From the CNRS FRE 2260, IFR 53 Biomolecules, Faculty
of Medicine, 51 Rue Cognacq Jay, F-51100 Reims, France, the
¶ Cell Biology Unit, Institute of Cellular Pathology and
Université catholique de Louvain, B-1200
Brussels, Belgium, the ** Institute of Enzymology, Biological Research
Center, Hungarian Academy of Sciences, H-1518 Budapest, Hungary, and
the §§ Laboratory of Biochemistry, Faculty of
Odontology, Paris V, F-92120 Montrouge, France
Received for publication, December 26, 2000, and in revised form, February 13, 2001
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ABSTRACT |
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The matrix metalloproteinases
gelatinase A (MMP-2) and gelatinase B (MMP-9) are implicated in the
physiological and pathological breakdown of several extracellular
matrix proteins. In the present study, we show that long-chain fatty
acids (e.g. oleic acid, elaidic acid, and cis-
and trans-parinaric acids) inhibit gelatinase A as well as
gelatinase B with Ki values in the micromolar range
but had only weak inhibitory effect on collagenase-1 (MMP-1), as
assessed using synthetic or natural substrates. The inhibition of
gelatinases depended on fatty acid chain length (with C18 > C16,
C14, and C10), and the presence of unsaturations increased their
inhibitory capacity on both types of gelatinase. Ex vivo experiments on human skin tissue sections have shown that micromolar concentrations of a long-chain unsaturated fatty acid (elaidic acid)
protect collagen and elastin fibers against degradation by gelatinases
A and B, respectively. In order to understand why gelatinases are more
susceptible than collagenase-1 to inhibition by long-chain fatty acids,
the possible role of the fibronectin-like domain (a domain unique to
gelatinases) in binding inhibitory fatty acids was investigated.
Affinity and kinetic studies with a recombinant fibronectin-like domain
of gelatinase A and with a recombinant mutant of gelatinase A from
which this domain had been deleted pointed to an interaction of
long-chain fatty acids with the fibronectin-like domain of the
protease. Surface plasmon resonance studies on the interaction of
long-chain fatty acids with the three individual type II modules of the
fibronectin-like domain of gelatinase A revealed that the first type II
module is primarily responsible for binding these compounds.
Matrix metalloproteinases
(MMPs)1 compose a family of
at least 23 related zinc-dependent endopeptidases (1) that
are collectively able to degrade extracellular matrix proteins such as
collagens, laminins, fibronectin, elastin, and proteoglycans. They are
consequently implicated in physiological remodeling of connective
tissue occurring in embryonic development and repair (2-4). Most of
them are secreted as inactive proenzymes and are then extra- or
pericellularly activated by other MMPs or serine proteinases (5). Their
catalytic activities are strictly controlled by endogenous specific
inhibitors designated as tissue inhibitors of metalloproteinases
(TIMPs) (6) and also Long-chain unsaturated fatty acids inhibit the expression and activity
of aggrecanases (17). They bind to serine proteinases such as
neutrophil elastase (18, 19) and plasmin (20, 21) and modulate their
catalytic activities. Studies by Suzuki et al. (22) show
that oleic acid, 18-carbon fatty acid with one double carbon bond in
the cis position, partially inhibits the formation of lung
metastases from subcutaneous implantation of colon carcinoma
cells in athymic mice. We recently reported that oleic acid inhibited
in a dose-dependent manner the hydrolysis of the
fluorogenic substrate
Mca-L-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (23) by
gelatinase A (24, 25). We report here an in vitro investigation showing that fatty acids, depending on their chain length
and degree of unsaturation, inhibit gelatinases A and B with similar
efficiency but only have low inhibitory capacity toward
collagenase-1.
We conclude that interaction between FN-II domain and unsaturated fatty
acids leads to gelatinase inhibition because of the following: (i)
oleic acid displayed markedly weaker inhibitory capacity toward
gelatinase A deleted in FN-II modules; (ii) fatty acids bound avidly to
the first FN-II module; and (iii) this module totally prevented the
oleic acid-mediated inhibition of full-length gelatinase A. The
physiological relevance of our findings was substantiated by ex
vivo experiments on human skin tissue sections demonstrating that
C18-unsaturated fatty acid efficiently impeded the collagenolytic and
elastolytic activities of gelatinases A and B, respectively.
Enzymes and Proteins--
Human recombinant progelatinases A and
B and natural procollagenase-1 were purchased from Calbiochem. The
recombinant pro- Recombinant FN-II Modules of Gelatinase A--
Recombinant
proteins coll 1, coll 2, coll 3, and coll 123 containing different
segments of the gelatin-binding site of human gelatinase A were
expressed in Escherichia coli and purified by gelatin-Sepharose 4B chromatography as described previously (14, 27).
