From the Department of Chemistry and Biochemistry,
Florida Atlantic University, Boca Raton, Florida 33431-0991, the
§ Department of Cell and Molecular Biology, Northwestern
University Medical School, Chicago, Illinois 60611, and the
¶ Kennedy Institute of Rheumatology Division, Imperial College of
Science, Technology, and Medicine, Hammersmith,
London W6 8LH, United Kingdom
Received for publication, November 6, 2002, and in revised form, March 11, 2003
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ABSTRACT |
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The role of proteases in the tumor cell invasion
process is multifaceted. Members of the matrix metalloproteinase (MMP)
family have been implicated in primary and metastatic tumor growth,
angiogenesis, and degradation of extracellular matrix (ECM) components.
Differentiating between the up-regulation of MMP production and the
presence of activated MMPs can be difficult but may well dictate which
MMPs are critical to invasion. Because the hydrolysis of collagens is
one of the committed steps in ECM turnover, we have investigated selective MMP action on collagenous substrates as a means to evaluate active MMPs. Two triple-helical peptide (THP) models of the MMP-9 cleavage site in type V collagen, The role of proteases, particularly metalloproteases, in the tumor
cell invasion process is multifaceted. Members of the matrix metalloproteinase (MMP)1
family have been implicated in primary and metastatic tumor growth, angiogenesis, and degradation of ECM components during tumor cell invasion (1-4). MMP expression is widespread in common human tumors,
with MMP-1, -2, -3, -7, -9, -11, and/or -14 found most often (3). Of
these MMPs, a great deal of attention has been focused on the two
gelatinases, MMP-2 and MMP-9. MMP-2 expression is found in the greatest
variety of stromal tissue surrounding neoplasms (5), and cleavage of
certain ECM components by MMP-2 may reveal "cryptic sites" that aid
cell migration and growth and angiogenesis (6, 7). Cell surface
activation and association of MMP-2 correlates to enhanced ECM
remodeling by tumors (8, 9). MMP-9 has also been shown to induce
angiogenesis during carcinogenesis (10). Activation of MMP-9 enhances
tumor cell invasion (11) and increases shedding of intercellular
adhesion molecule 1 (ICAM-1) from tumor cell surfaces (12), which in turn decreases tumor cell susceptibility to natural killer
cell-mediated cytotoxicity. MMP-9 expression has been shown to increase
in advanced-stage melanoma cells (13).
Although the vast majority of studies on MMP regulation examine factors
that up-regulate MMP production, the ability to differentially examine
actual MMP activity is necessary to discern which MMPs are critical to
the invasion process. The hydrolysis of collagen is one of the
committed steps in ECM turnover, and it has long been demonstrated that
tumor extracts can possess collagenolytic activity (14). The
triple-helical structure of collagen renders it resistant to most
mammalian proteases, with the exception of cathepsin K and MMPs. Thus,
triple-helical collagen-like substrates may provide an effective
screening tool for activated tumor cell proteases. Such substrates
could be used to determine whether different triple-helical sequences
can be cleaved selectively by MMP family members and, if so, how the
kinetics of hydrolysis compare.
Several approaches have been described for construction of THP
substrates (recently reviewed in Refs. 15 and 16). Non-covalent self-assembly of lipophilic molecules, N-terminal linked to a peptide,
can be used to form stable triple-helical "peptide-amphiphiles" for
mechanistic evaluation of MMP activity (17-19). Fluorescence resonance energy transfer triple-helical peptide-amphiphile substrates have been constructed by combining a sequence based on the type II
collagen 769-783 region, a fluorophore (Lys(Mca)) in the
P5 subsite, and a quencher (Lys(Dnp)) in the
P5' subsite (19). Individual kinetic parameters for MMP
hydrolysis were then determined using a continuous fluorometric assay
(19).
