From the
Institute for Physiological Chemistry,
Münster University Hospital, 48149 Münster, Germany and the
¶Trust Centre for Cell-Matrix Research, School of
Biological Sciences, University of Manchester, Manchester M13 9PT, United
Kingdom
Received for publication, February 21, 2003 , and in revised form, April 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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Laminins are major constituents of basement membranes
(9,
10), and their isoform
compositions vary with the tissue. The basement membrane of the myotendinous
junction is enriched in laminin-2
(11). Merely differing in its
chain, laminin-2 has the
1 and
1 chains in common with
laminin-1. The networks of both laminin and type IV collagen, together with
smaller proteins such as nidogen and proteoglycans, form the molecular basis
of the basement membrane. The laminin meshwork of the basement membrane allows
integrin-mediated anchorage of muscle fibers and other cells
(9,
10). In addition, basement
membranes separate the connective tissue from any other tissue. Only certain
cells such as leukocytes and invasively growing tumor cells are able to
penetrate through the basement membrane. Other processes such as wound healing
require cell movement along the basement membrane. Cell migration through and
along the basement membrane is mediated by laminin-binding integrins
(5,
12).
Some snake venoms contain components that are selectively directed against integrins. These so-called disintegrins interfere with the ability of integrins to bind to their cognate ECM ligands. Hence, cell/matrix interactions are disrupted, and tissue integrity is destroyed. However, snake venoms are known to exert several different effects on snakebite victims, among which are failure of the cardiovascular system and damages to muscle and neural tissue. In addition to disintegrins, these pleiotropic effects are caused by a variety of venom proteins. Being abundant in most snake venoms, phospholipase A2 is mainly responsible for myotoxic effects (13, 14). Various proteases of snake venoms specifically cleave blood-clotting factors or components of the ECM, thus resulting in bleeding dysfunctions and tissue necrosis (1517).
Most disintegrins known to date contain an RGD sequence mimicking the
ligands for the platelet IIb
3 and other
RGD-dependent integrins (18),
which cause failure of
IIb
3
integrin-mediated platelet aggregation and blood clotting. Moreover,
disintegrins are not limited to snake venoms, but are also found as domains in
ADAM (a disintegrin and a
metalloproteinase) family members, multidomain proteins that are
abundant in a wide range of animal species
(15,
19). ADAM proteins play
important roles in various biological processes such as fertilization and
cell/cell interactions and proteolytic processing
(19). Whereas few ADAM
proteins interact with integrins
(1921),
snake venom disintegrins interfering with integrin/laminin interaction have
not been described so far.
From the venom of Vipera lebetina, we have purified and
characterized two inhibitors that target laminin-binding integrins. One of
them is the recently described lebein. Although it has been proposed to be a
disintegrin for RGD-dependent integrins
(22), we show in this work
that lebein or lebein-1, as we refer to it, also avidly binds to the
laminin-binding 1 integrins in an RGD-independent manner. In
addition, we isolated a new disintegrin (lebein-2) with homology to lebein-1
and similar binding specificity. The interaction of both inhibitors with
laminin-binding
1 integrins both in vitro and in
in vivo cell attachment assays demonstrates their potential to
manipulate integrin-dependent cell/laminin interactions.
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MATERIALS AND METHODS |
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The construct pUC-hygMT-6X1-Fos, coding for the
6
integrin ectodomain splice variant X1 (amino acids 1989 of the
published sequence) (23), was
generated by isolating the HindIII- and XbaI-flanked cDNA
fragment encoding amino acids 1938 of the
6 integrin
ectodomain from pRC-CMV-
6X1 (kindly provided by Dr. A. Sonnenberg, The
Netherlands Cancer Institute, Amsterdam, The Netherlands) and by synthesizing
an XbaI- and SalI-flanked cDNA fragment encoding amino acids
939989 of the
6 integrin ectodomain by standard PCR.
Taking advantage of the SalI site, the two cDNA fragments were
directionally inserted into the pUC-hygMT-
3-Fos vector
(7) from which the
3 integrin ectodomain-coding cDNA had been removed.