Skin Tissue Sections--
Human foreskins from healthy young
children (10 months to 10 years old) were obtained on parental consent
during surgical operations and kept frozen at Chemicals--
The free acid forms of decanoic acid (capric
acid, 10:0), tetradecanoic acid (myristic acid, 14:0),
cis-9-tetradecenoic acid (myristoleic acid, c9-14:1),
hexadecanoic acid (palmitic acid, 16:0), cis-9-hexadecenoic
acid (palmitoleic acid, c9-16:1), octadecanoic acid (stearic acid,
18:0), cis-9-octadecenoic acid (oleic acid, c9-18:1), and
trans-9-octadecenoic acid (elaidic acid, t9-18:1), and an
alcohol analogue of oleic acid were purchased from Sigma. The
cis-9, trans-11, trans-13,
cis-15-octadecatetraenoic acid (cis-parinaric
acid, c9, t11, t13, c15-18:4), and all-trans-9, -11, -13, -15- octadecatetraenoic acid (trans-parinaric acid, all-t9,
11, 13, 15-18:4) were from Molecular Probes (Interchim, Montluçon, France). The hydroxamate derivative of oleic acid was
prepared as described previously (24). The quenched fluorescent substrate Mca-PLGL(Dpa)-AR-NH2 was from Bachem
(Voisins-le-Bretonneux, France).
Activation of Pro-MMPs--
Full-length and Inhibition of Matrix Metalloproteinase Activities by Long-chain
Fatty Acids--
The inhibitory effect of fatty acids (from C10 to
C18, either saturated or cis- or
trans-unsaturated) against gelatinase A (full-length or
FN-II-deleted forms), gelatinase B, or collagenase-1 was analyzed using
the fluorescent quenched substrate Mca-PLGL-(Dpa)-AR-NH2. Two hundred picomolar of each MMP species were preincubated for 15 min
at 22 °C with 0-40 µM fatty acid in a 50 mM HEPES buffer, pH 7.5, containing 150 mM
NaCl, and 5 mM CaCl2. The assays were initiated
by adding 2 µM Mca-PLGL-(Dpa)-AR-NH2. The
final concentration of dimethyl sulfoxide (Me2SO) used to
dissolve fatty acid and fluorogenic substrate never exceeded 1% (v/v).
The reaction was allowed to proceed at 22 °C for 20 min (gelatinase
A), 60 min (gelatinase B), or 180 min (collagenase 1) and then was
stopped by adding 10 mM EDTA. Under these experimental
conditions, MMPs generated a similar intensity of fluorescence,
allowing the comparison of the inhibition results. The effect of
substitution of the carboxylic end by alcohol or hydroxamate group on
the inhibitory potency of oleic acid against gelatinase A was similarly
evaluated. The rate of substrate cleavage was measured in quadruplicate
for each fatty acid or derivative concentration examined, using a
Perkin Elmer LS 50B spectrofluorimeter with excitation and emission
wavelengths of 325 and 387 nm, respectively. Less than 5% of the
substrate was hydrolyzed during the rate measurements. Addition of
fatty acid after the digestion of fluorogenic substrate had no effect on the fluorescent signal. Nonlinear regression analysis with the
Grafit computer software (R. J. Leatherbarrow, Erithacus
Software) allowed us to calculate the best estimates of the equilibrium dissociation constant of the enzyme-inhibitor complex or inhibition constant Ki, using the integrated Equation 1
(29),
The inhibitory effect of oleic, elaidic, or stearic acids (0-40
µM) was further evaluated against gelatinase A, using a
natural substrate, i.e. heat-denatured
[3H]collagen type I. Briefly, 1.6 nM
gelatinase A was first mixed with 0-40 µM fatty acid in
a 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl and 10 mM CaCl2 for 15 min
at 22 °C before incubation with radiolabeled natural substrate for
20 h at 37 °C (30). The 50% inhibitory concentrations
(IC50) of fatty acids for gelatinase A were determined
using the Grafit computer software.
Kinetic Analysis of Oleic Acid Inhibition of Full-length and
Determination of Dissociation Constants by Surface Plasmon
Resonance (SPR) Analysis--
The trimodular protein coll 123 contains
the entire fibronectin-related part of human gelatinase A (27). Oleic
acid and stearic acid-coll 123 interaction studies were performed using a BIAcore X system (Amersham Pharmacia Biotech). Sensor chip HPA (BIAcore AB) was used for all experiments. A flow rate of 5 µl/min was used, and the instrument was thermostated at 25 °C. The surface of sensor chip was washed for 5 min with 40 mM
n-octyl- Role of FN-II Modules in the Inhibition of Gelatinase A by Oleic
Acid--
Oleic acid (5 µM) and gelatinase A (200 pM) were preincubated for 15 min at 22 °C with molar
fold excess (1-100-fold in comparison with gelatinase A) of the
trimodular protein coll 123 or the single module proteins coll 1, coll
2, or coll 3, and residual enzymatic activity was measured with
Mca-PLGL- (Dpa)-AR-NH2 (2 µM), as described above. Whatever the concentration used, coll 123, coll 1, coll 2, or
coll 3 did not exhibit inhibitory effect against gelatinase A.