Fluorogenic substrates can be used to examine other triple-helical
sequences for selective MMP hydrolysis. As a starting point, we can
consider the five triple-helical collagen sequences hydrolyzed by MMPs
that have been identified.2
Types I-III collagens are hydrolyzed by MMP-1, -8, -13, -14, -18, and
-22 at the 775-776 bond (20-27). MMP-2 hydrolyzes type I collagen at
the same single locus (20). The Gly439-Val440
bonds in type V collagen are cleaved by MMP-9, along with a similar cleavage site sequence in type XI collagen (28).
For the present study, we have constructed THP models of the MMP-9
cleavage sites in types V and XI collagen. The THPs use the
Materials--
All standard peptide synthesis chemicals were
analytical reagent grade or better and purchased from Novabiochem (San
Diego, CA) or Fisher Scientific. 9-Fluorenylmethoxycarbonyl-amino acid derivatives were obtained from Novabiochem. Amino acids are of the
L-configuration (except for Gly). Hexanoic acid
(CH3-(CH2)4-CO2H, designated C6) was purchased from Aldrich. THPs were
synthesized and purified by methods previously described in our
laboratory (19, 29, 30).
Peptide Analyses--
Analytical RP-HPLC was performed on a
Hewlett Packard 1100 liquid chromatograph equipped with a Hypersil
small-pore, narrow-bore C18 reversed-phase column (5-µm
particle size, 120-Å pore size, 100 × 2.1 mm). Eluants were
0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid
in acetonitrile (B). The elution gradient was 0-100% B in 20 min with a flow of 0.3 ml/min. Detection was at Circular Dichroism Spectroscopy--
Circular dichroism spectra
were recorded over the range Cells--
M14 human melanoma cells were propagated as described
previously (33-35). Briefly, melanoma cells were cultured in Eagle's minimum essential medium or RPMI 1640 supplemented with 10% fetal bovine sera, 1 mM sodium pyruvate, 0.1 mg/ml gentamycin
(Roche Applied Science), 50 units/ml penicillin, and 0.05 mg/ml
streptomycin. Cells were passaged 8 times and then replaced from
frozen stocks of early passage cells to minimize phenotypic drift. All
cells were maintained at 37 °C in a humidified incubator containing 5% CO2. All media reagents were purchased from Fisher Scientific.
Matrix Metalloproteinases--
ProMMP-1 and proMMP-3 were
expressed in Escherichia coli and folded from the inclusion
bodies as described previously (36). ProMMP-2 was purified from the
culture medium of human uterine cervical fibroblasts (37). ProMMP-2 was
activated by reacting with 1 mM 4-aminophenylmercuric
acetate at 25 °C for 2 h. ProMMP-1 was activated by reacting
with 1 mM 4-aminophenylmercuric acetate and a 0.1 molar
amount of MMP-3 at 37 °C for 6 h. After activation, MMP-3 was
completely removed from MMP-1 by affinity chromatography using an
anti-MMP-3 IgG Affi-Gel 10 column. ProMMP-3 was activated to the 45-kDa
MMP-3 by reacting with 5 µg/ml chymotrypsin at 37 °C for 2 h.
Chymotrypsin was inactivated with 2 mM diisopropyl fluorophosphate. ProMMP-13 was a generous gift from Dr. Maureen Horrocks, AstraZeneca Pharmaceuticals. ProMMP-9 and MMP-14 were purchased from Chemicon (Temecula, CA). ProMMP-9 and -13 were activated
with 1 mM 4-aminophenylmercuric acetate. The amounts of
active MMP-1, -2, and -3 were determined by titration with recombinant
N-terminal domain of tissue inhibitor of metalloproteinases-1 (38) over
a concentration range of 0.1-3 µg/ml.
Assays--
Two different assay methods were utilized, the first
for discontinuous fluorometric analyses and the second for continuous fluorometric analyses. For the discontinuous assay method,
For continuous fluorometric analyses, substrate Cellular MMP Assay--
Melanoma cell adhesion to
substrate-coated non-tissue culture-treated plates (BD Biosciences) was
performed as described previously (35). The The first THP constructed as a possible MMP-2 and -9 substrate was
based upon the MMP-9 cleavage site in types V and XI collagen. It
incorporated the 1(V)436-450 THP and
1(V)436-447 fTHP, were hydrolyzed by MMP-2 and MMP-9 at the Gly-Val
bond, analogous to the bond cleaved by MMP-9 in the corresponding
native collagen. Kinetic analyses showed
kcat/Km values of 14,002 and 5,449 s
1M
1 for MMP-2 and -9 hydrolysis of
1(V)436-447 fTHP, respectively. These values, along
with individual kcat and Km
values, are comparable with collagen hydrolysis by MMP-2 and -9. Neither THP was hydrolyzed by MMP-1, -3, -13, or -14.