The vector pUC-hygMT-1-Jun was generated previously
(7). Together with the
pUC-hygMT-
1-Jun vector, the vectors pUC-hygMT-
7X2-Fos and
pUC-hygMT-
6X1-Fos were cotransfected into Drosophila
Schneider's cells, and single cell clones were established as described
previously (7,
24). Briefly, positive clones
secreting
6
1 and
7
1 integrins were detected in a sandwich
ELISA using monoclonal antibody GoH3 (kindly provided by Dr. A. Sonnenberg)
and affinity-purified anti-Fos peptide antiserum, respectively, as capturing
antibodies.
Clones G42 and
ASH6, producing soluble 7X2
1 and
6X1
1 integrins, respectively, were grown as
described previously (7). Both
integrins were isolated by affinity chromatography with the immobilized
cell-binding domain of invasin
(7). Protein concentration and
purity were determined by the BCA assay (Pierce) and SDS-PAGE,
respectively.
Soluble 2
1 and
3
1 integrins were generated and isolated as
described previously (7,
24). The generation and
isolation of soluble
1
1 integrin will be
described
elsewhere.2
Integrin Inhibition ELISATo screen for inhibition of
integrin-mediated laminin binding, microtiter plates were coated with 6
µg/ml murine laminin-1 (kindly provided by Dr. R. Timpl, Max Planck
Institute for Biochemistry, Martinsried, Germany) or 10 µg/ml human
laminin-2/4 (Merosin, Invitrogen, Karlsruhe, Germany) in Tris-buffered saline
(TBS), pH 7.4, containing 1 mM MgCl2
(TBS/MgCl2 buffer) overnight at 4 °C. Nonspecific
protein-binding sites were blocked with 1% heat-denatured bovine serum albumin
(BSA) in TBS/MgCl2 buffer for 2 h at room temperature. Together
with protease inhibitors, phenylmethylsulfonyl fluoride, and
1,10-phenanthroline (each at 1 mM) and aprotinin, leupeptin, and
pepstatin (each at 3 µg/ml), soluble 7
1
integrin was added at a concentration of 15 nM in 1% heat-denatured
BSA in TBS, pH 7.4, containing 2 mM MgCl2 and 1
mM MnCl2, either without any supplements (positive
control) or with 10 mM EDTA (nonspecific binding, negative control)
or with snake venom solutions of 2 mg/ml. Lyophilized snake venoms were
purchased from the Berchtesgadner Schlangenfarm (Berchtesgaden, Germany) and
Sigma. After 2 h of incubation at room temperature, wells were washed twice
with 50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM
MgCl2, and 1 mM MnCl2. Bound
7
1 integrin was fixed with 2.5%
glutaraldehyde in the same buffer for 10 min at room temperature and detected
in an ELISA procedure using rabbit antiserum directed against the human
1 integrin subunit (1: 400; kind gift of Dr. K. Kühn,
Max Planck Institute for Biochemistry) and an alkaline phosphatase-conjugated
anti-rabbit IgG antibody (1: 600; Sigma) as described previously
(7). Nonspecific binding was
measured in the presence of 10 mm EDTA and subtracted from all values. For the
calculation of the relative inhibitory activity, binding signals in the
presence of inhibitor were normalized to the non-inhibited control.
Isolation of Lebein-1 and Lebein-2Lyophilized venom of
V. lebetina was dissolved in 20 mM sodium phosphate, pH
6.5, 50 mM sodium chloride, and 1 mM EDTA and separated
by gel filtration on a Superose 6 column (Amersham Biosciences AB, Uppsala,
Sweden) in the same buffer. The fractions containing the
7
1 integrin-inhibiting fractions were
pooled; diluted with 20 mM MES, pH 6.0; and loaded onto a Mono S
column (Amersham Biosciences AB). The
7
1
integrin inhibitory activities of lebein-2 and lebein-1 were eluted in linear
sodium chloride gradients at 80 and 140 mM, respectively. The
eluate fractions were individually pooled and separated on a C8
reversed-phase column (Nucleosil, Macherey Nagel) in a linear gradient from
0.1% trifluoroacetic acid in water to 80% acetonitrile in 0.08%
trifluoroacetic acid/water. Lebein-1 and lebein-2 were eluted as individual
peaks, lyophilized, and dissolved in water. Protein concentration was
determined by the BCA assay. Purity was proven by SDS-PAGE.