Computerized Morphometric Analysis of Elaidic Acid Inhibition of
Skin Tissue Degradation by Gelatinases A and B--
A set of three
skin tissue sections was laid on a coated polylysine microscopic slide
(Bio-Rad) and overlaid with 10 µl of 50 mM Tris-HCl
buffer, pH 7.4, containing 150 mM NaCl and 5 mM CaCl2 (assay buffer) or the same buffer containing 50 nM APMA-activated gelatinase A or gelatinase B. The
preparations were then incubated for 4 h at 37 °C in a moist
chamber. After incubation, the tissue sections were rinsed and stained
with polyphenolic catechin-fuschin and red sirius for staining of
elastic and collagen fibers, respectively (32). Elastic fibers appeared
in deep blue-black, and the background was poorly stained and collagen
fibers were revealed in red-orange. This allowed a further quantitative
estimation of the area (AA) and volume
(VV) fractions occupied by these fibers. For elaidic
acid inhibition assays, gelatinase A or gelatinase B were first
incubated with 0, 1, or 10 µM fatty acid for 15 min at
22 °C; alternatively, tissue sections were preincubated with 1 or 10 µM elaidic acid or Me2SO-containing assay
buffer as negative control for 30 min at 37 °C before adding
gelatinase A or gelatinase B. The microscopic slides were then observed
under a Zeiss standard 14 microscope equipped with CF 126 PHR video
camera, and computerized analyses of the elastic and collagenic fibers
were carried out as described previously (32). Briefly, three skin
serial sections were used for each assay, and 10 fields of dimensions
0.7 × 0.7 mm were analyzed for each skin tissue section. Black
and white images generated by the video camera were converted into 256 different gray levels using a Sophretec NUM 600 image memory,
transferred to a BFM 186 microcomputer, and finally analyzed using a
software for mathematical morphology. The area fraction
(AA) occupied by the elastic and collagen fibers was
automatically calculated. The area fraction represents the surface of
fibers as a function of the tissue area analyzed. For the elastic
fibers, the volume fraction (VV) was also determined
and corresponds to AA × k, where
k <1 represents the Weibel correction factor (k = d/d + t, with d
and t, elastic fiber diameter, and section thickness,
respectively, in µm). The average diameter of the elastic fibers was
obtained semiautomatically using skin tissue sections treated or not
and a calibrated slide.
Relationship between Fatty Acid Structure and Inhibition of MMP
Activity--
The effect of fatty acids varying in chain length and
degree of unsaturation was first evaluated on gelatinases A and B using the fluorogenic Mca-PLGL-(Dpa)-AR-NH2 substrate. Table
I shows that both enzymes were inhibited
with a similar efficiency by fatty acids. Inhibition depended on their
alkyl chain length (with C18 > C16, C14, and C10). The presence
of unsaturations in fatty acids increased the inhibition of both
gelatinases. Furthermore, gelatinases A and B inhibition did not depend
on the cis-trans configuration of double bond(s)
since oleic acid and elaidic acid, its trans-counterpart,
displayed similar Ki values; also, no difference was
observed between cis- and trans-parinaric acids. Such polyunsaturated fatty acids were, however, more efficient as
inhibitors toward gelatinase B versus gelatinase A (Table
I).
When they were tested against collagenase-1, only unsaturated fatty
acids with 18 carbon atoms exhibited a low inhibitory effect, with a
Ki value equal to 59.6 ± 5.7 µM
for oleic acid. Contrary to data obtained with gelatinases, a
trans-configuration of the unsaturation slightly improved
collagenase-1 inhibition; 40 µM of oleic or elaidic acids
inhibited by 30 and 40%, respectively, the degradation of fluorogenic
substrate by collagenase-1.
Effect of Derivatization of the COOH-terminal End Group of
Oleic Acid on Its Gelatinase A Inhibitory Potential--
Changing
carboxylic end group of oleic acid to hydroxamic group, a more potent
bidentate ligand for catalytic zinc, did not dramatically improve
gelatinase A inhibition, with the Ki value only
decreasing from 4.3 ± 0.4 to 1.8 ± 0.2 µM
(Fig. 1). Also, replacement of carboxylic
group by an alcohol group, not considered as a zinc ligand, did not
strikingly impair the inhibitory activity of oleic acid, with the
Ki value only rising from 4.3 ± 0.4 to
6.3 ± 0.3 µM (Fig. 1).
