From CEA, Département d'Ingénierie et
d'Etudes des Protéines, CE-Saclay, 91191 Gif/Yvette Cedex,
France, § Institut de Génétique et de
Biologie Moléculaire et Cellulaire, INSERM/CNRS/ULP, BP 163, 67404 Illkirch Cedex, France, and ¶ Department of Organic
Chemistry, Laboratory of Organic Chemistry, University of Athens,
Panepistimiopolis, Zografou, Athens 15771, Greece
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ABSTRACT |
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The influence of the substrate
P1 position on the specificity of two zinc matrix
metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and
stromelysin-3 (ST3), was evaluated by synthesizing a series of
fluorogenic substrates of general formula
dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH2, where Xaa in the P1
position represents unusual amino acids containing
either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their
P1
position unusual amino acids with extremely long side
chains more efficiently than the corresponding substrates with natural
phenylalanine or leucine amino acids. In this series of substrates, the
replacement of leucine by S-para-methoxybenzyl cysteine
increased the kcat/Km ratio
by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with a
S-para-methoxybenzyl cysteine residue in the
P1
position displayed a
kcat/Km value of 1.59 106 M
1 s
1 and 1.67 104 M
1 s
1, when
assayed with MT1-MMP and ST3, respectively. This substrate is thus one
of the most rapidly hydrolyzed substrates so far reported for
matrixins, and is the first synthetic peptide efficiently cleaved by
ST3. These unexpected results for these two matrixins suggest that
extracellular proteins may be cleaved by matrixins at sites containing
amino acids with unusual long side chains, like those generated
in vivo by some post-translational modifications.
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INTRODUCTION |
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Matrix metalloproteases (MMPs),1 also known as matrixins, form a group of structurally related zinc endopeptidases collectively able to degrade all components of the extracellular matrix (1). MMPs are believed to be mediators of both normal and pathological tissue remodeling processes, and their increased expression has been observed in a variety of human disorders (2-4). In particular, membrane type-1 (MT1)-MMP, a progelatinase A activator (5), and stromelysin-3 (ST3), a matrixin with unusual functional properties (6), are expressed in most human carcinomas (7-9). MT1-MMP and ST3 both belong to a subgroup of MMPs which are believed to be intracellularly activated by furin or furin-like convertases, thereby suggesting that MT1-MMP and ST3 are present in tissues in an active form, in contrast to other MMPs which are secreted as inactive zymogens (10-12). As part of a program aimed at developing therapeutic inhibitors for these two MMPs, the specificity of these enzymes in cleaving synthetic substrates has been investigated. Current data on the specificity of MT1-MMP toward the degradation of synthetic substrates are extremely limited (13), while no synthetic substrate has yet been reported to be cleaved by ST3.
In contrast to MT1-MMP and ST3, several studies have been devoted to
the delineation of the specificity of other matrixin family members by
developing both synthetic substrates (14-18) and inhibitors for these
enzymes (19, 20). Such approaches have been greatly facilitated by the
resolution of the crystal structures of several MMP catalytic domains
(21-27), allowing structure-based design strategies to be used (28).
According to these structural studies, the S1 subsite of
these enzymes appears as a cavity of variable size, depending on the
nature of the amino acid residue located near the bottom of this
cavity. In stromelysin-1 (ST1), which contains leucine in this
particular position, the S1
pocket is a deep cavity,
forming a channel that extends through the whole body of the enzyme
catalytic domain (29, 30). X-ray structure analysis of a complex
between ST1 and a carboxylalkyl inhibitor harboring a homophenylalanine
in the P1
position has consistently shown that the
homophenylalanine side chain only fills half of the S1
pocket of ST1 (29). A comparable situation is likely to occur in most
other MMPs, including MT1-MMP, collagenase-2 (COL2), stromelysin-1
(ST1), stromelysin-2 (ST2), gelatinase A, and gelatinase B, due to the
presence in their S1
pocket of a leucine at the same
position. The high potency of several inhibitors, substituted in their
P1
position by side chains longer than homophenylalanine, toward this subgroup of matrixins is consistent with this proposal (28,
31-35). In contrast to this subgroup of matrixins, collagenase-1 (COL1), matrilysin, and ST3 possess in their S1
subsite a
residue other than leucine. COL1, matrilysin, and ST3 possess in this particular position an arginine, a tyrosine and a glutamine,
respectively. In the case of this matrixin subgroup, x-ray structures
of COL1 and matrilysin have demonstrated that the presence of either
arginine or tyrosine reduces the size of their S1
subsite
(24, 26, 27). While no crystal structure is presently available for
ST3, a similar situation has been suggested to occur in this matrixin (36).