Far-Western BlottingAfter separation by SDS-PAGE, snake
venom proteins were blotted onto nitrocellulose membrane (Schleicher &
Schüll). After nonspecific binding had been blocked with 1%
heat-denatured BSA in TBS/MgCl2 buffer, 50 nM soluble
7
1 integrin was added in the same solution
supplemented with 1 mM MnCl2 for 2 h at room
temperature. The membrane was washed twice with 50 mM HEPES, pH
7.5, 150 mM NaCl, 2 mM MgCl2, and 1
mM MnCl2. Bound
7
1
integrin was fixed with 2.5% glutaraldehyde for 10 min and detected by rabbit
antiserum against the human
1 integrin subunit (1:400) and
subsequently by an alkaline phosphatase-conjugated anti-rabbit IgG secondary
antibody (1:700). Both antibodies were applied in 1% heat-denatured BSA in
TBS/MgCl2 buffer, followed by three washes with
TBS/MgCl2 buffer. The overlay blot was developed with
5-bromo-4-chloro-3-indolyl phosphate /nitro blue tetrazolium solution
(Sigma).
Binding of Integrins to Lebein-1 and Lebein-2Lebein-1 and
lebein-2 (each at 3 µg/ml in 20 mM sodium phosphate buffer, pH
7.0), the CB4 fragment of human type IV collagen (10 µg/ml in
TBS/MgCl2 buffer), murine laminin-1 (10 µg/ml in
TBS/MgCl2 buffer), human laminin-5 (10 µg/ml in
TBS/MgCl2 buffer), and bovine type I collagen (20 µg/ml in 0.1
M acetic acid) were immobilized onto microtiter plates overnight at
4 °C. After a blocking step, soluble integrins were added at 50
nM in 1% heat-denatured BSA in TBS/MgCl2 buffer in the
presence of either 1 mM MnCl2 or 10 mM EDTA.
2
1 integrin was further activated by
addition of the
1 integrin-activating antibody 9EG7 (kindly
provided by Dr. D. Vestweber, Center for Molecular Biology of Inflammation,
Münster, Germany) (25).
Bound integrin was fixed and detected in an ELISA-type procedure as described
before.
RGD Peptide Inhibition AssayLebein-1 and lebein-2 (each at
3 µg/ml in 20 mM sodium phosphate buffer, pH 7.0) and murine
laminin-1 (at 6 µg/ml in TBS/MgCl2 buffer) were coated onto
microtiter plates overnight at 4 °C. After washing and blockage with 1%
BSA in TBS/MgCl2 buffer, soluble
7
1 integrin at 10.4 µg/ml (for lebein-1
and lebein-2 binding) and 1.3 µg/ml (for laminin binding) was added to the
wells in the presence of peptides GRGDS, GRGES, and GRDGS (Bachem Biochemica
GmbH, Heidelberg, Germany) for 2 h. Bound integrin was detected as described
above.
Cell Adhesion Inhibition TestsMicrotiter plates were coated with fibronectin (gift of Dr. M. Humphries, University of Manchester) and murine laminin-1 at 10 and 40 µg/ml, respectively, overnight at 4 °C. After blocking for 6 h with 1% BSA in water, 100 µl of a C2C12 cell suspension (0.8 x 106 cells/ml) in Dulbecco's modified Eagle's medium without fetal calf serum and containing a 1:3 serial dilution of lebein-1 and lebein-2 were added to the wells and incubated for 45 min at 37 °C in a humidified 5% CO2 incubator. After the supernatant had carefully been discarded, attached cells were washed with phosphate-buffered saline and fixed for 10 min at room temperature with 70% ethanol. After air-drying, cells were stained with 0.1% crystal violet. For 100% binding values, lebein-1 and lebein-2 were replaced by an equal volume of Dulbecco's modified Eagle's medium. Three experiments with duplicate measurements were carried out.