Involvement of FN-II Repeats in the Inhibition of Gelatinases A and
B by Fatty Acids--
Our data suggested that FN-II repeats, present
in gelatinases A and B but absent in collagenase- 1, could represent
selective targets for fatty acids. We therefore evaluated the
inhibitory capacity of oleic, elaidic, and stearic acids against
gelatinase A deleted in FN-II repeats. Fig.
2 illustrated the weak inhibition of
oleic and elaidic acids toward truncated gelatinase A; as shown above
for collagenase-1, oleic acid was less efficient than its trans-counterpart, elaidic acid, with Ki
values of 32.5 ± 3.0 and 12.3 ± 1.6 µM,
respectively. Again, stearic acid did not inhibit truncated
gelatinase A (Fig. 2).
Kinetic Analysis of Full-length and Binding of Fatty Acids to the FN-II Modules of Gelatinase
A--
Potential interactions between fatty acids and the gelatinase A
FN-II repeats were studied by SPR analyses using a recombinant peptide
corresponding to the three FN-II modules (coll 123) of gelatinase A and
oleic or stearic acids. Fatty acids were linked to the surface of a
sensor cell as described under "Experimental Procedures." Solutions
of coll 123 were allowed to bind to the immobilized oleic acid or
stearic acids, and data were analyzed as a function of time. The
results of typical binding assays are shown in Fig.
4, A and B. The
association rate constant (ka), dissociation rate
constant (kd), and dissociation equilibrium constant
(KD) of coll 123 for oleic acid or stearic acid were
calculated from SPR analyses, using the nonlinear data fitting software
BIAevaluation (Table II). Oleic and
stearic acids bound coll 123, with KD of 41 ± 1 and 36.1 ± 2 µM, respectively. Furthermore, SPR
analysis demonstrated that oleic acid interacted with the first FN-II
module, coll 1, with a KD value of 62 ± 4 µM, close to that obtained with the entire FN-II domain, coll 123; on contrary, the second and third FN-II modules weakly bound
oleic acid with dissociation constants of 210 ± 90 and 3900 ± 1400 µM, respectively.
We next investigated whether the ability of oleic acid to bind FN-II
modules was related to its gelatinase inhibitory capacity. Gelatinase A
activity was measured in the presence of increasing concentrations of
trimodular protein coll 123 and an oleic acid concentration producing
50% inhibition, i.e. 5 µM. Increasing concentrations of coll 123 relieved the inhibition of gelatinase A, and
the inhibitory effect of oleic acid was totally abolished by a 100-fold
molar excess of coll 123 (Fig.
5A). In the same way, we
analyzed the effect of isolated FN-II modules, i.e. coll 1, coll 2, and coll 3, on gelatinase A inhibition by oleic acid. A 50-fold
molar excess of coll 1 restored up to 95% gelatinase A activity in the
presence of 5 µM oleic acid (Fig. 5B). In
contrast, only a slight or no restoration of gelatinase A activity was
observed in the presence of coll 2 or coll 3.
Effect of Fatty Acids on the Degradation of Gelatin by Gelatinase
A--
Denatured collagen (gelatin) represents physiological substrate
for gelatinase A. We also evaluated the effect of oleic, elaidic, and
stearic acids on gelatinolytic activity of gelatinase A (Fig. 6). The IC50 values of oleic
and elaidic acids, determined using heat-denatured type I collagen,
were equal to 1.6 ± 0.3 and 1.3 ± 0.3 µM,
respectively, versus 9.6 ± 0.9 µM
obtained for stearic acid, their saturated counterpart (Fig. 6). By
using the synthetic fluorogenic substrate, at such concentrations,
oleic and elaidic acids inhibited ~25% of the activity of gelatinase
A, whereas 10 µM stearic acid caused any detectable
inhibition.
Effect of Elaidic Acid on the Degradation of Human Skin Collagen
and Elastin by Gelatinases A and B--
We previously showed, by
quantitative morphometric analysis, that gelatinase A preferentially
degraded collagen fibers, whereas gelatinase B displayed elastolytic
activity (32). By using a similar approach, the inhibitory effect of
elaidic acid on the ex vivo collagenolysis (Fig.
7, A-C) and elastolysis (Fig.
7, D-F) by gelatinases A and B, respectively, has been
investigated on human skin tissue sections. In control sections (Fig.
7A), skin collagen fibers appeared as thick homogenous
bundles. When skin sections were incubated with 50 nM
gelatinase A, the number of collagen fibers decreased (Fig.
7B); they often appeared as scattered bundles, with most of
them exhibiting teased extremities. When gelatinase A was preincubated
with 1 or 10 µM elaidic acid, a
dose-dependent inhibition of collagen fiber hydrolysis was
observed (Fig. 7C and Table
III). Pretreatment of skin tissue
sections by elaidic acid concentration as low as 1 µM
impeded degradation of collagen fibers by gelatinase A (Table III).