While the particular shape of the S1 pocket in matrixins
has been extensively exploited for the design of matrixin inhibitors, only one study so far examined the influence of this S1
cavity on the cleavage of synthetic substrates with unusual amino acids in their P1
position (37). It is worth remembering that
most of the matrixin inhibitors developed to date and harboring long side chains in their P1
position are not transition-state
analogues. Therefore, the ability of matrixins to cleave substrates
containing in their P1
position amino acids with unusual
side chains cannot be predicted from these inhibitor studies. To
address this issue more systematically in the case of MT1-MMP and ST3,
synthetic heptapeptides containing in their P1
position
amino acids with arylalkyl or alkyl side chains of varying size were
synthesized. The amino acid sequence covering the P3 to
P3
positions of these substrates was selected according to
the canonical -Pro-Leu-Gly(Ala)-Leu-Trp-Ala- sequence previously
established for matrixins (38, 39). In these substrates, the cleavage
site has been demonstrated to occur between the Gly(Ala)-Leu residues.
Accordingly, several fluorogenic substrates were prepared by
substituting the N-terminal side of this canonic sequence with a dansyl
group (dns), as a quencher, the tryptophan in the P2
position of this sequence being retained as a fluorophore. An arginine
residue was added at the C-terminal extremity of this sequence to
improve peptide solubility. In the present report, these fluorogenic
peptides were evaluated as substrates for MT1-MMP and ST3. Based on
this study, a fluorogenic compound, containing dinitrophenyl-coumarin
as a quencher-fluorophore pair, was also synthesized and examined for
comparison.
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MATERIALS AND METHODS |
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Rink amide resin, 2-(1H-benzotriazol-1-yl)-1,1,3,3,tetramethyluronium hexafluorophosphate (HBTU) and all Fmoc natural amino acid derivatives were purchased from Novabiochem. The unusual Fmoc amino acids were from Novabiochem and Advanced Chemtech. 7-Methoxycoumarin-2-acetic acid (McaOH) and 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride, dnsCl) were from Aldrich.
Chemistry-- Unusual Fmoc amino acids not commercially available (namely, 2-aminoheptanoic acid and 2-amino-5-phenyl-pentanoic acid) were synthesized by catalytic phase transfer alkylation of ethyl diphenylmethyleneglycinate with the appropriate alkyl bromides followed by hydrolysis, as described by O'Donnell et al. (40) and O'Donnell and Eckrich (41). 2-Aminoheptanoic, 2-aminooctanoic (Aldrich) and 2-amino-5-phenyl-pentanoic acids were converted into their Fmoc derivatives by the action of Fmoc-Cl in water/Na2CO3/dioxane solution according to the standard literature procedure (42). The Fmoc amino acids were obtained as white, crystalline compounds after their purification by flash chromatography on silica gel using ethyl acetate/hexane/acetic acid eluents. Their structure and the purity were confirmed by NMR and mass spectroscopy analysis. These unusual Fmoc amino acids were used for peptide synthesis as mixtures of enantiomers. For these peptides, the diastereoisomers were separated by HPLC. N2-Fmoc-N3-2,4-dinitrophenyl-L-2,3-diaminopropionic acid (Fmoc-DpaOH) was synthesized starting from Fmoc-L-AsnOH, as described by Knight et al. (14).