1(V)436-447 fTHP and a general fluorogenic THP were used to screen
for triple-helical peptidase activity in
2
1 integrin-stimulated melanoma cells. Binding of the
2
1 integrin resulted in
the production of substantial triple-helical peptidase activity, the
majority (>95%) of which was non-MMP-2/-9. THPs were found to
provide highly selective substrates for members of the MMP family and
can be used to evaluate active MMP production in cellular systems.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1(V)436-450 sequence
Gly-Pro-Pro-Gly
Val-Val-Gly-Glu-Gln-Gly-Glu-Gln-Gly-Pro-Pro as a
template. We have compared the susceptibility of the THPs to several
MMP family members (MMP-1, -2, -3, -9, -13, and -14) and determined
individual kinetic parameters by continuous monitoring of a fluorogenic
THP. Mechanistic aspects of MMP hydrolysis have been examined by
comparing activities toward THPs and the analogous SSPs. Ultimately,
fluorogenic THPs were used to assess MMP-2/-9 and general MMP activity
in
2
1 integrin-stimulated metastatic melanoma cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
= 229, 324, and 363 nm. Edman degradation sequence analysis was performed on an
Applied Biosystems 477A protein sequencer/120A analyzer as described
(31) for "embedded" (non-covalent) sequencing. MALDI-MS was
performed on a Hewlett-Packard G2025A mass spectrometer using either a
sinapinic acid or 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxy-benzoic acid (9:1, v/v) matrix (32). Single-chain mass values were as follows:
1(V)436-450 THP, [M+H]+ 3636.2 Da (theoretical 3639.9 Da);
1(V)436-450 SSP, [M+H]+ 1402.9 Da
(theoretical 1403.5 Da);
1(V)436-447 fTHP,
[M+H]+ 4487.4 Da (theoretical 4490.7 Da); and
1(IV)402-413, [M+H]+ 3609.3 Da (theoretical 3609.0 Da).
= 190-250 nm on a JASCO J-600
using a 10-mm path length quartz cell. Thermal transition curves were
obtained by recording the molar ellipticity ([
]) at
= 225 nm while the temperature was continuously increased in the range of
5-80 °C at a rate of 0.2 °C/min. Temperature was controlled
using a JASCO PTC-348WI temperature control unit. For samples
exhibiting sigmoidal melting curves, the reflection point in the
transition region (first derivative) is defined as the melting
temperature (Tm).
1(V)436-450 THP was prepared as 270 µM stock solution
in "fluorometric assay" buffer (50 mM Tricine, pH 7.5, 50 mM NaCl, 10 mM CaCl2, 0.05% Brij-35). A 0.5-M stock solution of
o-phenanthroline was prepared in Me2SO, followed
by dilution with assay buffer to a concentration of 20 mM.
MMP assays were carried out in assay buffer by incubating a range of
substrate concentrations with 40 nM enzyme at 30 °C. Enzymatic activity was terminated by the addition of 20 µl of the
enzyme/substrate solution to 30 µl of o-phenanthroline (20 mM) at appropriate times. Rates of hydrolysis were
monitored by the addition of 200 µl of fluorescamine solution.
Fluorescamine solution was prepared by first dissolving fluorescamine
in acetone at a concentration of 40 mM, then diluting to 5 mM with assay buffer minus Brij-35. Fluorescamine reacts
with free amino groups, resulting in a fluorophore with
excitation = 387 nm and
emission = 480 nm. Fluorescence was measured on a Molecular Devices SPECTRAmax Gemini
dual-scanning microplate spectrofluorometer.