As shown in Fig. 7D, control skin sections stained with
polyphenolic catechin dye revealed an intact elastic fiber system around the vessel. When skin sections were incubated with 50 nM gelatinase B, an important alteration of the elastic
fiber network could be evidenced (Fig. 7E). Elastolytic
activity was markedly diminished when gelatinase B was preincubated
with 1 or 10 µM elaidic acid (Fig. 7F), as
assessed by morphometric analyses (Table III). Also, preincubation of
skin tissue with 1 µM elaidic acid partially protected
tissue against elastolytic activity of gelatinase B (Table III).
We recently reported that oleic acid inhibited in a
dose-dependent manner gelatinase A activity (24, 25). In
this study, we first compared the effect of various fatty acids toward
gelatinases A and B and collagenase-1. By using a synthetic fluorogenic
substrate, the extent of MMP inhibition by fatty acids was related to
both fatty acid chain length and to the presence of at least one double carbon bond, as previously evidenced for neutrophil elastase (18). Similar gelatinase inhibition was obtained with
cis-unsaturated fatty acids and their trans
isomers, in contrast with the stringent conformational requirement
observed for neutrophil elastase inhibition (18).
The fatty acid carboxylate end group, a potential zinc-coordinating
group, was initially suspected to be the main driving force in fatty
acid-mediated gelatinase inhibition (24). However, the hydroxamate
derivative of oleic acid did not significantly improve its inhibitory
capacity (24), whereas 200- and 500-fold enhancements in inhibitory
capacity from carboxylate to hydroxamate inhibitors were previously
reported for matrilysin and collagenase-1, respectively (33, 34); in
addition, the alcohol analogue of oleic acid, a compound unable to
chelate the catalytic zinc, was also found nearly as effective as oleic
acid in inhibiting gelatinase A, suggesting that, at least for
gelatinases, zinc chelation was not the main determinant in enzyme
inhibition by fatty acids.
We further examined the possibility that, as for other hydrophobic
inhibitors, fatty acids could occupy the catalytic site of MMPs. The
catalytic domains of all MMPs display structural homology (35).
However, at the vicinity of the catalytic zinc, S1' invagination was
found to differ in size and shape among the various MMPs. Based on
x-ray crystallographic and homology modeling studies (36), MMPs may be
classified into two broad structural subgroups depending on the depth
of S1'. Gelatinases A and B possess a deep S1' pocket with leucine at
position 214; in collagenase-1, leucine residue is replaced by arginine
that partly occludes that enzyme subsite (37). Differences observed in
fatty acid inhibition efficiency between gelatinases and collagenase-1
could therefore result from the difference in the depth of the S1'
pocket. However, the marked difference in extent of inhibition between
full-length gelatinase A and The FN-II domain of gelatinases consists of three FN-II homology units
(11, 12). Comparison of secondary structures between FN-II modules of
gelatinase A and kringle domains in plasmin revealed a remarkable
concordance in the position of Fatty acids were more potent in inhibiting gelatinase activity on
protein substrate than on a synthetic substrate. A direct involvement
of the FN-II domain in binding of gelatinases A and B to gelatin and
native collagen has been extensively reported (14, 15, 42-45). The
stearic acid efficiency in inhibiting denatured type I collagen
degradation by gelatinase A was thus in agreement with the ability of
the FN-II domain to bind to the saturated acid. Computerized
morphometric analysis of skin tissue sections has shown that gelatinase
B degraded elastic fibers and fibrillin, whereas gelatinase A
preferentially degraded type III collagen fibers (32). By using a
similar experimental model, elaidic acid efficiently impaired
degradation of collagen and elastic fibers by gelatinase A and
gelatinase B, respectively, by both inhibiting gelatinase activity and
protecting fibers against hydrolysis. These data strongly suggest that
besides their direct inhibitory effect toward MMPs, fatty acids could
also bind and protect extracellular matrix macromolecules, as
demonstrated previously (40) for oleic acid protection of fibronectin
against neutrophil elastase or cathepsin G.
Hydrolysis of elastic fibers in the arterial walls is a critical step
in the development of atherosclerosis (46, 47). By using an ex
vivo model of aortic explants cultured with or without
monocyte/macrophage-like cells, Katsuda et al. (48) reported
a fragmentation of elastic fibers of the aortic explants by gelatinase
B. Targeted gene disruption of gelatinase B suppressed development of
abdominal aortic aneurysms in mice (49), emphasizing the role of this
MMP in such a degenerative condition associated with aging and
atherosclerosis. The gelatinase inhibition by fatty acids as we have
disclosed in an in vitro study and then confirmed in an
ex vivo model on human skin tissue (32) could thus explain, at least partly, the relationship between a Mediterranean-style diet,
in which olive oil is the main source of fat, and protection from
cardiovascular disease (50); it could also be involved in the reduction
of lung metastasis formation by colon carcinoma cells in athymic mice
after treatment by oleic acid (22).
