a first test of this idea. This
comparison reveals considerable distinction in the residues that occupy
the P2, P1, and P2' subsites. Nearly one-third of all group I substrates for MMP-9 contain Arg at
P2. Although Arg can also be found at P2 in
peptide substrates for MMP-13, its frequency is much lower than in the
MMP-9 substrates we selected (47). Furthermore, Arg is rarely, if ever,
found at P2 in MMP-3 or -7 peptide substrates (32). Ser or
Thr most frequently occupies the P1 position of the MMP-9
substrates. However, a Gly residue is preferred at this position by
MMP-13, and Asp or Glu are preferred by MMP-3 and -7 (32, 47).
Significant differences are also observed at P2'. In the
group I MMP-9 substrates, 23 of 28 substrates have Ser or Thr at
P2', but neither residue is prevalent at this subsite in
the substrates selected for the other MMPs (32, 47). These observations
support the contention that subsite interactions other than
P3 and P1' have a significant impact on
substrate selectivity among the MMP family.
Here we observed two additional families of substrates for MMP-9 that
are distinct from the P-X-X-Hy family. Group II
substrates were selected least frequently and contain only three
members, making it difficult to identify subsite preferences outside of the Gly-Leu-(Lys/Arg) consensus motif that occupies the P2,
P1', and P2' positions. Group III represents a
novel substrate preference for MMP-9. As a whole, group III substrates
were cleaved far less efficiently than either group I or group II.
Nevertheless, in the context of the whole phage, at least one of the
phages in this family (C11) was cleaved nearly as well as most of the group I substrates. An overriding feature of the group III substrates is the presence of Arg at P2 and often at P1.
Although this recognition specificity is unexpected, it is corroborated
by three other pieces of data. First, eight substrates from group I,
including the four best peptides in this study, also have Arg at the
P2 position. Second, when peptide A11 from group I was
mutated to contain Arg at both P2 and P1, the
kcat/Km ratio for the peptide nearly tripled. Third, docking studies with a representative group III
peptide illustrate the favorable interaction of Arg with the S2 subsite (Fig. 1).
A walk across the model of the catalytic pocket of MMP-9, subsite by
subsite, illustrates many of the features that are likely to guide
substrate interactions, and confer recognition specificity. On the
non-primed sides of the scissile bond, the structural features of the
S3 and S2 subsites are consistent with the corresponding substrates
that we selected. As with most MMPs, the S3 subsite is a
hydrophobic pocket that introduces a deviation from linearity into the
active-site cleft. This deviation explains why Pro is favored at
position P3. At S2, one finds the side chain of
Asp-410 contributing its hydrophilic characteristics, and providing a potential salt bridge for the guanidinium group of Arg that is often
found at P2. MMP-13 also has an Asp residue at this
position, and Arg occasionally occupies the P2 positions of
its substrates. In contrast, however, MMP-7 displays an Ala at this
site rather than an Arg (3, 42), and none of the MMP-7 peptide
substrates contain Arg at P2 (32).
Moving across the cleft, one encounters the S1 and
S1' pockets. The S1 space of MMP-9 is an
extended hydrophobic surface that can accommodate a wide variety of
amino acids (41). In fact, this is what we find. Like other MMPs, the
S1' subsite is a deep hydrophobic pocket, and it is likely
to be the most important substrate recognition point in the active
site. This notion is supported by the fact that Leu and Ile residues
dominate the P1' position. As discussed previously, the
depth of this subsite also guides the binding of most of the small
molecule antagonists of the MMPs (1, 3, 42).