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RESULTS |
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The biological activity of the soluble
7
1 integrin was tested qualitatively and
quantitatively on different ECM substrates. As shown in
Fig. 1B,
7
1 integrin showed a higher binding signal
for laminin-1 than for laminin-2/4. Laminin-2 is the most abundant laminin
isoform of basement membranes in muscle tissue. In contrast, laminin-5 was not
recognized. As expected from the purification protocol, invasin, a surface
protein of Yersinia bacteria, also interacted with soluble
7
1 integrin
(Fig. 1B). Like other
integrins,
7
1 integrin is activated for
ligand binding by Mn2+ ions. However, unlike other
integrins, this activation seems sufficient, as addition of the
integrin-activating antibody 9EG7, directed against the
1
integrin subunit (25), did not
increase the binding further. In the presence of EDTA, binding to any ligand
was completely abolished, thus confirming the dependence of
7
1 integrin on divalent cations
(Fig. 1B).
Binding of soluble 7X2
1 integrin to
immobilized laminin-1 and laminin-2/4 was further studied by an ELISA-type
titration assay, from which apparent Kd values
could be calculated according to Heyn and Weischet
(26). As shown in
Fig. 1C, specific
binding of soluble
7
1 integrin to the
immobilized laminin isoforms reached saturation. In the presence of
Mn2+ ions,
7
1
integrin bound more avidly to laminin-1 (Kd = 0.2
nM) than to laminin-2/4 (Kd = 2.3
nM). Addition of the activating antibody 9EG7 did not shift the
titration curves to lower
7
1 integrin
concentrations and did not alter its apparent affinity constants
significantly. However, Ca2+ ions reduced
7
1 integrin binding to both laminin-1 and
laminin-2/4, thus increasing the apparent Kd
values by
15-fold (Fig.
1C).
Screening Various Snake Venoms for Their Capability to Interfere
with 7
1 Integrin
Binding to Laminin-1Playing a key role in skeletal muscle,
7
1 integrin conceivably is a target for
myotoxic snake venoms. Because of its high affinity for
7
1 integrin, we chose laminin-1 as its
interaction partner to search for a snake venom inhibitor to this interaction
(Table I). Crude snake venoms
and soluble
7
1 integrin were added to
laminin-1-coated microtiter plates. Protease inhibitors were also supplemented
to avoid proteolytic degradation of integrin or its ligand by the numerous
proteases that are abundant in snake venoms. Of 33 snake venoms tested that
may cause severe muscular dysfunctions, such as venoms of the Elapidae,
Viperidae, and Crotalidae families, only the venom of V. lebetina
showed a drastic abolition of
7
1 integrin
binding to laminin-1 (Table I). Titration of the V. lebetina venom demonstrated that it suppressed
7
1 integrin binding entirely to both
laminin-1 and laminin-2/4 in a dose-dependent manner
(Fig. 2).
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Purification of the
7
1 Integrin
Inhibitory Activity of the V. lebetina VenomTo purify the
7
1 integrin inhibitor, the venom was
separated by gel filtration, ion-exchange chromatography, and reversed-phase
chromatography. From the gel filtration column, the
7
1 integrin inhibitory activity was eluted
in a single peak (Fig.
3A, gray bar). However, further purification on
a Mono S column separated the inhibitory activity into two different
fractions, called MS-I and MS-II (Fig.
3B). Each of these two inhibitory peaks, MS-I and MS-II,
showed a characteristic elution profile upon reversed-phase chromatography
(Fig. 4, A and
B). Fraction MS-I could not further be separated by
reversed-phase chromatography and contained only one inhibitor that eluted as
a single peak (Fig.
4A).
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After reversed-phase chromatography, the major inhibitory peak of fraction
MS-II (Fig. 4B) was
identified as lebein, a recently discovered disintegrin
(22), which we refer to as
lebein-1. Edman degradation of lebein-1 revealed two staggered N-terminal
sequences, MNGSNPXXD and NGSNPXXD, at a 2:1 ratio. Except
for the initial methionine residues, this sequence is identical to the
published primary sequence of lebein (Swiss-Prot accession number P83253
[GenBank]
)
(22). Furthermore, mass
spectrometry determined its mass to be 14,083 Da and proved its identity to
lebein. Upon SDS-PAGE, the heterodimeric lebein-1 showed an apparent molecular
mass of 18 kDa. After reduction, it was separated by SDS-PAGE into two highly
homologous subunits, and
, with apparent molecular masses of
14 kDa (Fig. 5A).
