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J. Biol. Chem., Vol. 277, Issue 52, 50326-50332, December 27, 2002
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§,
,,, and
From the Medizinische Klinik III and the
** Institut für Biochemie der
Rheinisch-Westfälischen Technischen Hochschule Aachen,
Pauwelsstrasse 30, 52057 Aachen, Germany, the ¶ Biochemisches
Institut der Christian-Albrechts-Universität Kiel,
Olshausenstrasse 40, 24118 Kiel, Germany, and Amersham
Biosciences Europe, Munzinger Strasse 9, 79111 Freiburg, Germany
Received for publication, July 24, 2002, and in revised form, October 11, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Matrix metalloproteinases (MMPs) are involved in
the remodeling processes of the extracellular matrix and the basement
membrane. Most MMPs are composed of a regulatory, a catalytic, and a
hemopexin subunit. In many tumors the expression of MMP-9 correlates
with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain
(MMP-9-PEX) was expressed as a glutathione
S-transferase fusion protein in Escherichia
coli. After proteolytic cleavage, the isolated PEX domain was
purified by size exclusion chromatography. In a zymography assay,
MMP-9-PEX was able to inhibit MMP-9 activity. The association and
dissociation rates for the interaction of MMP-9-PEX with gelatin were
determined by plasmon resonance. From the measured rate constants, the
dissociation constant was calculated to be Kd = 2,4 × 10 Matrix metalloproteinases
(MMPs)1 are a family of zinc
metallo-endopeptidases secreted by cells. They are responsible for most of the turnover of matrix components. The MMPs are produced as zymogens
with a signal sequence and propeptide segment that has to be removed
during activation. The propeptide domain contains a conserved cysteine
that chelates the zinc in the active site. The gelatinases MMP-2 and
MMP-9 contain fibronectin type II domains that are inserted in the
middle of the catalytic domain, presumably to enhance substrate binding
(1). MMP-9 also has a collagen type V-like domain located between the
catalytic and the C-terminal hemopexin domain (Fig. 1). All but two
MMPs (MMP-7 and MMP-26) contain a regulatory subunit, the hemopexin
domain, separated from the catalytic domain by a variable hinge region
(2). This domain is thought to confer much of the substrate specificity to the MMPs (3). It is involved in activation as well as inhibition of
MMPs (3, 4) and may enhance substrate binding and specificity (5). The
hinge region also confers specificity to the MMPs either by direct
binding of the substrate or by setting the orientation of the hemopexin
domain and the catalytic domain (6). The hemopexin domain of MMP-2 is
known to bind heparin (7). Heparin has been shown to potentiate the
activities of some MMPs, and MMPs are often found associated with
heparin sulfate glycosaminoglycans on the cell surface (8). The overall
three-dimensional structure of the hemopexin domain is a four-bladed
propeller with a calcium binding site nestled in the folds (3). A
fragment of MMP-2, which comprises the C-terminal hemopexin domain, has
been shown to inhibit MMP-2 activity by preventing enzyme binding to
Since its discovery in 1974 (10), MMP-9 has been implicated in the
degradation of the extracellular matrix in a variety of physiological
and pathological processes. MMP-9 is important for the migration of
different cell types (e.g. leukocytes and cancer cells)
because of its ability to degrade basement membranes and components of
the extracellular matrix such as collagens, elastin, and aggrecan.
MMP-9 is present in large amounts in the granules of neutrophils and
plays an important role in inflammation diseases (11). Tumor cells
themselves induce MMP-9 production in the neighboring cells and use
MMP-9 for their migration when becoming invasive and metastatic (12,
13). In addition, MMP-9 is important for the formation of new blood
vessels essential for tumor growth and triggers the angiogenic switch
during carcinogenesis (14). MMP-9 is also responsible for the
processing of cytokines, e.g. pro-interleukin-1 Furthermore, MMPs participate in cell migration (18) and may be
expressed on the surface of cells, thus allowing for localized proteolysis (19, 20). MMP-9 is necessary for the migration of
Langerhans cells, on which it is expressed on the cell surface, and can
be inhibited by a broad spectrum inhibitor of MMPs (21).