Substrate Synthesis and Characterization-- Solid phase synthesis of the substrates was performed in a model 357 Advanced Chemtech multiple peptide synthesizer on a Rink amide resin. Typically, three equivalents of an Fmoc amino acid, three equivalents of HBTU, and five equivalents of diisopropylethylamine in N-methylpyrrolidone were added to the resin, and the coupling reaction was allowed to proceed for 30 min. The Fmoc N-protection group was removed with a 30% solution of piperidine in N-methylpyrrolidone. N-terminal acylation of the peptides was achieved either with excess dansyl chloride (20 equivalents) in the presence of diisopropylethylamine or with triple coupling of McaOH, under the conditions described above. Cleavage of the peptides from the resin, together with the cleavage of the side chain protection groups, were performed by the action of trifluoroacetic acid containing 5% triisopropylsilane.
All peptides were purified by preparative HPLC column (Vydac, 218TP1022) performed on a Gilson system equipped with a variable wavelength detector. Gradient elutions were performed using solutions A (10% acetonitrile in 0.1% trifluoroacetic acid in water) and B (90% acetonitrile in 0.1% trifluoroacetic acid in water). All peptides were recovered by lyophilization. Peptide purities were checked by amino acid analyses, analytical HPLC (Vydac, 218TP104 column) and mass spectroscopy.Enzymes--
cDNAs corresponding to the catalytic domains of
mouse ST3 (Phe-102 to Ser-276) and human MT1-MMP (Tyr-111 to Arg-298)
were introduced into the expression vector pET-3b, expressed in
Escherichia coli BL21 (DF3) cells after
isopropyl-1-thio--D-galactopyranoside induction and
purified essentially as described in Noël et al. (43).
Briefly, both MMP catalytic domains were solubilized from bacterial
inclusion bodies with 8 M urea in the presence of 100 mM dithiothreitol and purified on a Q-Sepharose
anion-exchange column (Pharmacia Biotech Inc.). Purified catalytic
domains were then slowly refolded at a protein concentration of 50 µg/ml by dialysis to dilute out the urea. The refolding step was
followed by size exclusion chromatography, using a gel filtration
column (Superdex-gf 200, Pharmacia) to eliminate the aggregates, and to
retain the active monomeric protein alone.
Enzyme Kinetics--
Substrate specificity assays were performed
in 50 mM Tris/HCl buffer, pH 7.5, 10 mM
CaCl2, in the absence (MT1-MMP) or presence of 0.2 M NaCl (ST3), at 25 °C. Substrate concentrations were
determined spectrophotometrically using 340 nm = 4300 M
1 cm
1 for dansyl peptides (44)
and
328 nm = 12900 M
1
cm
1 for coumarin peptides (14). Substrates were prepared
as 1 mM stock solutions in dimethyl sulfoxide. Enzyme
concentrations were determined from optical density, using the method
of Gill and von Hippel (45) to calculate the extinction coefficient of
these two matrixins. In the case of MT1-MMP, values of
kcat/Km were determined from
first-order full-time course reaction curves obtained at [S]
Km (S = 0.2 µM), at 10 nM final enzyme concentration. These progress curves were
monitored by following the increase in fluorescence at 340 nm
(
ex = 280 nm), induced by the cleavage of the dns
substrates, in a Biologic PMS 200 spectrophotometer. Due to the lower
efficiency of ST3 in cleaving this series of substrates, the
observation of full-time course reactions for ST3 has required the use
of higher enzyme concentrations, leading to a high fluorescence
background. Thus, the kinetic parameters for ST3 were based on HPLC
(Thermo Separation Products system) allowing the separation of the
unreacted substrate from the cleavage products and its quantification.
Immediately after the initiation of the reaction, aliquots were
withdrawn from this reaction solution by the autosampler (Spectra
System AS300), at predetermined time intervals, and injected onto the
column. Substrate and products (S = 0.2 µM, ST3
concentration from 50 to 200 nM) were separated on a C18
column (Vydac, 218TP104), eluted with a linear acetonitrile gradient in
0.1% trifluoroacetic acid. Products were detected using an FL300
Spectra System fluorescence detector (
ex, 280 nm;
em, 340 nm) and were identified by mass spectroscopy
analysis. Data analysis was performed with a Thermo Separation Products datajet integrator.