1(V)436-447 fTHP
was prepared as 270 µM stock solution in fluorometric
assay buffer. MMP assays were carried out in assay buffer by incubating a range of substrate concentrations (1-50 µM) with 40 nM enzyme at 30 °C. Fluorescence was measured using
excitation = 325 nm and
emission = 393 nm. Initial velocities were obtained from plots of fluorescence
versus time, using only data points corresponding to less
than 40% full hydrolysis. The slope from these plots was divided by
the fluorescence change corresponding to complete hydrolysis and then
multiplied by the substrate concentration to obtain initial velocity in
units of µM/s. The fluorogenic substrates NFF-1, NFF-3, and fTHP-4 were used for control assays of MMP activity as described (19, 39, 40).
1(IV)402-413 ligand was
dissolved in phosphate-buffered saline, diluted in 70% ethanol, added
to the 96-well plate, and allowed to adsorb overnight at room
temperature with mixing. Plates were rinsed three times with sterile
phosphate-buffered saline to remove all traces of ethanol. Cells
were released with 5 mM EDTA in phosphate-buffered saline
and washed two times with adhesion medium (20 mM HEPES,
RPMI 1640). Cells were then resuspended in adhesion medium and added to
the plate. The plate was incubated for 60 min at 37 °C. Non-adherent
cells were removed by washing three times with adhesion medium, and
fresh medium was added. Conditioned media were collected at various
time points, centrifuged to remove cell debris, and stored at
20 °C. One volume of the appropriate fluorogenic substrate was
added to each well in a 96-well plate. Where applicable, EDTA was added
to each well. The plate was incubated at 30 °C in a humidified
atmosphere for 30 min, and one volume of conditioned media, adhesion
medium (as a negative control), or MMP (as a positive control) was
added to each well. The plate was incubated at 30 °C in a humidified atmosphere for at least 18 h. Fluorescence readings
(
excitation = 325 nm and
emission = 393 nm) were taken and a standard curve created by plotting the increase in
fluorescence versus concentration of MMP standard. The
standard curve was used to calculate the active enzyme concentration in
the conditioned media.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1(V)436-450 sequence between N- and C-terminal (Gly-Pro-Hyp)4 repeating tripeptides and an N-terminal
C6 alkyl chain
(C6-(Gly-Pro-Hyp)4-Gly-Pro-Pro-Gly
Val-Val-Gly-Glu-Gln-Gly-Glu-Gln-Gly-Pro-Pro(Gly-Pro-Hyp)4-NH2). The
1(V)436-450 THP had a Tm value of
49.5 °C in 1.0% (v/v) fluorometric assay buffer (Fig.
1), which is a desirable thermal
stability for an MMP substrate. The composition and homogeneity were
confirmed by Edman degradation sequence analysis (prior to addition of
the C6 alkyl chain), RP-HPLC, and MALDI-MS.
View larger version (9K):
[in a new window]
Fig. 1.
Thermal transition curves for
purified (A) 1(V)436-450
THP and (B)
1(V)436-447 fTHP in 1.0% (v/v) fluorometric assay
buffer at substrate concentrations of 10 µM. Molar ellipticities ([
])
were recorded at
= 225 nm while the temperature was increased
from 10 to 80 °C. Inserts are the first derivative of the
transition curves, from which Tm values are
determined.
