From the Institut für Physiologische Chemie und
Pathobiochemie, Waldeyerstrasse 15 and the ¶ Institut für
Physikalische Chemie, Schlossplatz 7, Universität Münster,
48149 Münster, Germany
Received for publication, October 12, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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We have recombinantly expressed a
soluble form of human Integrins are cell adhesion molecules that consist of two
noncovalently associated subunits, Integrin-mediated cell adhesion not only anchors the cell mechanically
within the extracellular matrix of the tissue but also elicits several
cellular responses, such as cell spreading and migration, cell
proliferation, and differentiation (for review see Ref. 2). A well
studied example of cellular response triggered by integrin-ligand
interaction is platelet activation and aggregation (8, 9). Thrombocytes
abundantly possess the platelet integrin Furthermore, snake, leeches, and ticks have developed natural
inhibitors of integrin-ligand interactions, called disintegrins, that
target at the integrin-mediated platelet adhesion to fibrinogen/fibrin and collagen (9). By inhibiting blood clotting, their venoms lead to
severe bleeding, hemorrhages, or even death of their victims. Besides
proteolytic enzymes, disintegrins are mainly responsible for these
poisonous effects. Most of the known disintegrins contain a linear RGD
sequence placed within a rather flexible loop, which prevents the
RGD-dependent platelet integrin
Using a recombinantly expressed, soluble human
Of even more general importance, Production of the cDNA Constructs of a Recombinant Human
Soluble Establishing a Stable,
To screen hygromycin B-resistant clones for their ability to secrete
soluble Isolation of Recombinant Human Soluble
Diluted with Mono Q buffer A (20 mM Tris/HCl, pH 8.0, 1 mM MgCl2), the Binding of Soluble Separation of the Snake Venom Proteins from C. rhodostoma--
Snake venom lyophilizate from C. rhodostoma
(Sigma) was dissolved in TBS, pH 7.4, containing 1 mM EDTA
(TBS/EDTA) at a protein concentration of about 200 mg/ml. The proteins
were separated by gel filtration on a Superose 6 column HR30/30
(Amersham Pharmacia Biotech) using TBS/EDTA at 0.3 ml/min. Two distinct
pools of fractions were able to inhibit the binding of soluble
Protein concentration was determined by bichinonic acid. Purity of
LMW-CI and the apparent molecular masses of its subunits were assessed
by SDS-PAGE and Coomassie staining.
Inhibition ELISA: Inhibition of Titration of Immobilized Rhodocetin with Soluble
RGD Peptide Inhibition Assay of Circular Dichroism Spectroscopy of Rhodocetin--
The buffer of
the rhodocetin solution was changed to 20 mM sodium
phosphate, pH 7.0, 50 mM NaCl by gel filtration on a TSK G3000SWXL column (TosoHaas, Stuttgart, Germany). The rhodocetin containing eluate fractions were concentrated in a Centricon 10 tube by
centrifugal ultrafiltration to reach a concentration of about 0.3 mg/ml. The CD spectrum was recorded from 190 to 260 nm in a
0.1-mm cuvette in a CD spectrophotometer type CD6 (Jobin Yvon, Paris,
France). Temperature was controlled by a self-constructed Peltier
element cuvette holder. The relative amount of secondary structures
( Inhibition of Cell Adhesion to Collagen by
Rhodocetin--
Monomeric bovine type I collagen at a concentration of
0.2 µg/ml in 0.1 M acetic acid was immobilized onto a
microtiter plate at 4 °C overnight. After being washed with
TBS/MgCl2 for three times, the plate was blocked with
BSA/TBS/MgCl2 for 2 h at room temperature. HT1080
fibrosarcoma cells at a density of 500,000 cells/ml in Dulbecco's
modified Eagle's medium were plated onto the plate for 35 min in a
tissue culture incubator at 37 °C in both absence and presence of
various concentrations of rhodocetin. Adherent cells were detected by
staining with crystal violet (24). Briefly, adherent cells were fixed
with 70% (v/v) solution of ethanol for 7 min and stained with a 0.1%
(w/v) solution of crystal violet in destilled water. After washing the
wells, cell bound dye was extracted with a 0.2% (v/v) Triton X-100
solution, and its amount was measured in an ELISA reader at 560 nm.