In the present work we demonstrate that MMP-9 activity (in gelatin
zymography) can be inhibited by a recombinant MMP-9 hemopexin (PEX)
domain. Because this construct lacks the known gelatin binding region
of MMP-9, i.e. the fibronectin type II domain T2HU-2 within the catalytic domain, we assumed a second gelatin binding domain to be
present within the MMP-9 hemopexin domain. We demonstrate a high
affinity binding between MMP-9-PEX and gelatin. Furthermore, we show
that MMP-9-PEX inhibits the migration of MMP-9-expressing melanoma cells.
Material and Methods--
Culture reagents were from Sigma,
Invitrogen, or Sarstedt (Berlin, Germany). All chemicals were
purchased from Sigma (Germany), Amersham Biosciences, or ICN
(Meckenheim, Germany). An affinity-purified goat polyclonal antibody
raised against a peptide at the C terminus of murine MMP-9 (C-terminal
domain, M-17) was purchased from Santa Cruz Biotechnology. A sheep
polyclonal antibody against murine MMP-9 was a generous gift from G. Murphy (Cambridge, UK). A polyclonal rabbit GST antiserum, GST (Z-5)
horseradish peroxidase, was from Santa Cruz Biotechnology. Purified
MMP-2/MMP-9 gelatinases zymography standards (CC073) and purified
murine MMP-9 (100 kDa, CC069) were from Chemicon International
(Hofheim, Germany), and murine MMP-2 (444227) was from Calbiochem
(Schwalmbach, Germany). The BD BioCoat Matrigel invasion chamber was
purchased from BD Biosciences. Standard cloning procedures were
performed as described (22). A control peptide (H-GIPETKKLM-OH) of
>90% purity with a molecular weight of 1015.6 g/mol was produced by
Jerini AG (Berlin, Germany).
Construction of Murine MMP-9-PEX-GST Fusion Proteins--
GST
fusion proteins were constructed using primers based on the published
murine MMP-9 sequence (23). An antisense primer specific for the
C-terminal nucleotides 1558-2187 was engineered to contain an internal
XbaI site (5'-GTGGTCTCTAGAGACTTGCACTGCACGG-3'). A sense primer was engineered with an internal HindIII site
(5'-ACTGCGAAGCTTATGGAGGCCTCTACAGAG-3'). A full-length MMP-9
cDNA was a generous gift from S. Masure (Leuven, Belgium) and was
used as a PCR-template. Products were digested and ligated into
pGEX-5X-1 plasmid (Amersham Biosciences).
Expression and Purification--
The GST-MMP-9-PEX fusion
protein was expressed in bacteria (BL21).
Isopropyl-1-thio- Western Blot--
For antibody-based detection, buffer samples
were separated by SDS-polyacrylamide gel electrophoresis utilizing
separating gels of 10% polyacrylamide and stacking gels of 3%
polyacrylamide. Lanes were loaded with 2 µg of total protein each.
Following electrophoresis at 30 V, the proteins were transferred to
nitrocellulose membrane. Membrane transfer was monitored by staining
with Ponceau S. Blots were blocked with TBS-N (pH 7.6) containing 10%
BSA, 20 mM Tris, 137 mM NaCl, and 0.1% Nonidet
P40, and were washed and incubated with antibodies against the C
terminus of MMP-9 or GST (dilution 1:1000). Signals were visualized by
enhanced chemiluminescence according to the manufacturer's
instructions (Amersham Biosciences). Experiments were done in triplicate.
Circular Dichroism Spectroscopy--
Circular dichroism (CD)
measurements were carried out on an AVIV (Lakewood, NJ) 62DS CD
spectrometer equipped with a temperature control unit calibrated with a
0.1% aqueous solution of D-10-camphersulfonic acid
according to Chen and Yang (24). The spectral bandwidth was 1.5 nm. The
time constant ranged between 1 and 4 s, and the cell path length
was between 0.1 and 10 mm.
Calculation of Protein Concentrations--
Protein
concentrations were calculated from absorption spectra in the range of
240-320 nm using the method of Waxman et al. (25).