MMP-1 and MMP-9 hydrolysis of 1(V)436-450 THP was studied at
30 °C using the discontinuous fluorometric assay with 40 µM substrate. MMP-9 rapidly hydrolyzed the substrate
within 1 h, whereas MMP-1 did not cleave the substrate even after
24 h. MMP-9 hydrolysis of the
1(V)436-450 THP was analyzed by
MALDI-MS. Each intact chain of the
1(V)436-450 THP has a mass of
3638.9 Da. If the Gly-Val bond is cleaved, the two products generated
in assay buffer are the single-stranded peptides
C6-(Gly-Pro-Hyp)4-Gly-Pro-Pro-Gly ([M+Na]+ = 1515.6 Da) and
Val-Val-Gly-Glu-Gln-Gly-Glu-Gln-Gly-Pro-Pro-(Gly-Pro-Hyp)4-NH2 ([M+Na]+ = 2187.3 Da). Mass spectrometric analysis of
MMP-9 hydrolysis showed two products, one of [M+Na]+ = 1516.2 Da and one of [M+Na]+ = 2189.8 Da. No hydrolysis
of the substrate by MMP-1 was detected using MALDI-MS analysis. The
1(V)436-450 THP is the first synthetic substrate that shows
complete selectivity between MMP-1 and MMP-9. Subsequent treatment of
1(V)436-450 THP with MMP-2 and MALDI-MS analysis indicated cleavage
at the Gly-Val bond. Edman degradation sequence analysis of the
1(V)436-450 THP cleavage products also showed that MMP-2 hydrolysis
occurred exclusively at the Gly-Val bond, because the only amino acids
seen in the first two cycles are PTH-Val and PTH-Val (emanating from
the C-terminal fragment of the cleaved THP). Thus, both Edman
degradation sequence analysis and MALDI-MS analyses indicated that
MMP-2 and MMP-9 cleaved the
1(V)436-450 THP exclusively at the
Gly-Val bond. This is the analogous bond cleaved by MMP-9 in types V
and XI collagen (28). Although the cleavage sites for MMP-2 hydrolysis
of type V collagen have not been determined, the digestion pattern is
very similar to that of MMP-9 (41), suggesting that the
Gly439-Val440 bond could be cleaved by MMP-2 in
intact type V collagen.
To determine whether primary structure alone accounted for the observed
MMP-2/-9 selectivity, an SSP analog of the 1(V)436-450 sequence
(Gly-Pro-Pro-Gly
Val-Val-Gly-Glu-Gln-Gly-Glu-Gln-Gly-Pro-Pro-NH2) was constructed and incubated with several different MMPs. RP-HPLC and
MALDI-MS indicated that the
1(V)436-450 SSP was very slowly cleaved
by MMP-3 and -9. Interestingly, MMP-9 hydrolyzed the
1(V)436-450 SSP at a considerably slower rate than the
1(V)436-450 THP. Thus, primary structure was not the sole determinant for MMP-2/-9 selectivity of the
1(V)436-450 THP, and substrate triple-helical
conformation greatly enhanced hydrolysis rates. These results suggest
that MMP-9 acts as a true "collagenase," preferentially cleaving a specific sequence in triple-helical conformation, whereas MMP-3 has
limited triple-helical peptidase activity.
Based on the results with 1(V)436-450 THP, a fluorogenic substrate
was designed and designated
1(V)436-447 fTHP
((Gly-Pro-Hyp)5-Gly-Pro-Lys(Mca)-Gly-Pro-Pro-Gly
Val-Val-Gly-Glu-Lys(Dnp)-Gly-Glu-Gln-(Gly-Pro-Hyp)5-NH2). The Gln in the P5' subsite from
1(V)436-450 THP was
replaced with Lys(Dnp), whereas Hyp in the P5 subsite was
replaced with Lys(Mca). Prior studies indicated that MMP-2 and -9 well
tolerate substitution of a P5 subsite Hyp by a bulky
aromatic residue (42). To improve substrate solubility while not
sacrificing thermal stability, (a) the C6 alkyl
chain was replaced by two Gly-Pro-Hyp repeats on the N terminus and
(b) an additional Gly-Pro-Hyp repeat was added to the C
terminus. The
1(V)436-447 fTHP has a Tm
value of 45.0 °C in 1.0% (v/v) fluorometric assay buffer (Fig. 1),
which is a desirable thermal stability for an MMP substrate. The
composition and homogeneity were confirmed by Edman degradation
sequence analysis, RP-HPLC, and MALDI-MS.