Experiments with cells were done in triplicates. The adhesion signal of
HT1080 cells measured on BSA was considered nonspecific background and subtracted from the adhesion signals of cells on type I collagen. Adhesion signals in the presence of rhodocetin were normalized to the
adhesion signal of the noninhibited cell adhesion to type I collagen
without any inhibitor.
Production and Isolation of Recombinant Soluble Human
Characterization of the Recombinant Soluble
The soluble Whole Snake Venom of C. rhodostoma Inhibits Binding of Soluble
Rhodocytin/Aggretin Does Not Inhibit Binding of Soluble
Because immobilization may have caused inactivation of
rhodocytin/aggretin, binding of soluble
Searching for the Component of C. rhodostoma Venom That Inhibits
the Interaction of Soluble Characterization of LMW-CI--
Under nonreducing conditions,
LMW-CI shows an apparent molecular mass of 27 kDa in SDS-PAGE (Fig. 3,
lane 2). LMW-CI is a heterodimer, which upon reduction falls
apart in two subunits of 16 and 14 kDa (Fig. 3, lane 5). The
N-terminal sequences of both LMW-CI subunits were identified by Edman
degradation with the N terminus of the 16-kDa subunit being
D[-/(F)]PD[G/S]WSSTKSYYR[P/(R)][F/(P)][K/(F)][E/(K)][K/(E)]3
and the N terminus of the 14 kDa subunit being
DFRPTTWSMSKLY [-/(S)]YKPF(K). These N-terminal sequences clearly
showed that the LMW-CI is distinct from the rhodocytin/aggretin.
However, these sequences disclosed that LMW-CI is identical to
rhodocetin, a recently published inhibitor of collagen-induced platelet
aggregation (16).
On a molecular level, rhodocetin inhibited binding of soluble
Rhodocetin Is a Disintegrin That Directly and Specifically Binds to
It is noteworthy that the ability of LMW-CI/rhodocetin to interact with
To determine the binding affinity of rhodocetin to
Rhodocetin Does Not Contain a Triple Helical Collagen
Domain--
Being essential for its inhibitory activity, the native
conformation of LMW-CI/rhodocetin was further studied by CD. We were especially interested in whether or not LMW-CI contains any triple helical collagenous motifs, because high affinity ligands of
Rhodocetin Is an RGD-independent Integrin--
Many disintegrins
bind to RGD-dependent integrins in an RGD peptides
inhibitable manner. However, the linear GRGDSP peptide failed to
inhibit the interaction of Effect of Rhodocetin on Here, we report that rhodocytin, an inducer of platelet
aggregation from the hemorrhagic snake venom of C. rhodostoma, does not interact with
Identification of LMW-CI/rhodocetin as disintegrin, which specifically
binds to A great advantage of the soluble Having sufficient amounts of a stable, soluble
Being the initial step of collagen-triggered platelet activation and
aggregation, Possessing various collagen receptors and being easily activated by
several stimuli other than collagen, such as ADP, thrombin, etc., whole platelets are rather coarse targets to screen
hemorrhagic snake venoms for specific inhibitors to the
Without using whole platelets, we have established a cell-free
inhibition assay as a tool to search for an inhibitor of the integrin-collagen interaction on the molecular level without any interfering cellular reactions that occur on platelets during or after
their activation. Furthermore, the use of soluble
LMW-CI/rhodocetin is a heterodimer with an apparent molecular mass
of 27 kDa consisting of two subunits of 16 and 14 kDa, which are firmly
attached. Despite lacking any covalent, intercatenary disulfide bridges
(16), the two subunits remained associated under the harsh denaturation
conditions of the SDS-PAGE sample buffer, containing 2% SDS. However,
reduction of intracatenary disulfide bridges leads to destruction of
the tertiary structure and subsequentially to the dissociation of the
two subunits. Judging the native tertiary structure by its capability