Mass Spectrometry--
The purity and correct molecular mass of
the protein were proven by ESI and MALDI-TOF MS. ~80 pmol of
MMP-9-PEX was injected into an LC-MS-coupled electrospray single
quadruple mass spectrometer (Ettan ESI ToF, Amersham Biosciences).
The protein was first desalted on a MicroTrap column (Michrom
BioResources) before spraying into the ionization chamber. For
determining the mass by MALDI-TOF (Ettan Maldi ToF Pro, Amersham
Biosciences), ~3 pmol of MMP-9-PEX was crystallized in an excess of
3,5-dimethoxy-4-hydroxycinnamic acid. Deconvolution of mass spectra was
performed using MagTran software
(www.ionsource.com/links/programs.htm).
Zymography--
MMP activity was assessed by gelatin zymography
following the methods described previously (26, 27). Lanes were loaded with a definite amount of recombinant protein or serum-free supernatant as indicated in Figs. 3 and 5. Samples were preincubated with 4-aminophenyl mercuric acetate (APMA) at a final concentration of 2 mM for 2 h at 37 °C. Proteins were run on
non-reducing SDS/polyacrylamide gel (10%) containing 1 mg/ml gelatin
either with or without 0.41 µg of recombinant hemopexin domain (or
albumin or peptide GIPETKKLM as indicated in the Fig. 3 legend) per
milliliter of gel. After electrophoresis in 25 mM Tris, 250 mM glycine, and 1% SDS, the gel was washed at room
temperature with 2.5% Triton X-100, 5 mM CaCl2, 50 mM Tris/HCL, pH 7.5, and incubated
again in the same buffer twice for 1 h. After rinsing the gel
extensively with six changes of distilled water, it was incubated
overnight at 37 °C in 5 mM CaCl2, 50 mM Tris/HCL, pH 7.5, followed by Coomassie Blue staining
(0.5% w/v) and destaining in methanol/acetic acid/water (10:10:80).
Gelatin zymography depicts MMPs as negatively staining bands of
gelatinolytic activity. Zymographic bands were scanned, and the optical
density was determined using the Bio-Rad Gel-Doc system. The activity
data of all samples analyzed by zymography were normalized by setting
the activity of a defined sample to 1.0 as indicated in the Fig. 3 legend.
Surface Plasmon Resonance Studies--
Gelatin was covalently
immobilized to a carboxymethyl dextran matrix (Fisons, Loughborough,
UK) at 10 µg/ml for 2 min in 10 mM sodium acetate buffer,
pH 3.9, as recommended by the manufacturer. Binding experiments were
performed at controlled temperature (15 °C) with 12 different
concentrations of the purified hemopexin domain using the IASYSTEM
(Fisons, Loughborough, UK) optical biosensor. Association was monitored
for at least 2 min, the sample was replaced by 10 mM sodium
acetate buffer, pH 3.9, and dissociation was monitored accordingly
before the cuvette was regenerated with phosphate buffer/1
M sodium chloride, pH 7.4, and equilibrated again with 10 mM acetate buffer, pH 3.9. Association and dissociation
affinograms were analyzed by nonlinear regression with the FASTfit
software (Fisons, Loughborough, UK), which uses the Marquardt-Levenburg algorithm for iterative data fitting.
Cells and Cell Treatment--
Human malignant melanoma A375
cells were obtained from ATCC (CRL-1619), and rat mesangial cells were
a generous gift from P. Mertens (RWTH, Aachen, Germany). Cells were
maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 5% (v/v) fetal-calf serum (FCS), 100 mg/liter streptomycin, and
60 mg/liter penicillin. Cells were grown at 37 °C in a
water-saturated atmosphere in air/CO2 (19:1). Cells were
assessed for the expression of MMP-2 and MMP-9 by zymography after
culturing under serum-free conditions for 24 h.
Boyden Chamber Experiments--
Transwell 0.64-cm2
filter inserts (8-µm pores) coated with a thin layer of Matrigel
basement membrane matrix (125 µg/cm2) were rehydrated
according to the customer's instructions either with or without
recombinant MMP-9-PEX (1-500 µg/ml) and placed in wells of 24-well
plates containing 0.5 ml of serum-free DMEM. Prior to seeding into the
Transwell inserts, cells were released using trypsin-EDTA and
sequentially rinsed with DMEM containing 10% FCS and serum-free DMEM.