In conclusion, our data provide in vitro and
ex vivo evidence that fatty acids could contribute to
regulate the extracellular matrix breakdown by inhibiting gelatinases A
and B. This inhibition was related to fatty acid chain length and to
the presence of double carbon bond(s) and was driven mainly by
interaction of fatty acid with the first FN-II module of gelatinase A.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin (7). The balance
between activated MMPs and TIMPs determines the overall MMP proteolytic
activity and consequently the extent of extracellular matrix
degradation. Local disruption of the MMP-TIMP balance can lead to
pathological degradative processes including rheumatoid arthritis,
atherosclerosis, tumor growth, and metastasis (8-10). MMPs are
multidomain enzymes containing propeptide, catalytic and, except
matrilysin (MMP-7), MMP-23, and endometase/matrilysin-2 (MMP-26),
hemopexin-like domains. Gelatinase A (MMP-2) and gelatinase B (MMP-9)
contain in addition three tandem copies of a 58-amino acid fibronectin
type II (FN-II) module (11, 12) inserted within their catalytic domain
(13). The basic fold of the FN-II modules is composed of a pair of
sheets that form a hydrophobic pocket accessible to solvent (13). The
FN-II repeats confer high affinity binding of these enzymes to gelatin
and insoluble elastin (14-16), a prerequisite for their efficient proteolysis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Val-191
Gln-364 gelatinase A
(pro-
FN-II gelatinase A or truncated progelatinase A), corresponding
to progelatinase A with FN-II domains deleted (15), was kindly provided
by Dr. G. Murphy (University of East Anglia, Norwich, UK). Human
recombinant TIMP-2 was a gift from Prof. Y. A. DeClerck
(Children's Hospital of Los Angeles, Los Angeles, CA). Acid-soluble
type I collagen was extracted and purified from guinea pig skin as
described previously (26).
80 °C. Serial
sections of 8 µm thickness were prepared with a cryostat microtome at
20 °C.
FN-II
progelatinases A were fully activated by incubating the proenzymes in
150 mM NaCl, 10 mM CaCl2, 50 mM Tris-HCl, pH 7.4, with 1 mM
p-aminophenylmercuric acetate (APMA) for 18 h at
4 °C. Active gelatinase B and collagenase-1 were obtained using 2 mM APMA in Tris buffer at 37 °C for 1 h. Full
activation of each enzyme was assessed by gelatin zymography or Western
blots. Each enzyme was further active site-titrated using a
standard preparation of TIMP-2 (28).
where vi is the rate of substrate hydrolysis
in the presence of inhibitor; vo is the rate in its
absence; and [E]o and [I]o are the
initial concentrations of enzyme and inhibitor, respectively.
(Eq. 1)
FN-II Gelatinases A--
For each enzyme, the mode of inhibition of
oleic acid was analyzed with the Grafit computer software, using the
graphical methods of either Dixon or Cornish-Bowden (see Ref. 31).
-D-glucopyranoside in water. Fatty
acid (20 µl of an 8 mM solution in Me2SO) was then injected. The process of spontaneous fatty acid adsorption on the
sensor chip was monitored. When the sensorgram reading began to level
out, the flow rate was briefly increased to 100 µl/min to suppress
the multiple lipid layers formed on the sensor chip surface. An
additional injection of 10 mM NaOH (10 µl) was performed
to regenerate the sensor chip and to reach a stable base line. To
assess the extent of coverage of the sensor chip surface by both fatty
acids, we injected 10 µl of 0.1 g/liter bovine serum albumin in 4.4 mM Na2HPO4 buffer, pH 7.4, 130 mM NaCl, 3 mM KCl. The amount of bovine serum
albumin bound to the sensor chip surface corresponded to 43 resonance
units, a value much lower than that typically obtained with a surface
fully coated with dimyristoyl phosphatidylcholine or palmitoyloleoyl
phosphatidylcholine. Binding experiments with coll 123 were performed
in the same buffer. After each cycle, the sensor chip was regenerated
by injecting 10 µl of 10 mM NaOH. These regeneration
conditions allowed to restore the base-line level observed before each
injection. The coll 123 binding kinetics were measured at six different
concentrations of analyte, i.e. 3.125, 6.25, 12.5, 25, 50, and 100 µM. We similarly studied oleic acid interactions
with the isolated FN-II modules of gelatinase A, i.e. coll
1, coll 2, or coll 3. The binding curves were analyzed using the
nonlinear data fitting software BIAevaluation to obtain the rate
constants of association (ka) and dissociation
(kd) and the equilibrium constants of dissociation (KD).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Inhibition of gelatinases A and B activities by fatty acids
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Fig. 1.