The difference in molecular mass determined by SDS-PAGE and mass spectrometry
is likely due to the fact that the molecular masses of proteins below 20 kDa
show a nonlinear behavior on polyacrylamide gels. Furthermore, the
nonlinearity of the apparent molecular masses of the heterodimeric protein and
its subunits upon SDS-PAGE can be explained by the fact that the Stoke radius
of the two closely associated subunits within the nonreduced heterodimeric
disintegrin is likely to be smaller than the sum of the Stoke radii of the
individual unfolded subunits after reductive cleavage of disulfide
bridges.
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The inhibitory activity of fraction MS-I
(Fig. 4A) was
identified as a novel protein from V. lebetina venom. Despite its
high sequence identity, its N-terminal sequence (MNSANPXXDDI),
especially residues alanine and isoleucine in positions 4 and 11,
respectively, clearly discriminated this inhibitor from lebein-1. Furthermore,
its molecular mass of 14,735 Da, as determined by mass spectrometry, differed
from that of lebein-1. The different molecular mass of lebein-2 was also
observed upon SDS-PAGE (Fig.
5A). Upon reduction, the novel V. lebetina
inhibitor with an apparent molecular mass of 20 kDa dissociated into two
subunit chains ( and
) with apparent molecular masses of 15 and 7
kDa, respectively (Fig.
5A). Edman degradation revealed that its
subunit
was N-terminally blocked, whereas the N-terminal amino acid sequence of the
subunit was proven to be identical to the N terminus of the nonreduced
protein. This amino acid sequence has not been published yet. Because of its
sequence homology to lebein and similar binding affinities, we propose the
name lebein-2 for it. Thus, lebein-2 is a 14,735-Da heterodimeric protein
consisting of two subunits,
and
, the latter one of which has
the N-terminal amino acid sequence MNSANPXXDDI.
Lebein-1 and Lebein-2 are the
7
1 Integrin-binding
Components of V. lebetina Snake VenomA far-Western blot was
established to test the fractions of V. lebetina snake venom for
7
1 integrin-binding components
(Fig. 5B). After
electrophoretic separation, snake venom components were transferred onto a
nitrocellulose membrane. Soluble
7
1
integrin was allowed to bind to the blotted snake venom proteins and detected
immunologically. The strongest signal was observed with purified lebein-2
(Fig. 5B). Remarkably,
an intense signal was detected not only with the lebein-2 monomer at 20 kDa,
but also with a band at
30 kDa, which may be the aggregate of two
lebein-2 molecules. In contrast, this band with an apparent molecular mass of
30 kDa was hardly visible on the Coomassie-stained SDS-polyacrylamide
(Fig. 5A). The
additional band at
43 kDa, which was seen only on the far-Western blot,
could not be identified yet, but might be an even higher aggregate of
lebein-2. The far-Western blot (Fig.
5B) provided the first evidence that lebein-2 binds
7
1 integrin directly. This interaction
seemed to be specific and depended on the disulfide bridge-stabilized
quaternary and/or tertiary structure of lebein-2 because reduction of lebein-2
entirely abolished the binding signal on the far-Western blot (data not
shown).
Lebein-1 also bound 7
1 integrin in the
far-Western assay only under nonreducing conditions
(Fig. 5B), albeit with
a weaker binding signal than lebein-2. However, far-Western blot assays can be
assessed only qualitatively, as protein interaction may be weakened because of
only partial renaturation of the blotted proteins after SDS treatment. The
Mono S fraction MS-II, which contains lebein-1
(Fig. 5B, MS-II
lane), included an additional band at an apparent molecular mass of 44
kDa, which was also recognized by
7
1
integrin. This may be the precursor of lebetase, a snake venom
metalloproteinase that also possesses a disintegrin domain highly homologous
to lebein-1 (22,
27). Indeed, this band showed
proteolytic activity in a gelatin zymogram (data not shown). Our data suggest
that the lebetase precursor is also able to interact with
7
1 integrin presumably via its disintegrin
domain.