The rinsed cells were resuspended in DMEM (5.0 × 104
cells/ml) and 250 µl were added to the upper chambers. DMEM
containing 5% FCS was used as chemoattractant. Chambers were incubated
for 22-24 h at 37 °C, 5% CO2 in a humidified tissue
culture incubator. The cells on the upper surface of the filters were
then removed using a cotton swab, and those remaining on the lower
surface of the filter were fixed with 10% methanol and stained with
hematoxylin and eosin.
The filters were rinsed with deionized water, dried, and examined using
light microscopy. The number of cells in five random optical fields
(400× magnification) from triplicate filters were averaged to obtain
the number of migrating cells.
Molecular Cloning, Purification, and Characterization of
MMP-9-PEX--
To obtain a soluble MMP-9 PEX domain we generated a GST
fusion protein. The cDNA encoding the PEX domain (MMP-9 amino acids Glu-20 to Pro-729) was inserted into pGEX downstream of the sequence encoding GST and a recognition site for factor Xa (Fig.
1).
The GST fusion protein was expressed in Escherichia coli.
Aliquots of bacterial lysates were subjected to SDS-PAGE. In Western blots the protein was detected with two different polyclonal antisera against the C terminus of murine MMP-9 (Fig.
2A, right, and
not shown) or GST (Fig. 2A, left). A protein band
representing the GST-MMP-9-PEX fusion protein was detectable with both
antisera and corresponds to a molecular mass of about 52 kDa. Bacterial lysates were then applied to a glutathione-Sepharose column. After digestion with factor Xa, a protein of 25 kDa could be detected using
the MMP-9 antiserum (Fig. 2A), and a protein of 27 kDa was detectable with a polyclonal antiserum against GST (open
arrow).
The recombinant protein was further purified by size exclusion
chromatography (Fig. 2B). The three fractions containing
detectable amounts of proteins (fractions 9, 20, and 34) were analyzed
by SDS-PAGE and Western blotting using a sheep polyclonal antiserum directed against the C-terminal part of MMP-9. Only fraction 34 contained considerable amounts of a protein with a molecular mass of 25 kDa (Fig. 2C).
We then identified and characterized MMP-9-PEX by mass spectrometry and
circular dichroism, respectively. The purified protein was
automatically desalted and analyzed on an Ettan LC-MS (Fig. 2D). The MS run presented in the lower
panel of Fig. 2D corresponds to the main peak in
the ion chromatogram (not shown). The determined experimental molecular
mass for PEX is 25,034 Da (MALDI and ESI MS) and exhibits an offset of
42.58 Da compared with theoretical mass (24,991.42 Da, M + H+), which could be in line with an additional acetyl group
(+ 42.03 Da). The CD spectrum of the purified recombinant protein in
the far UV is indicative of a protein in a folded state (Fig.
2E). Secondary structure analysis (28) of the far UV CD
spectrum reflects the Inhibition of MMP-9 Activity by MMP-9-PEX--
MMP-9-PEX itself
did not have any gelatinolytic activity in the zymography assay or the
MMP-9 ELISA (data not shown). Next, we examined whether MMP-9-PEX had
any influence on the enzymatic activity of MMP-9 toward gelatin.
Gelatin zymography was used to determine MMP-9 activity in a
semiquantitative assay. Zymography bands (Fig.
3A) were quantified by
densitometry (Fig. 3B). The activity of 0.2 ng of
recombinant murine MMP-9 was given the arbitrary value of 1 in standard
gelatin zymography (Fig. 3B). In the presence of 0.41 µg
of MMP-9-PEX per milliliter of gelatin gel, the activity of MMP-9 (0.2 ng and 1 ng) was reduced by 71 and 61%, respectively (Fig. 3,
A, second panel from the
left, and B). As negative controls, albumin and
the peptide GIPETKKLM, respectively, were polymerized into the gel
instead of MMP-9-PEX, and no effect on the MMP-9 activity was observed
(Fig. 3A, far right panel
and second panel from the
right). No effect of MMP-9-PEX could be detected on the activity of MMP-2 by zymography (data not shown).