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(Eq. 1) |
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RESULTS |
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The different side chains in the P1 position of our
series of fluorogenic substrates are described in Fig.
1. Arylalkyl side chains longer than that
of phenylalanine were selected to assess the effect of the side
chain's length on MT1-MMP1 and ST3 cleaving activities. In addition,
substrates with arylalkyl side chains containing an oxygen or sulfur
heteroatom were also examined. The effects of alkyl side chains longer
than leucine were also studied.
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Products resulting from the cleavage of this series of substrates by
MT1-MMP or ST3 were characterized both by HPLC and mass spectroscopy.
For each substrate, we observed a single cleavage site between the
expected Ala-Xaa peptide bond, with no inhibition of the reaction by
the released products (Fig. 2, and data
not shown). Values of the specificity constant
kcat/Km that were determined
from the first-order progress curves for the hydrolysis of substrates
by MT1-MMP are reported in Table I. The
most rapidly cleaved substrate by this enzyme was that harboring the
longest side chain in the P1 position (Cys(OMeBzl)). In
the arylalkyl series, the lengthening of the side chain by one (hPhe)
and two (pPhe) methylene(s) resulted in a marked increase in catalytic efficiency, as compared with the substrate containing phenylalanine in
the P1
position. The comparison of substrates with a side chain of equal length (pPhe, Ser(Bzl), Cys(Bzl)) reveals the
significant role played by a sulfur atom in the
position of the
side chain, which was associated with a much faster cleavage rate of
the substrate by MT1-MMP. In the alkyl series, the rate of hydrolysis
was also found to depend on the length of the side chain in the
P1
position. Comparison between the arylalkyl and alkyl
series of substrates reveals that, in addition to the side chain
length, the presence of the phenyl aromatic group is a determinant
factor for the optimization of the rate of hydrolysis, since the
substrates in the alkyl series were always cleaved at a slower rate
than their counterparts in the arylalkyl series (Table I). The
substitution Leu
Cys(OMeBzl) in these substrates led to a 37-fold
increase in the kcat/Km ratio
(Table I). In the case of ST3, the preferred side chain in the
P1
position of the substrate was also the longest
arylalkyl one (Cys(OMeBzl)) (Table I). However, the substrates
containing leucine, methionine or n-pentyl in the
P1
position were cleaved more rapidly than that with an
n-hexyl side chain. As compared with MT1-MMP, the former
three substrates were rather well cleaved by ST3, while the
n-hexyl compound was a poor substrate.
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The Km, kcat and
kcat/Km values determined for
two substrates (Xaa = Leu and Cys(OMeBzl)) are reported in Table
II. For these matrixins, the substitution
Leu Cys(OMeBzl) was found to increase both the substrate affinity
and the kcat value. However, while these two
substrates displayed similar Km for MT1-MMP and ST3,
the kcat values determined for ST3 on these substrates were about two orders of magnitude lower than those measured
for MT1-MMP. The free energy difference
(
G
) associated with the substitution
Leu
Cys(OMeBzl) was evaluated for each enzyme from the ratio of the
kcat/Km values determined for
these two substrates. For MT1-MMP, this substitution corresponds to a
free energy change of 2 kcal/mol, while the same modification causes a
free energy change of 1.55 kcal/mol for ST3.
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The substitution of alanine by phenylalanine in the P1 position, as well as the replacement of the Pro-Leu sequence by alanine residues in the P3 and P2 positions, led to substrates which were poorly hydrolyzed by MT1-MMP (Table I). In contrast, these two substrates were rather well cleaved by ST3. Thus, based on these preliminary data, the specificity requirement of the non-primed subsites of ST3 appears quite different from that observed for MT1-MMP.
The finding that the best substrate in this series was that containing
a Cys(OMeBzl) residue in the P1 position led us to develop
a fluorogenic substrate,
Mca-Pro-Leu-Ala-Cys(OMeBzl)Trp-Ala-Arg-Dpa-NH2, characterized by the presence of the Mca-Dpa fluorophore-quencher pair.
Such a fluorophore-quencher pair has been demonstrated to provide more
sensitive fluorescent assays than those based on the dns-Trp pair.