Edman degradation sequence analysis of the 1(V)436-447 fTHP
cleavage products showed that MMP-2 hydrolysis occurred exclusively at
the Gly-Val bond, because the only amino acids seen in the first three
cycles are PTH-Val, PTH-Val, and PTH-Gly (emanating from the C-terminal
fragment of the cleaved THP). MMP-2 and -9 hydrolysis of the
1(V)436-447 fTHP was also examined by MALDI-MS. Each intact chain
of the
1(V)436-447 fTHP has a mass of 4489.7 Da. If the Gly-Val
bond is cleaved, the two products generated are the single-stranded
peptides (Gly-Pro-Hyp)5-Gly-Pro-Lys(Mca)-Gly-Pro-Pro-Gly ([M+H]+ = 2162.3 Da) and
Val-Val-Gly-Glu-Lys(Dnp)-Gly-Glu-Gln-(Gly-Pro-Hyp)5-NH2 ([M+H]+ = 2347.5 Da). Mass spectrometric analysis of
MMP-2 or MMP-9 hydrolysis showed two products of [M+H]+ = 2159.6 and 2346.4 Da. Thus, both Edman degradation sequence analysis
and MALDI mass spectrometric analyses indicated that MMP-2 and -9 cleaved the
1(V)436-447 fTHP exclusively at the Gly-Val bond. No
hydrolysis of the substrate by MMP-1, -3, -13, or -14 was detected
using MALDI-MS analysis.
Individual kinetic parameters for MMP
hydrolysis of 1(V)436-447 fTHP were
evaluated by Lineweaver-Burk (Fig. 2), Hanes-Woolf, and Eadie-Hoftsee
analyses. The relative order of apparent
kcat/Km values is MMP-2 > MMP-9
MMP-14 ~MMP-13 > MMP-1 ~MMP-3 (Table I). MMP-2 has the highest
kcat/Km value for hydrolysis of
1(V)436-447 fTHP, because of both a lower Km
value (4.4 µM for MMP-2 versus 8.1 for MMP-9)
and a higher kcat value. For MMP-2,
1(V)436-447 fTHP is a 13-fold better substrate than the previously
described triple-helical substrate fTHP-3 (Table II), which is modeled after the type II
collagen MMP cleavage site. MMP-2 prefers
1(V)436-447 fTHP to type
I collagen (Table II). MMP-2 cleaves type V collagen very
efficiently, although at a 4-fold lower rate than MMP-9 (41). Thus,
some differences exist between triple-helical peptidase and
collagenolysis specificity, as previously observed (18, 19).
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Incubation of 1(V)436-447 fTHP with MMP-14 or -13 resulted in a
slight increase in fluorescence (Table I). However, MALDI-MS analysis
(see above) indicated that neither of these MMPs cleaves
1(V)436-447 fTHP. The slight increase in fluorescence appears to
result from perturbation of the substrate structure by MMP-13 and -14, but not from hydrolysis. MMP-1 and -3 do not cleave
1(V)436-447 fTHP under the conditions studied here. Simultaneous studies using the
fluorogenic substrates NFF-1, NFF-3, and fTHP-4 indicated that MMP-1,
-3, -13, and -14 were active. For example, treatment of NFF-3 with
MMP-3 resulted in a rapid increase in substrate fluorescence and
MALDI-MS detection of two peptide products corresponding to
Mca-Arg-Pro-Lys-Pro-Val-Glu and
Nva-Trp-Arg-Lys (Dnp)-NH2.