Lebein-2 and Lebein-1 Are Disintegrins That Bind to the
Laminin-binding 1 IntegrinsTo
further analyze whether binding of lebein-2 and lebein-1 is specific for
7
1 integrin, we investigated their
integrin-binding spectra. To this end, lebein-2 and lebein-1 were immobilized,
and the soluble integrins (
1
1,
2
1,
3
1,
6
1, and
7
1) were tested for their binding. Although
all integrins showed binding activity for their cognate ECM ligands such as
the CB3 fragment of type IV collagen, type I collagen, laminin-5, and
laminin-1, only the laminin-binding integrins
(
3
1,
6
1,
and
7
1) showed a clear binding signal for
immobilized lebein-2 and lebein-1, which depended on the presence of divalent
cations (Fig. 6). Soluble
1
1 integrin also showed a slight binding
signal for both disintegrins, which, however, was considered nonspecific due
to its persistence in the presence of EDTA
(Fig. 6, open bars).
Antibody 9EG7 increased the binding signals of
3
1 and
6
1 (but not
7
1) integrins for both disintegrins
(Fig. 6). These binding
analyses demonstrated that lebein-2 and lebein-1 are disintegrins that
recognize the laminin-binding
1 integrins, but not the
collagen-binding integrins (
1
1 and
2
1), in a divalent cation-dependent
manner.
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Titration of immobilized lebein-2 and lebein-1 with soluble
7
1 and
3
1 integrins in the presence of divalent
cations yielded titration curves that showed saturation (data not shown).
Furthermore, linearization of these titration curves according to Heyn and
Weischet (26) provided
apparent affinity constants in the nanomolar range
(Table II). Identical to
laminin-1, both lebein-2 and lebein-1 bound avidly to soluble
7
1 integrin in the presence of 1
mM Mn2+ ions without any marked alteration of
affinities after addition of the activating antibody 9EG7.
Ca2+ ions increased the apparent dissociation constants
significantly. In addition, the presence of Ca2+ showed
more clearly that lebein-1 bound more avidly to
7
1 integrin compared with lebein-2.
Although the affinities of
3
1 integrin for
both disintegrins are lower, the tendency of Mn2+ and
Ca2+ ions to increase and to decrease, respectively, the
affinity of soluble
3
1 integrin for
lebein-2 and lebein-1 resembled the effects on soluble
7
1 integrin. In contrast to soluble
7
1 integrin, binding of soluble
3
1 integrin to both disintegrins could be
substantially improved by the activating antibody 9EG7
(Table II).
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Inhibition of Binding of
7
1 and
3
1 Integrins to Their
Cognate Laminin Isoform LigandsBoth lebein-2 and lebein-1 bound to
the laminin-binding
1 integrins with the same divalent cation
dependences as to the laminin ligands (Fig.
1C and Table
II). Furthermore, both disintegrins completely and efficiently
blocked binding of
7
1 and
3
1 integrins to their respective laminin
isoform ligands in a dose-dependent manner
(Fig. 7). Because of its higher
affinity for both integrins, lebein-1 showed half-maximal inhibition at up to
3-fold lower concentrations than lebein-2
(Fig. 7 and
Table III).
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RGD Peptides Interfere Only Weakly with
3
1 and
7
1 Integrin Binding
to Lebein-1 and Lebein-2Because at least lebein-1 is known to
contain RGD sequences in both chains, we investigated whether the inhibitory
activities of lebein-1 and lebein-2 on
7
1
integrin depend on an RGD peptide sequence. To this end, binding of soluble
7
1 integrin to immobilized laminin-1,
lebein-1, and lebein-2 was challenged by increasing concentrations of RGD
peptides or their derivatives (Fig.
8). The
7
1 integrin/laminin-1
interaction was not affected by the GRGDS peptide even at a 1.6 million-fold
molar excess (8 mM) to the integrin. In contrast, the binding of
7
1 integrin to both lebein-1 and lebein-2
decreased at GRGDS peptide concentrations above 1 mM. However, this
decline in binding was incomplete even at the very high peptide concentration
of 8 mM. Furthermore, control peptides such as GRGES and the
scrambled sequence GRDGS showed a similar, yet less pronounced decline in
integrin binding to both disintegrins at high peptide concentrations above 1
mM. At RGD peptide concentrations below 1 mM, lebein-2
and lebein-1 bound to soluble
7
1 integrin
in an RGD-independent manner, similar to the natural integrin ligand. Hence,
both lebein-2 and lebein-1 must mechanistically be considered RGD-independent
disintegrins when interacting with laminin-binding
1
integrins.