Using the Mca-Pro-Leu-Ala-Cys(OMeBzl)Trp-Ala-Arg-Dpa-NH2 substrate, the enzymatic activity of a 5 nM ST3 solution
can be determined with high accuracy, in less than 20 min. On this time scale, only a very small fraction of the commercially available Mca-Dpa
substrate for matrixins
(Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) was cleaved with the
same amount of ST3, even at a 5 µM final concentration of
this substrate. The
Mca-Pro-Leu-Ala-Cys(OMeBzl)Trp-Ala-Arg-Dpa-NH2 displayed a
kcat/Km value of 3.6 104 M
1 s
1 and 7.3 105 M
1 s
1, when
assayed with ST3 and MT1-MMP, respectively.
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DISCUSSION |
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In this study, we demonstrate that the substitution of the
P1 position in synthetic substrates by unusual long
arylalkyl side chains leads to peptides which are cleaved by MT1-MMP
and ST3 much more rapidly than the corresponding substrates containing natural phenylalanine or leucine amino acids. The dns substrate with a
Cys(OMeBzl) residue in the P1
position exhibited a
kcat/Km value of 1.59 106 M
1 s
1 when
tested with MT1-MMP, at 25 °C. This substrate is therefore one of
the most rapidly cleaved synthetic substrates so far reported for
matrixins. For example, a
kcat/Km value of 1.57 105 M
1 s
1 has been
determined for the cleavage of the
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by MT1-MMP,
at the same pH, but at 37 °C (13). Furthermore, the first synthetic
substrates cleaved by ST3 were identified in this series. The cleavage
of the Mca-Pro-Leu-Ala-Cys(OMeBzl)-Trp-Ala-Arg-Dpa-NH2 fluorogenic peptide provides a simple test for routine assay of ST3,
useful for ST3 inhibitor screens. The influence of unusual amino acid
substitutions in the P1
position of synthetic substrates was previously evaluated for two matrixins, COL1 and GEL B (37). A
small increase (4-fold) in the
kcat/Km value was observed for substrates containing
-S substituted cysteine in the
P1
position, as compared with the parent compound with
leucine in the P1
position. Based on the present work,
this modest effect may be due to the fact that only
-S substituents
of reduced size, with a methyl and an ethyl group, were investigated in
this study.
The substitution Leu Cys(OMeBzl) in the P1
position of
the substrate improved the Km value of the substrate
for MT1-MMP, a finding in agreement with x-ray data (25, 29, 30) and
inhibitors studies (31-35). This suggests that in all matrixins containing a leucine residue in their S1
pocket, this
pocket should be a deep cavity able to accommodate extremely long amino acid side chains. In contrast, the increase in substrate affinity resulting from the Leu
Cys(OMeBzl) substitution for ST3 is
unexpected. ST3 is characterized by the presence of glutamine in its
S1
subsite, a property which was predicted to confer a
shallow S1
pocket to ST3 (36), paralleling the situation
observed in the x-ray structures of COL1 and matrilysin (24, 26, 27). A
possible interpretation of our results is that the glutamine side chain in the ST3 S1
pocket can move, allowing this matrixin to
bind and cleave substrates harboring long side chains in the
P1
position. Such a scenario has been proposed during
inhibitor binding for the arginine side chain of COL1, since this
matrixin was unexpectedly found to be potently inhibited by synthetic
compounds with long side chains at the P1
position (34). A
shift of the glutamine side chain in ST3, allowing substrate binding,
may explain why the free energy change associated with the Leu
Cys(OMeBzl) substitution is lower for ST3 than MT1-MMP. In the case of
ST3, the interactions engaged between the P1
position of
the substrate and the S1
pocket would have to compensate
for the energy associated with the displacement of the glutamine side
chain.