The present work has demonstrated that triple-helical sequences are
selectively hydrolyzed by members of the MMP family. Thus, depending
upon the invasion pathway and the type of collagen encountered, different MMPs may be required to facilitate metastasis. Similar conclusions could be drawn from prior studies on collagenolysis. For
example, a subset of the MMP family are capable of cleaving type I
collagen (see earlier discussion). In similar fashion, type IV collagen
is hydrolyzed by MMP-2, -3, -7, -9, -10, and -12, but not by MMP-1, -8, or -13 (43). However, the prior collagenolysis studies consider several
MMP actions, including (a) the binding and manipulation of
collagen, (b) unwinding of the triple-helix, and
(c) hydrolysis of individual collagen strands. The
utilization of THPs eliminates effects due to the binding and
manipulation of collagen, allowing for direct comparisons of
triple-helical sequences and MMP susceptibility. Thus, studies with the
1(V)436-450 THP and
1(V)436-447 fTHP are the first to show
discrimination of substrate hydrolysis by collagenolytic MMPs
(i.e. MMP-1 and -2) based on triple-helical sequence
specificity. These results complement prior studies showing that
selectivity between collagenolytic MMPs (i.e. MMP-1, -2, -8, etc.) and non-collagenolytic MMPs (i.e. MMP-3) can be based
on triple-helical sequence specificity (18, 44). Interestingly, the
selectivities of
1(V)436-450 THP and
1(V)436-447 fTHP are not
solely because of the primary structure of the substrates. A
single-stranded analog of the
1(V)436-450 sequence is cleaved by
several MMPs. Also, MMP-3 cleaves the single-stranded substrate
Gly-Pro-Gln-Gly
Val-Ala-Gly-Gln 2-fold faster than MMP-2 or -9 (45).
Thus, triple-helical structure appears to effect the susceptibility of
Gly-Val bonds to cleavage by MMP-3.
1(V)436-447 fTHP is the first truly selective fluorogenic substrate
described for gelatinases (MMP-2 and -9). Of the previously studied
gelatinase substrates,
Dnp-Pro-cyclohexylalanyl-Gly
Cys(CH3)-His-Ala-Lys(N-methylanthranilic acid)-NH2 is cleaved by MMP-1 and -9, Dnp-Pro-Leu-Gly
Leu-Trp-Ala-D-Arg-NH2 is
cleaved by MMP-1, -2, -3, and -9, and
Mca-Arg-Pro-Lys-Pro-Tyr-Ala
Nva-Trp-Met-Lys(Dnp)-NH2 is
cleaved by MMP-2, -3, and -9 (45). The (cyanine
fluorochrome)-Gly-Pro-Leu-Gly
Val-Arg-Gly-Lys(fluorescein isothiocyanate)-Cys-NH2 substrate, used for
near-infrared fluorescent imaging of MMP-2 positive tumors, is
additionally hydrolyzed by MMP-1, -7, -8, and -9 (46).
The results described herein suggest that triple-helical substrates
could be utilized to discriminate among MMP family members for a given
cell or tissue type. We have previously described a general MMP
fluorogenic THP substrate (19) and now complement this with a selective
gelatinase substrate. To test whether these substrates can be used to
discriminate between MMP family members, we have examined MMP
production by highly metastatic M14 melanoma cells. Melanoma cells are
known to express a variety of MMPs, including MMP-1, -2, and -9, when
in a highly metastatic state (47, 48). In addition, the
2
1 integrin, which is abundant on M14
melanoma cell surfaces (49), is a positive regulator for MMP-1 gene
expression (50) and has been implicated in MMP-9 regulation (51). It
has not been determined previously whether
2
1 integrin-mediated signaling results in
the production of active MMPs nor whether specific MMP-2/-9 activity is
present. To induce
2
1 integrin signaling,
we constructed a triple-helical model of
1(IV)402-413. This region
of type IV collagen contains the Gly-Phe-Hyp-Gly-Glu-Arg motif which,
in triple-helical conformation, binds to the
2
1 integrin (52-54). The
1(IV)402-413 THP ligand was used to engage the
2
1 integrin of M14 melanoma cells, and MMP production was quantitated. Significant triple-helical
peptidase activity was detected in melanoma cell-conditioned media,
because an increase of ~1140 fluorescence units was observed (Table
III). This activity was completely
inhibited by EDTA, indicating metalloproteinase activity. At an enzyme
concentration of 5 nM, MMP-1 hydrolysis of fTHP-3 results
in an increase of ~1100 fluorescence units, whereas MMP-2 shows an
increase of ~950 fluorescence units (Table III). Thus, although