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Lebein-2 and Lebein-1 Affect Muscle Cell Interactions Not Only with
Laminin, but also with FibronectinTo examine the efficiency of the
purified V. lebetina venom components on muscle cells, attachment of
myoblasts to laminin and fibronectin was assessed in the presence of either
lebein-2 or lebein-1 (Fig. 9).
Both disintegrins inhibited cell attachment to laminin-1, presumably by
blocking the interaction of 7
1 integrin,
although complete inhibition could not be achieved even at high disintegrin
concentrations. Interestingly, cell adhesion to immobilized fibronectin was
even more severely compromised by both lebein-2 and lebein-1. Most likely,
this was caused by RGD sequences that are located in both chains of lebein-1
(22). The complete primary
structure of lebein-2 (and hence the presence of RGD sequences within
lebein-2) is not known yet.
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DISCUSSION |
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In this study, we have searched for an inhibitor that interferes with
integrin-mediated adhesion to laminin. With the help of soluble
7X2
1 integrin and its high affinity binding
to laminin-1, we could screen various snake venoms for their inhibitory
capabilities in a protein interaction assay. Several venomous snakes from the
Elapidae, Viperidae, and Crotalidae families are considered to exert myotoxic
effects. However, in most of them, proteolytic enzymes, phospholipase
activities, and other factors may account for their destructive effects on
muscle tissue (13,
14), as only one of 33 tested
venoms entirely inhibited interaction of
7
1
integrin with its laminin ligands. In addition to abundant phospholipase
activity and numerous proteases
(14,
17), the venom of V.
lebetina contains two disintegrins (lebein-2 and lebein-1) that inhibit
7
1 integrin binding to its laminin ligands.
In this study, we have established and optimized their purification from the
crude toxin and have characterized their integrin-inhibiting function.
Lebein-1 and its primary structure have been published recently, as lebein
(22). Because both subunits
bear RGD sequences, lebein-1 was considered an RGD-dependent disintegrin.
However, our study provides experimental evidence not only that lebein-1
interferes with the RGD-dependent cell attachment to fibronectin, but also
that it additionally binds to laminin-binding 1 integrins in
an RGD-independent manner. Therefore, it efficiently inhibits the interaction
of laminin-binding integrins with their respective laminin isoforms.
During the purification of lebein-1, we also found a 44-kDa protein that
showed proteolytic activity. This protein might be a precursor of lebetase, a
Zn2+ ion-containing metalloprotease that has been
identified at the cDNA level
(27). This precursor also
contains a disintegrin domain
(27), in good agreement with
our observation that the 44-kDa protein bound
7
1 integrin on the far-Western blot.
We isolated lebein-2 as a novel protein of the V. lebetina venom.
It has a molecular mass of 14,735 Da, as determined by mass spectrometry, and
consists of two subunits, and
. Whereas the
subunit of
lebein-2 was N-terminally blocked, its
subunit shows an N-terminal
amino acid sequence similar to, yet distinct from, those of the lebein-1
subunits, thus proving that lebein-2 is an independent gene product. Lebein-2
and lebein-1 strongly bound to the laminin-binding
1
integrins (
7
1,
6
1, and
3
1), but did not recognize the
collagen-binding integrins (
1
1 and
2
1) in a divalent cation-dependent manner.
Although we have tested only the X1 splice variant of the
6
integrin subunit, it can be assumed that
6
1
integrin is, in general, a target of lebein-2 and lebein-1, as all its splice
variants have similar ligand-binding specificities
(30). We also limited our
experiments to the X2 splice variant of
7 integrin. However,
this is the predominant
7
1 integrin isoform
in skeletal muscle and thus the potential target of the snake venom.
Furthermore, because of their broad binding specificity for all
laminin-binding
1 integrins, we assume that both disintegrins
are also functional against the X1 splice variant of
7
1 integrin, which is mainly expressed
during myogenesis.