Binding of MMP-9-PEX to Gelatin--
To specifically address the
ability of MMP-9-PEX to directly bind gelatin, we performed surface
plasmon resonance studies. Gelatin was immobilized to a carboxymethyl
dextran matrix as described under "Experimental Procedures."
Binding experiments were performed at a controlled temperature with 12 different concentrations of purified hemopexin protein using the
IASYSTEM optical biosensor. Association and dissociation affinograms
(Fig. 4) were analyzed by nonlinear
regression with FASTfit software (Marquardt-Levenburg algorithm for
iterative data). From these data, an association rate of
kon = 3.83 × 105
M Invasion of MMP-9-expressing Cells in the Presence of
MMP-9-PEX--
These results prompted us to examine whether the
recombinant MMP-9-PEX is able to interfere with the
MMP-dependent invasion of mammalian tumor cells. Human
malignant melanoma A375 cells expressing MMP-9 and MMP-2 and rat
mesangial cells expressing MMP-2 as demonstrated by gelatin zymography
(Fig. 5A) were cultured on
Matrigel-coated membranes in Boyden chambers under serum-free conditions. FCS-containing medium was used as a chemoattractant. Half
of the chambers were preincubated with recombinant MMP-9-PEX domain in
a concentration of 1, 10, 100, and 500 µg/ml (0.04-20 µM) media as indicated in Fig. 5B. A375
incubated with MMP-9-PEX showed decreased invasion and migration
through the coated filters compared with control cells. Incubation with
GIPETKKLM at 20 µM as an additional control did not alter
the migration pattern of A375 melanoma cells (Fig. 5B). Rat
mesangial cells, expressing MMP-2 exclusively, however, showed no
difference in migration in the presence of MMP-9-PEX (data not shown).
Thus, with respect to gelatinases, recombinant MMP-9-PEX inhibited
migration of predominantly MMP-9 expressing cells but not the migration
of exclusively MMP-2 expressing cells.
Reported here is the gelatin binding property of the C-terminal
hemopexin domain of murine gelatinase B, the first such report for any
of the MMPs. In 1995, Li et al. (3) described the crystal structure of porcine synovial collagenase (MMP-1) consisting of a
catalytic domain and a second domain of ~200 amino acids homologous to hemopexin, a heme-binding glycoprotein. The MMP-1 hemopexin domain
contains four units of four-stranded antiparallel Since 1992 it has been known that the gelatin binding of MMP-9 is
mediated by the second fibronectin-type II domain (T2HU-2), although
the presence of another gelatin binding site could not be excluded.
Although T2HU-2 mediates binding, it is not the rate-limiting step in
the hydrolysis of gelatin by the enzyme (29). Because Me2SO inhibits gelatin binding but not its
degradation (29), and the hemopexin structure depends on the joining of
the two cystein residues susceptible to Me2SO, this would
suggest that Me2SO inhibits gelatin binding by destroying
the hemopexin structure. Therefore, we postulated a second gelatin
binding site within the MMP-9 hemopexin domain.
To obtain high amounts of protein, the MMP-9 hemopexin domain was
expressed as a GST fusion protein in E. coli. A short
sequence of buffer amino acids at the N terminus of the protein
(GIPETKKLM) was attributed to the cloning procedure and most probably
does not influence the binding properties in a significant way. MMP-9 activity in gelatin zymography and the migration of MMP-9-producing tumor cells was not influenced by the peptide GIPETKKLM (Figs. 3 and
5). The precise mass of the hemopexin domain, including the buffer
amino acids, was measured to be 25,034 Da by electrospray mass
spectrometry (Fig. 2D), which is close to the estimated
molecular mass. The difference of 42.58 Da compared with the
theoretical mass of 24,991.42 Da (M + H+) might be
explained by acetylation, a rather common phenomenon also observed for
proteins from E. coli (30, 31). The difference between the
experimental and theoretical mass of the protein is 0.55 Da, which is
an acceptable error of the instrument. However there may also be
differences due to oxidation reactions occurring somewhere in the
protein. MMP-9-PEX contains three cysteines and two methionines, which
may all be candidates to undergo oxidation. Immunoreactivity with two
different polyclonal antibodies further verified the identity of the
purified protein, and circular dichroism spectra indicated the correct
folded protein. When the enhanced chemiluminesence (ECL) reaction was
allowed to proceed, a few bands of higher molecular weight turned up on
the Western blots, which might represent cross-linked multimeric forms
of the recombinant protein.