The S2' subsite, a small hydrophobic surface, is the final
significant recognition point for substrate in the cleft of MMP-9. The
S2' subsite provides opportunity for flexible amino acids
to adapt conformations that would allow interactions with the solvent
on substrate binding at this position, including amino acids with small
hydrophobic or large hydrophilic side chains. We find that Ser/Thr are
preferred at this position. Even though two-thirds of the MMP-9
substrates derive their P2' position from an invariant Thr
encoded by the vector, the remaining one-third derive Thr or Ser at
their P2' positions by selection. Interestingly, the
character of the S2' subsite differs among MMP-9, MMP-7, and MMP-13. In MMP-9 this subsite essentially comprises Asp-188, Gly-189, Leu-190, and Met-422 (D-G-L-M) (3, 42). The corresponding residues in MMP-7 are G-N-T-T, and in MMP-13 are S-G-L-I. These distinctions are consistent with the fact that the substrates for each
protease also differ at P2' (see above).
Given this new understanding of substrate recognition by MMP-9, one can
arrive at an optimal substrate consensus
(Pro-Arg-(Ser/Thr)-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-proteinase inhibitor (5). Finally, recent studies in the RIP1-Tag2
transgenic mouse model of multistage carcinogenesis indicate that MMP-9
is part of the angiogenic "switch" that is essential for tumor
growth (11, 12). Other reports suggests that MMP-9 may play a role in
inflammation in the nervous system. MMP-9 is elevated in
encephelomyelitis (7, 8), in the cerebrospinal fluid of patients with
multiple sclerosis (13), and in patients with AIDS-related dementia
(14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
). Several phage were selected for sequencing to confirm randomness in the insert sequences and the
correct reading frame.
ex 320 nm and
em 405 nm.
ex 355 nm and
em 460 nm. The data were
transformed to double-reciprocal plots (1/[S] versus
1/vi) to determine Km and
kcat (21, 31, 34, 39). Similar results were
obtained using different batches of protease. For some substrates,
Km and kcat could not be
determined individually, but the specificity constant, kcat/ Km, was derived by the
equation: kcat/Km = vi/(E0)(S0)
(22).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sequence alignment and grouping of phage selected by MMP-9
Kinetic analysis of MMP-9 peptide substrates based on phage clones
. Amino acids
to the right of the
are primed residues (...P1',
P2', etc.), and amino acids to the left are non-primed residues
(...P2, P1) (40). **, measurements were
not made; *, not detected. Standard deviations of triplicate
experiments ranged from 11% to 22%.
1 to 703 s
1, and
Km values were generally in the high micromolar to
low millimolar range. Overall, peptides from group I appeared to
represent the best substrates, with peptide C15 having a
kcat/Km ratio of 180,000 M
1 s
1,
the highest of any of the substrates. Peptide A11, which closely matches the consensus recognition motif of group I, also exhibits a
high kcat/ Km ratio (67,500 M
1 s
1)
and considerable selectivity for MMP-9.
Arg substitution at P1 within the context of the
sequence of the A11 peptide. Hence, the mutant peptide contained
elements of both group I (Pro at P3 and Leu at
P1') and group III (Arg at P2 and
P1). Peptide A11m1 had a
kcat/Km twice that of the
parent peptide, an effect resulting primarily from an increase in
kcat. This finding suggests that the
substitution of Arg at P1 lowers the transition state
energy of the protease-substrate interaction. This observation also
indicates that, within the Pro-X-X-Hy-(Ser/Thr)
motif, Arg residues at P2 and P1 are favored for MMP-9, and that the substrates from groups I and III may have a
related binding mechanism.
ATA-NH2). This
substitution had deleterious effects on both
kcat and Km, and reduced the
kcat/Km ratio nearly 30-fold.
These results are consistent with the idea that the S1'
subsite of MMP-9 coordinates substrate binding and also influences the
rate of hydrolysis. Importantly, however, even this mutant peptide had a measurable kcat/Km ratio
(2000 M
1
s
1), indicating that efficient hydrolysis can
be enacted by MMP-9 if the rest of the substrate sequence is optimal.
View larger version (74K):
[in a new window]
Fig. 1.