Having identified lebein-2 and lebein-1 in a protein interaction assay, we
also proved their inhibitory activities on myoblasts in cell attachment
studies. Interestingly, both lebein-2 and lebein-1 inhibited integrin-mediated
cell adhesion not only to laminin, but also to fibronectin. The inhibition
mechanism for fibronectin is probably caused by RGD sequences, although so
far, only lebein-1 is known to be an RGD-dependent disintegrin. Our in
vitro binding data support the conclusion that inhibition of cell
attachment to laminin-1 is caused by a direct inhibitory interaction of
lebein-2 or lebein-1 with laminin-binding 1 integrins on the
cell surface that is independent of an RGD sequence.
Laminin-binding 1 integrins avidly bound to both lebein-2
and lebein-1, with Kd values similar to those of
the natural laminin ligands (Fig.
1C) (7).
Like the binding to laminin, integrin binding affinities for the two V.
lebetina disintegrins increased in the presence of
Mn2+ ions and decreased in the presence of
Ca2+ ions. Furthermore, EDTA completely abolished
integrin binding to both laminin and disintegrins. Additionally, the fact that
binding of laminin and that of lebein-1 or lebein-2 to integrins are mutually
exclusive suggests that both disintegrins act as laminin mimetics that bind to
or close to the ligand-binding site of laminin-binding integrins. The
interaction of
7
1 integrin with lebein-1
and lebein-2 did not depend on an RGD sequence, although at least lebein-1
bears two RGD sequences, nor did soluble
7
1
integrin interact with laminin-1 in an RGD-dependent manner in our study, in
agreement with earlier findings
(31). However, binding of
laminin-1 to its integrin receptors not only requires a yet undefined
three-dimensional recognition site within the laminin G module, but also
depends on the presence of the coiled-coil domain consisting of all three
laminin chains,
1,
1, and
1
(3235).
Further mechanistic and structural studies will answer the question of how the
small size disintegrins lebein-2 and lebein-1 can mimic the large laminin
molecules and how they can achieve this inhibition.
Both lebein-1 and lebein-2 belong to the rare group of disintegrins that
interact with laminin-binding 1 integrins in an
RGD-independent manner. To our knowledge, fertilin (ADAM-2) and
meltrin-
(ADAM-9) are the only examples of disintegrins that interact
with the laminin-binding
6
1 integrin
(20,
21,
36). Like lebein-1 and
lebein-2, ADAM-2 is functional only as a heterodimeric molecule together with
ADAM-1, thus taking an essential part in sperm-oocyte fusion during
fertilization (20). Lebein-1
shows some sequence similarities to the disintegrin domains of these ADAM
proteins. However, neither ADAM-2 nor ADAM-9 contains an RGD peptide sequence
(21,
36). In contrast to ADAM-2 and
ADAM-9, which are anchored in the plasmalemma, snake venom disintegrins are
highly soluble and are able to inhibit cell/matrix interaction, thus leading
to tissue dissipation.
In this study, we have identified and characterized two disintegrins from
V. lebetina venom (lebein-1 and the novel lebein-2) that interact
with laminin-binding 1 integrins in a divalent
cation-dependent and RGD-independent manner both in vitro and in
vivo. Through their potential to inhibit integrin-mediated cell
attachment to laminins, both lebein-1 and lebein-2 may be valuable tools to
influence laminin-dependent cell functions. Among these are the attachment,
mechanical force transduction, and migration of cells such as myoblasts
(5,
12) and
7
1 integrin-bearing melanoma cells
(31) along or through the
laminin-rich basal membranes. These cell functions are of paramount importance
in complex physiological processes such as wound healing and tumor
invasion.
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FOOTNOTES |
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To whom correspondence should be addressed: Münster University Hospital,
Inst. for Physiological Chemistry, Waldeyerstr. 15, 48149 Münster,
Germany. Tel.: 49-251-835-2289; Fax: 49-251-835-5596; E-mail:
eble{at}uni-muenster.de.
1 The abbreviations used are: ECM, extracellular matrix; ELISA, enzyme-linked
immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin;
MES, 4-morpholineethanesulfonic acid.
2 S. Niland and J. A. Eble, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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