Secondary structure analysis (28) of the far UV CD spectrum reflects
the For MMP-2 it could already be shown that the C-terminal domain exhibits
strong binding properties for fibronectin and heparin and that binding
depends on the structural Ca2+ but not Zn2+ ion
in this domain (7). In these studies, however, the influence of the
hemopexin domain on enzymatic activity was not tested. By gelatin
zymography, an easily practicable in vitro assay to determine gelatinase activity, we demonstrated a reduction of MMP-9
activity as a consequence of the presence of recombinant MMP-9-PEX by
60-71%. A greater reduction in the case of smaller amounts of MMP-9
may be explained by reaching the nonlinear range of zymography and
densitometry when loading high amounts like 1 ng of MMP-9 protein (Fig.
3, A and B).
To explain a reduction of MMP-9 activity in the presence of recombinant
MMP-9-PEX, we carried out a comprehensive study to determine the
kinetic parameters for the binding of MMP-9-PEX to gelatin. By plasmon
resonance, we measured a dissociation constant of Kd = 2.64 × 10 We extended our characterization of MMP-9-PEX with respect to its
impact on cell migration and found that migration of malignant human
melanoma cells (A375) was reduced in the presence of recombinant MMP-9-PEX (Fig. 5). Because MMP-9-PEX was able to inhibit the migration
of MMP-9-expressing A375 cells but not of mesangial cells known to
express MMP-2 (but no detectable amounts of MMP-9), we speculate that
MMP-9-PEX interferes specifically with the MMP-9-dependent migration. MMP-9 is not crucial for only migration and metastasis but
also for hematopoiesis. Recently, it has been published (38) that
recruitment of stem and progenitor cells from the bone marrow requires
MMP-9-mediated release of serum Kit ligand (sKitL). The relative
deficiency of serum Kit ligand at baseline or after myelosuppression in
MMP-9 We have presented MMP-9-PEX as an inhibitor of MMP-9
activity in vitro and in cell culture, which raises many
questions and perspectives for further investigations. One important
issue is the identification of the gelatin binding epitope within the
MMP-9 hemopexin domain. In addition, the recombinant MMP-9-PEX offers the possibility for determining other substrate specificities. Another
important question relates to the effects of MMP-9-PEX in
vivo. Because the catalytically inactive MMP-9-PEX binds with high
affinity to gelatin, a substrate of MMP-9, we consider MMP-9-PEX as a
candidate to antagonize MMP-9-activity in the context of tumor cell
invasion and metastasis. Recombinant MMP-9-PEX may be used as a new
approach in the treatment of diseases with high MMP-9 activity such as
melanomas or colorectal carcinomas.
8 M, demonstrating a high
affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments
the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma
cells secreting high amounts of MMP-9 in a dose-dependent
manner. These data demonstrate for the first time that the hemopexin
domain of MMP-9 has a high affinity binding site for gelatin, and the
particular recombinant domain is able to block MMP-9 activity and tumor
cell invasion. Because MMP-9 plays an important role in metastasis,
this antagonistic effect may be utilized to design MMP inhibition-based
cancer therapy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3 integrin and blocks cell
surface collagenolytic activity (9).