Three-dimensional models of distinct binding
modes between substrate and MMP-9. To ascertain whether
peptides from different substrate families exhibit different
modes of binding in the active site of MMP-9, models with two
representative peptides binding to MMP-9 were constructed. The stereo
view represents the energy-minimized complexes of peptide A10
(SGPLFYSVTA, panel A) and peptide C11
(SGRRLLSRTA, panel B) in the active site of the
MMP-9 catalytic domain. The active site of MMP-9 is shown as a
green Conolly surface, and the peptide atoms are colored
according to type (carbon, white; oxygen, red;
nitrogen, blue). The catalytic zinc ion is represented as a
yellow sphere, and the backbone of MMP-9 outside
the active site is shown in magenta. The orange
arrows in both panels show the deep hydrophobic pocket at
the S1' subsite. In panel A, the
white arrow shows the Pro of substrate A10
penetrating well into the S3 pocket. In contrast, a Gly
from substrate C11 occupies this subsite (panel
B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
h
1. These values are ~100 fold lower than
those exhibited by the best peptides reported here. Consequently, the
added diversity afforded by phage libraries allowed us to identify
better substrates and a wider range of substrates. Interestingly, many
different MMPs recognize the Pro-X-X-Hy core
sequence. Can subsite interactions at positions other than
P3 and P1' generate selectivity within this
family of sequences?
-(Leu/Ile)-(Ser/Thr)). A comparison of this motif
to previously reported substrates for MMP-9 reveals some interesting
similarities and distinctions (Table III). For example, all of the collagen
substrates and even aggrecan, tissue factor pathway inhibitor and
galectin 3 contain a Pro at the P3 position and a
hydrophobic amino acid at the P1' position (6, 48, 50-52).
However, none of the substrates contain residues found to be optimal at
other positions. The remaining substrates have significantly lower
homology to the MMP-9 consensus recognition motif of group I (8,
53).
A comparison of the MMP-9 substrate consensus with cleavage sites in
known protein substrates
.
These comparisons raise questions as to whether all of the
physiologically relevant substrates for MMP-9 have been identified. We
reasoned that a query of the protein data bases with the optimal recognition motif might reveal a short list of putative substrates. Indeed, this search revealed only nine human proteins that contain this
sequence within their extracellular domain (Table
IV). Although at this juncture these
proteins can only be considered hypothetical substrates for MMP-9, many
of them are functionally linked to MMP-9-related pathology. For
example, MMP-9 influences skin blistering in the autoimmune disorder
bullous pemphigoid (5, 55). Interestingly, two of the hypothetical
substrates for MMP-9, ladinin 1 and desmoglein 3, are also associated
with autoimmune skin blistering disorders (56, 57). Similarly, MMP-9
has been suggested as a regulator of the angiogenic switch in tumor
development (11). Two of the hypothetical substrates for MMP-9,
integrin 5 and endoglin 1, are also involved in
angiogenesis (49, 54, 58). Along with the presence of a potential MMP-9
cleavage site, these functional associations lead to important and
testable hypotheses about physiologic substrates for MMP-9.
|
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FOOTNOTES |
---|
¶ Current address: Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 2S2, Canada.
** To whom correspondence should be addressed: Program on Cell Adhesion and Cancer Research Center, Burnham Inst., 10901 N. Torrey Pines Rd., La Jolla, CA 92037. E-mail: jsmith@burnham-inst.org.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M100900200
This workstudy was supported by California Breast Cancer Research Program Grant 5JB0033, National Institutes of Health Grants AR42750 and CA69036, Cancer Center Support Grant CA30199 (to J. W. S.), United States Army Grant DAMD17-97-17174 (to S. M.), Fellowships AR08505 from the National Institutes of Health and 2PD0182 from the California Cancer Research Program (both to S. J. K.), and a graduate fellowship from the United States Breast Cancer Research Program (to E. C.).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.
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ABBREVIATIONS |
---|
The abbreviations used are: MMP, matrix metalloproteinase; PCR, polymerase chain reaction; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; TBS-T, Tris-buffered saline with Tween 20; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; APMA, p-Aminophenylmercuric acetate.
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