Interestingly, the modification Leu Cys(OMeBzl) not only improves
the Km, but also the kcat
value of the substrate for both MT1-MMP and ST3 enzymes. Therefore, for
these two matrixins, the binding energy associated with the interaction
of substrates containing an unusual amino acid side chain in the
P1
position with the enzyme catalytic site is used to
stabilize the energy of the substrate-enzyme complex both in the ground
and transition-states. This last effect, as mentioned in the
introduction, could not be predicted from previous studies aimed at
evaluating matrixin-inhibitor interactions, since most inhibitors so
far developed were not analogues of the substrate in the
transition-state (20). For a long time, the functional significance of
a deep S1
subsite in matrixins able to accommodate long
P1
groups of inhibitors was a subject of debate. In this
respect, our data clearly indicate that the filling of this
S1
subsite by an appropriate group in the P1
position yields substrates with high
kcat/Km values.
The kcat values reported in this study for two
substrates are two orders of magnitude lower for ST3 than those
observed for MT1-MMP. These low turnover numbers are in agreement with
the very weak proteolytic activities reported for ST3 against most usual matrixin substrates, with the exception of 1 proteinase inhibitor (43, 47, 48). Arguments pointing to ST3 as a particular member of the matrixin family have been presented before and include, in addition to unusual proteolytic properties, both unusual proform processing (10, 49) and regulation of gene expression (50, 51).
However, our observations do not rule out the possibility that ST3 may
rapidly hydrolyze in vivo some, as yet unexamined, substrates. Our preliminary data on the specificity of ST3 for the
nonprimed residues of synthetic substrates, in comparison to MT1-MMP,
support the possibility that ST3 could cleave substrates which are not
presently considered as matrixin substrates. Many reports have
documented the preference of matrixins for small residues in the
P1 position of the substrate, as well as the key role of
proline in their P3 position (15, 16), a feature apparently not conserved in the case of ST3 (Table I). It is therefore important to define more exhaustively the specificity requirements of ST3 for the
non-primed positions of the substrate. As illustrated in the present
study, where only one position of the substrate was exhaustively
investigated, such optimization could dramatically increase the
kcat/Km value, as this
involves a gain of only a few kilocalories/mol in the enzyme-substrate
binding energy (Table II). An important issue is to determine whether
it is possible to identify specific peptide sequences that are
hydrolyzed by ST3 with an efficiency similar to that reported for the
other matrixins. Our data showing that ST3 efficiently cleaves
substrates harboring unusual long side chains in their sequence suggest
that this matrixin may hydrolyze, in vivo, proteins also
containing unusual amino acids. In this respect, extracellular proteins
undergoing particular post-translational modifications may represent
potential targets for ST3. Although a large number of protein
substrates have already been described for MT1-MMP, as well as for the
other matrixins containing a leucine residue in their S1
subsite, our observations raise the possibility that these matrixins
may also cleave, in vivo, proteins at sites characterized by
the presence of amino acids with unusual long side chains.
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ACKNOWLEDGEMENT |
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We thank I. Stoll for technical assistance.
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FOOTNOTES |
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* This work was supported by funds from the Commissariat à l'Energie Atomique, the Institut National de la Santé et de la Recherche Scientifique, the Center National de la Recherche Scientifique, the Center Hospitalier Universitaire Régional, the Bristol-Myers Squibb Pharmaceutical Research Institute, the Association pour la Recherche sur le Cancer, the Ligue Nationale Française contre le Cancer, the Comité du Haut-Rhin, the Fondation de France, the BIOMED 2 (contract no. BMH4CT96-0017) and BIOTECH 2 (contract no. ERBBIO4CT96-0464) Program, and a grant to P. Chambon from the Fondation Jeantet.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. Tel.:
33-0169083585; Fax: 33-0169089071; E-mail: dive{at}dsvidf.cea.fr.
1 The abbreviations used are: MMP, matrix metalloprotease; MT1, membrane type-1; ST, stromelysin; COL, collagenase; dns, dansyl, 5-dimethylaminonaphthalene-1-sulfonyldansyl; Mca, (7-methoxycoumarin-4-yl)acetyl; Dpa, N3-(2,4-dinitrophenyl)-L-2,3-diamino propionyl; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3,tetramethyluronium hexafluorophosphate; Bzl, benzyl; Cys(OMeBzl), S-para-methoxybenzyl cysteine; HPLC, high performance liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl.
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REFERENCES |
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