Inhibition of gelatinase A by
COOH-terminal end-modified oleic acids. 0-40
µM hydroxamate ( ) or alcohol (
) derivatives were
mixed with 200 pM gelatinase A for 15 min at 22 °C
before addition of 2 µM fluorogenic substrate in a 50 mM HEPES buffer, pH 7.5, containing 150 mM NaCl
and 5 mM CaCl2. Reaction mixtures were
incubated at 22 °C for 20 min before being stopped by 10 mM EDTA. The data obtained for each fatty acid were fitted
to Equation 1 by nonlinear regression, and lines represent
the best fit curves. Ordinates,
vi/vo, initial reaction rate in
the presence of fatty acid/initial reaction rate in its absence.
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Fig. 2.
Inhibition by fatty acids of
FN-II gelatinase A. 0-40 µM
oleic (
), elaidic (
), or stearic (
) acids were mixed with 200 pM truncated gelatinase A for 15 min at 22 °C before
addition of 2 µM fluorogenic substrate in a 50 mM HEPES buffer, pH 7.5, containing 150 mM NaCl
and 5 mM CaCl2. Reaction mixtures were
incubated at 22 °C for 20 min before being stopped by 10 mM EDTA. The data obtained for each fatty acid were fitted
to Equation 1 by nonlinear regression, and lines represent
the best fit curves. For vi/vo,
see legend to Fig. 1.
FN-II Gelatinases A
Inhibition by Oleic Acid--
The mode of inhibition of each enzyme by
oleic acid was evaluated. In both cases, Dixon plots (data not shown)
were consistent with oleic acid acting as either competitive or mixed
inhibitor, with Ki values graphically estimated to
1.6 and 26.3 µM for full-length and truncated gelatinases
A, respectively. Cornish-Bowden plot allowed us to distinguish between
these two possibilities (Fig. 3). The
intersection of lines above the abscissa in the Cornish-Bowden plot was
consistent with a mixed mode of inhibition of oleic acid against the
full-length gelatinase A (Fig. 3A), whereas it appeared to
act as a weak competitive inhibitor toward truncated gelatinase A (Fig.
3B).
View larger version (13K):
[in a new window]
Fig. 3.
Comparison of the inhibition of full-length
and FN-II gelatinases A by oleic acid.
Kinetic analyses were performed, as described under "Experimental
Procedures." The fluorogenic substrate concentrations were as
follows: 1.25 µM (
), 2 µM (
), and 4 µM (
). The lines were determined by linear
regression using the GrafitTM software. A,
Cornish Bowden plot showing inhibition of full-length gelatinase A by
oleic acid. B, Cornish Bowden plot showing inhibition of
FN-II gelatinase A by oleic acid.
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[in a new window]
Fig. 4.
Sensorgrams of recombinant FN-II domain (coll
123) binding to immobilized fatty acids. The trimodular protein
coll 123 was allowed to bind to oleic acid (A) or stearic
acid (B) and was examined by SPR, as described under
"Experimental Procedures." Six concentrations of coll 123, 3.125 µM (a), 6.25 µM (b),
12.5 µM (c), 25 µM
(d), 50 µM (e), and 100 µM (f) have been used. The asterisk
indicates the end of the association phase. One representative overlay
of sensograms among two independent experiments is shown.
RU, resonance units.
Kinetic and equilibrium constants of FN-II repeats of gelatinase A
with oleic or stearic acids
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Fig. 5.
Effect of FN-II modules on the inhibition of
gelatinase A by oleic acid. Oleic acid (5 µM) and
gelatinase A (200 pM) were preincubated for 15 min at
22 °C with molar fold excess (1-100-fold in comparison with
gelatinase A) of trimodular protein coll 123 (A) or isolated
FN-II modules, coll 1 ( ), coll 2 (
), or coll 3 (
)
(B); residual enzymatic activity was measured with
Mca-PLGL-(Dpa)-AR-NH2 (2 µM). The
vi/vo ratio was determined by
measuring the enzyme velocity in the absence (vo) or
the presence (vi) of oleic acid for each FN-II
module concentration. All experiments were done in duplicate.
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Fig. 6.
Inhibition by fatty acids of denatured type I
collagen hydrolysis by gelatinase A. 0-40 µM oleic
( ), elaidic (
), or stearic (
) acids were mixed with 1.6 nM gelatinase A for 15 min at 22 °C before addition of
3H-denatured type I collagen in a 50 mM
Tris-HCl buffer, pH 7.4, containing 150 mM NaCl and 10 mM CaCl2. Reaction mixtures were incubated at
37 °C for 20 h.
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[in a new window]
Fig. 7.
Ex vivo effect of elaidic
acid on ex vivo collagenolysis and elastolysis.