fTHP-3 indicates significant activity, the nature of MMP(s) present is
unknown. Based on comparison with standard curves (data not shown) and
correction for dilution, the increase in fluorescence generated by the
melanoma-conditioned media correlates to 24.3 nM MMP-2 or
11.7 nM MMP-1 if either of these enzymes was the sole MMP
present. Subsequent analysis of melanoma-conditioned media with the
1(V)436-447 fTHP showed an increase of ~135 fluorescence units
(Table III). This activity was also completely inhibited by EDTA. MMP-2
hydrolysis of
1(V)436-447 fTHP results in an increase of ~300
fluorescence units at an enzyme concentration of 1 nM
(Table III). Based on comparison with a standard curve (data not shown)
and correction for dilution, the increase in fluorescence generated by
the melanoma-conditioned media correlates to 1.02 nM MMP-2
if this enzyme was the sole MMP present. To a very rough approximation,
this indicates that only ~4% of the triple-helical peptidase
activity in melanoma cell-conditioned media was because of MMP-2. These
results suggest that melanoma binding via the
2
1 integrin produces active
metalloproteinases and that only a small percentage of the activity is
because of gelatinases. The relative distribution of specific MMPs
produced in response to
2
1 integrin
binding, at both the gene and protein level, will be described
elsewhere.3
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By varying the sequence within THPs, we can hope to design additional
selective fluorogenic substrates that incorporate triple-helical structures. It may also be possible to manipulate the P2
subsite to obtain selectivity between MMP-2 and -9 (45, 55). Selective THP substrates would allow for a better understanding of the specific functions of MMP domains and subsequently could be used to design specific inhibitors of these enzymes. Finally, the fluorogenic triple-helical substrate described herein allows for continuous monitoring of MMP-2 and MMP-9 activity and thus has distinct advantages over enzyme-linked immunosorbent assay and zymographic methods for
analysis for gelatinases (56).
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ACKNOWLEDGEMENT |
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We thank Dr. Jeff Borgia for performing the Edman degradation sequence analyses.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR 39189 (to H. N.) and CA 77402 and CA 98799 (to G. B. F.) and by the Wellcome Trust (reference number 057508, to H. N.).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.
To whom correspondence should be addressed: Dept. of
Chemistry and Biochemistry, Florida Atlantic University, 777 Glades
Rd., Boca Raton, FL 33431-0991. Tel.: 561-297-2093; Fax: 561-297-2759; E-mail: fieldsg@fau.edu.
Published, JBC Papers in Press, March 17, 2003, DOI 10.1074/jbc.M211330200
2 Although MMP-3 and -9 cleavage sites within the triple-helical region of type IV collagen have been identified (57), it was not determined whether these sites were cleaved while the triple helix was intact or after the triple-helical region had denatured because of other MMP-3 or MMP-9 cleavages.
3 J. A. Borgia, J. L. Lauer-Fields, and G. B. Fields, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
MMP, matrix
metalloproteinase;
1(IV)402-413, C16-(Gly-Pro-Hyp)4-Gly-Ala-Hyp-Gly-Phe-Hyp-Gly- Glu-Arg-Gly-Glu-Lys-(Gly-Pro-Hyp)4-NH2;
Dnp, 2,4-dinitrophenyl;
ECM, extracellular matrix;
fTHP-3, (C6-(Gly-Pro-Hyp)5-Gly-Pro-Lys(Mca)-Gly-Pro-Gln-Gly-Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-(Gly- Pro-Hyp)5-NH2)3;
fTHP-4, ((Gly-Pro-Hyp)5-Gly-Pro-Lys(Mca)-Gly-Pro- Gln-Gly-Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-(Gly-Pro-Hyp)5-NH2)3;
Hyp, 4-hydroxy-L-proline;
MALDI-MS, matrix-assisted laser
desorption ionization-mass spectrometry;
Mca, (7-methoxycoumarin-4-yl)acetyl;
NFF-1, Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys(Dnp)-Gly;
NFF-3, Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2;
Nva, norvaline;
PTH, phenylthiohydantoin;
RP-HPLC, reversed-phase high
performance liquid chromatography;
SSP, single-stranded peptide;
THP, triple-helical peptide;
Tricine, N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine.
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