and
pro-tumor necrosis factor- into their active forms (15, 16). In
hepatocellular carcinomas the expression of MMP-9 is correlated with
local tumor growth, invasion, and intrahepatic metastases (17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (IPTG)-induced log phase bacterial cultures containing these constructs were shock frozen
and sonicated several times. After lysis, the insoluble parts were
removed by centrifugation. GST fusion proteins were purified on
Sepharose 4B-coupled glutathione beads (Amersham Biosciences). Beads
were washed extensively with PBS, immobilized fusion proteins were
digested with 5 mM factor Xa in 50 mM Tris/HCl,
pH8, and the cleavage product was eluted with PBS according to the
supplier's instructions. The amino acids GIPETKKLM at the neo-N
terminus after digestion resulted from cloning procedures and the
factor Xa recognition site. Fractions were pooled, concentrated, and subjected to size exclusion chromatography, which was performed with an
equilibrated Superdex 75 (16/60) column (Amersham Biosciences) at a
constant flow rate of 1 ml/min. The column was calibrated using a
mixture of four proteins of known molecular mass, i.e. bovine serum albumin (69 kDa), ovalbumin (46 kDa), myoglobin (17 kDa),
and aprotinin (6 kDa). The column was equilibrated with PBS and loaded
with 1.0 ml of protein solution. 3-ml fractions were collected and
analyzed by SDS-PAGE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the functional
domains of MMP-9. The amino acids (one-letter code) corresponding
to the N- and C-terminal borders of the MMP-9-PEX domain after the GST
fusion protein are indicated. *, factor Xa cleavage site.
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Fig. 2.
Purification and characterization of
MMP-9-PEX. A, for Western blot analysis 2-µg
aliquots of bacterial lysates were separated by SDS-PAGE before ( 
)
and after (+) digestion with factor Xa. The blots were
stained with a sheep polyclonal antiserum against MMP-9
(right) or a rabbit polyclonal antiserum against GST
(left). GST-MMP-9-PEX has a molecular mass of ~52 kDa,
MMP-9-PEX has one of ~25 kDa, and the GST protein has one of 27 kDa.
B, size exclusion chromatography elution profile of the
recombinant MMP-9 hemopexin domain monitored at 280 nm. Absorption is
given in arbitrary units (AU). C, aliquots of the
different fractions were separated by SDS-PAGE. A Western blot was
stained with a polyclonal antiserum against MMP-9 (left).
Coomassie staining of a gel run in parallel is shown on the
right. Only fraction 34 contains significant amounts of
MMP-9-PEX protein. D, the MMP-9-PEX protein was
automatically desalted and analyzed on an Ettan LC-MS. The MS run is
shown in the lower panel. The deconvoluted MS
spectrum in the upper panel exhibits the correct mass of the protein,
which may be modified by an acetylation (+ 42.03 Da). Deconvolution of
the mass spectrum was performed using MagTran software. E,
CD spectrum of the recombinant MMP-9 hemopexin domain in the far
UV.
-sheet character of the protein
(-helix = 21%, -sheet = 35%, turn = 17%), which
is typical for a hemopexin domain.
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Fig. 3.
Inhibition of MMP-9 activity by
MMP-9-PEX. A, gelatin zymography was used to
demonstrate the inhibition of MMP-9 activity by MMP-9-hemopexin domain.
0.2 and 1.0 ng of recombinant MMP-9 were separated by SDS-PAGE in a gel
with (second panel from the left) or without (far
left panel) 0.41 µg of MMP-9-PEX or in the presence of
0.41 µg of albumin (second panel from the right) or 20 ng
of peptide GIPETKKLM (far right panel) per milliliter of gel
as additional controls. B, bands were quantified by
densitometry. For each zymography gel run, the activity of 0.2 ng of
MMP-9 in standard gelatin zymography was given the arbitrary value of
1.
1 s1 and a dissociation rate
of koff = 1.01 × 102
s1 could be deduced. With these values, the dissociation
constant of Kd = 2,64 × 108
M was calculated (Fig. 4, and Table
I). Evidently the MMP-9-PEX domain is
able to bind to gelatin.
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Fig. 4.
Kinetics of binding of MMP-9-PEX to
gelatin. Association of MMP-9-PEX to immobilized gelatin led to
the increase of the resonance angle as a function of time. The
association phase was recorded for different nanomolar MMP-9-PEX
concentrations. The decrease of the resonance angle indicates the
dissociation phase resulting from replacement of the MMP-9-PEX solution
from the solid phase by triplicate washing with 10 mM
sodium acetate buffer, pH 3.9. The data obtained from the curves
allowed the determination of the dissociation rate constant
koff from the ordinate intercept and the
association rate constant kon from the slope of
the graph (see Table I). All kinetic experiments were performed at a
controlled temperature (25 °C).