Human skin tissue sections were overlaid with buffer alone as control
(A and D), or containing 50 nM
gelatinases A or B (B and E), or 50 nM gelatinases A or B preincubated with 10 µM elaidic acid (C and
F) and then incubated 4 h at 37 °C before staining
by red sirius to visualize collagen fibers (A-C) or
polyphenolic catechin-fuschin for elastin fibers (D-F).
e, epidermis; d, dermis; v, vessel;
arrowhead, collagen bundles; arrow, mature
elastic fibers.
Inhibition of ex vivo collagenolytic and elastolytic activities by
elaidic acid (EA)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FN-II gelatinase A suggests that
inhibition must involve additional mechanism(s). For
FN-II
gelatinase A, the weak oleic acid inhibition was characterized as fully
competitive by different analyses, indicating that unsaturated fatty
acid could lie on to the enzyme-extended active site. In contrast, oleic acid behaved as a more potent partially competitive inhibitor toward full-length gelatinase A, pointing to other binding modes possibly implicating the FN-II domain of the enzyme.
sheets, suggesting an overall
structural homology between those structures (38). The kringles mediate
the binding of multidomain proteins to other proteins. The presence of
FN-II modules allows gelatinases A and B to bind and degrade insoluble
elastin, a highly hydrophobic extracellular matrix macromolecule (16,
39). Binding of long-chain fatty acids to kringles was found to
modulate plasmin activity toward both synthetic substrate and
fibrinogen (20, 21). The involvement of the FN-II modules of gelatinase
A in its inhibition by fatty acids was therefore investigated. Binding
of oleic acid to plasma fibronectin has been documented previously
(40). SPR experiments showed that coll 123, a recombinant protein
containing the three FN-II repeats of gelatinase A (14, 27), equally bound oleic and stearic acids, suggesting that such an interaction did
not involve the double carbon bond of the fatty acid but somehow contributed to the higher inhibition efficiency of unsaturated fatty
acids versus their saturated forms. The FN-II domain could act as a "helping hand" in terms of additional binding or orienting the fatty acid. According to the x-ray structure of progelatinase A
(13), the three FN-II modules turn outward to form a "three-pronged fishhook." The basic structure of each module is composed of a pair
of
sheets connected with a short
helix. The
sheets form a
hydrophobic pocket allowing substrate binding. Interestingly, although
the three FN-II modules were shown to act cooperatively to bind gelatin
(14, 27, 41), oleic acid specifically bound the first FN-II module of
gelatinase A. In addition, at a concentration as low as 15 nM, the first FN-II module, but not the second or third
FN-II modules, completely abolished gelatinase A inhibition by 5 µM oleic acid. Preliminary data (not shown) demonstrated that the first FN-II module, but not the second and third modules, served as a nucleating site to form oleic acid micromicelles. Altogether, these results suggest that more than 1 mole of oleic acid
could bind the first FN-II module of gelatinase A.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. Jacques Cohen (Faculty of Medicine, Reims, France) for assistance with the BIAcore X system. We thank Dr. Patrick Henriet (Institute of Cellular Pathology, Brussels, Belgium) for critical reading of this manuscript. Dr. Marie-Paule Mingeot (Faculty of Pharmacy, Brussels, Belgium) provided valuable advice about fatty acid chemistry.
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FOOTNOTES |
---|
* This work was supported in part by a CNRS grant, France, and grants from the Fondation pour la Recherche Médicale, ARERS, Ligue Régionale des Ardennes Contre le Cancer, Région Champagne-Ardennes, the Belgian Fonds de la Recherche Scientifique Médicale, Interuniversity Attraction Poles and Concerted Research Actions of the Université catholique de Louvain.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.
§ Recipient of a fellowship from Shiseido International France.
Postdoctoral fellow of the Christian de Duve Institute of
Cellular Pathology (Brussels, Belgium) and of the Association de la
Recherche Contre le Cancer, France.
Supported by International Center for Genetic
Engineering and Biotechnology Grant CRP/HUN98-03 and Hungarian
National Research Fund Grant T0022949.
¶¶ To whom correspondence should be addressed: CNRS FRE 2260, Faculté de Médecine, 51 rue Cognacq Jay, 51095 Reims Cedex, France. Tel.: 33-326-91-80-56; Fax: 33-326-91-80-55; E-mail: herve.emonard@univ-reims.fr.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M011664200
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ABBREVIATIONS |
---|
The abbreviations used are:
MMP, matrix
metalloproteinase;
TIMP, tissue inhibitor of metalloproteinases;
FN-II, fibronectin type II;
pro-Val-191
Gln-364 or pro
FN-II gelatinase A, progelatinase A mutant with amino acids
Val191-Gln364 deleted;
Mca, (7-methoxycoumarin-4-yl) acetyl;
Dpa, [3-(2',4'-dinitrophenyl)-l-2,3-diaminopropionyl];
APMA, p-aminophenylmercuric acetate;
Me2SO, dimethyl
sulfoxide;
SPR, surface plasmon resonance.
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