Kinetic (kon, association; koff, dissociation)
and equilibrium (Kd, dissociation) constants for the binding of
the MMP-9 hemopexin domain to gelatin
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Fig. 5.
MMP-9-PEX inhibits invasion of A375 melanoma
cells. A, zymography of the serum free supernatants of
human malignant A375 melanoma cells, which secrete MMP-2 and MMP-9, and
rat mesangial cells, which secrete MMP-2 only. An MMP-standard for
human MMP-2 and MMP-9 is shown (left lane). B,
invasion and migration of A375 melanoma cells were measured in a Boyden
chamber in the presence of MMP-9-PEX at different concentrations as
indicated (500 µg/ml equivalent to 20 µM). Cells were
cultured with or without MMP-9-PEX for 24 h. For control, cells
were incubated with GIPETKKLM (20 µg/ml equivalent to 20 µM) as indicated. Additional control cells were incubated
with equal amounts of phosphate-buffered saline (0). Cells
on the lower surface of the membranes were counted after staining with
hematoxylin and eosin. y axis, number of cells in [%].
Each bar represents the mean ±S.D. of three chambers. This
is a representative result of three experiments. *, p < 0.01 compared with control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets stabilized
on its 4-fold axis by a cation (3). The domain constitutes a
four-bladed -propeller structure and was assumed to control the
specificity of MMPs, affecting both substrate and inhibitor binding
(3). Two cysteines at either end are conserved in all hemopexin domains
of the MMP family.
-sheet character of the protein (-helix = 21%, -sheet = 35%, turn = 17%). Inspection of the x-ray
structures of the hemopexin domains of MMP-2 and MMP-13 revealed
-sheets only and no helical secondary structure (32, 33). The
overestimation of the calculated -helical content of the MMP-9-PEX
is due to the negative band around 228 nm, which might originate from
aromatic side chain contributions (34).
8 M. The
Kd values of TIMP-1 for the MMP-9 latent and active
species are 35 and 23.9 nM for the high affinity site (35). That means that the binding affinity of gelatin to MMP-9-PEX is as high
as the binding of MMP-9 to TIMP-1. These results indicated that the
association of MMP-9-PEX and gelatin was rather rapid (kon ~3.8 × 105
M1 s1). Furthermore, the
dissociation of the complex was quite slow (koff
~102 s1), resulting in a very effective
binding. Collectively, these findings support the importance of the
C-terminal domain of MMP-9 for an efficient binding to the substrate
gelatin. Others (36) have also characterized the binding parameters of
pro-MMP-9 and demonstrated that the proenzyme binds with high affinity
(Kd ~20-30 nM) to the surface of a
variety of cell types, and the Kd values of the
2(IV) chain of collagen IV for pro-MMP-9 were calculated to be ~45
nM. However, because a pro-MMP-9-TIMP-1 complex and MMP-9
binds to 2(IV), the authors suggested that neither the C-terminal
(binds to TIMP-1) nor the N-terminal domain of MMP-9 (which is
processed during activation) is directly involved in 2(IV) binding,
but the domain was not identified further. From our results, we
concluded that the C-terminal domain of MMP-9, although known to bind
TIMP-1 with high affinity (35, 37), is also able to bind to gelatin
with a comparable high affinity.
/ mice strongly suggests that MMP-9 plays a
physiological role in releasing sKitL, setting up the stage for
hematopoietic reconstitution. Thus, inhibition of MMP-9
(e.g. by MMP-9-PEX) may provide a novel mechanism for
regulating hematopoiesis in sKitL-dependent
myeloproliferative disorders.
| |
ACKNOWLEDGEMENT |
|---|
We thank M. Roderfeld for critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 542.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.: 49-241-8089507; Fax: 49-241-8082455; E-mail: eroeb@ukaachen.de.
Published, JBC Papers in Press, October 15, 2002, DOI 10.1074/jbc.M207446200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MMP, matrix metalloproteinase; CD, circular dichroism; DMEM, Dulbecco's modified Eagle's medium; ESI, electrospray ionization; FCS, fetal calf serum; GST, glutathione S-transferase; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; PEX, hemopexin.
| |
REFERENCES |
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RESULTS
DISCUSSION
REFERENCES
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