to bind to Interestingly, both rhodocytin/aggretin and LMW-CI/rhodocetin belong to
a family of snake venom proteins that bear similarity to the
carbohydrate recognition domain of C-type lectins (16, 31). However,
platelet-derived wild-type It is noteworthy that LMW-CI/rhodocetin does not bind to
As a prerequisite for its binding activity to
Having found and characterized LMW-CI/rhodocetin as snake venom
disintegrin that specifically recognizes
2
1 integrin
that lacks the membrane-anchoring transmembrane domains as well as the
cytoplasmic tails of both integrin subunits. This soluble
2
1 integrin binds to its collagen ligands
the same way as the wild-type
2
1
integrin. Furthermore, like the wild-type form, it can be activated by
manganese ions and an integrin-activating antibody. However, it does
not bind to rhodocytin, a postulated agonist of
2
1 integrin from the snake venom of
Calloselasma rhodostoma, which elicits platelet
aggregation. Taking advantage of the recombinantly expressed, soluble
2
1 integrin, an inhibition assay was
established in which samples can be tested for their capability to
inhibit binding of soluble
2
1 integrin to
immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay,
we found a component of the snake venom that inhibits the interaction
of soluble
2
1 integrin to type I collagen
efficiently. N-terminal sequences identified this inhibitor as
rhodocetin, a recently published antagonist of collagen-induced
platelet aggregation. We could demonstrate that its inhibitory effect
bases on its strong and specific binding to
2
1 integrin, proving that rhodocetin is a
disintegrin. Standing apart from the growing group of
RGD-dependent snake venom disintegrins, rhodocetin
interacts with
2
1 integrin in an
RGD-independent manner. Furthermore, its native conformation, which is
stabilized by disulfide bridges, is indispensibly required for its
inhibitory activity. Rhodocetin does not contain any major collagenous
structure despite its high affinity to
2
1
integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking
2
1 integrin as the major collagen
receptor on platelets, rhodocetin is responsible for hampering
collagen-induced,
2
1 integrin-mediated
platelet activation, leading to hemorrhages and bleeding disorders of
the snakebite victim. Moreover, having a widespread tissue
distribution,
2
1 integrin also mediates
cell adhesion, spreading, and migration. We showed that rhodocetin is
able to inhibit
2
1 integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
(for review see Refs. 1
and 2). The subfamily of integrins sharing the
1 subunit are well known receptors for extracellular matrix molecules, such as
collagens, laminins, and fibronectin. The subfamily of
3
subunit containing cytoadhesins comprise the platelet integrin,
IIb
3 which binds fibrinogen/fibrin (3)
and the vitronectin receptor
V
3. The
latter ones, along with several
1 integrin, such as the
fibronectin receptor
5
1 integrin,
recognize a linear arginyl-glycyl-aspartyl sequence within their
respective ligands, such as fibrinogen, fibronectin, and vitronectin
(4). In contrast, the collagen binding integrins
1
1 and
2
1
recognize arginine and aspartate/glutamate residues of different
collagen chains, which are in close proximity to each other within the
triple helical collagenous framework of the collagen (5-7), thus
forming a completely different spatial structure than the linear RGD peptide.
IIb
3 on their surface, which unless
activated does not bind to fibrinogen/fibrin (3). Ablation of
endothelial cells from the blood vessel wall or other injuries of blood
vessels make type IV and type I collagen of the basement membrane and
the underlying connective tissue, respectively, accessible to
platelets. Once getting in contact with collagen, platelets avidly bind
to collagen via their collagen receptors (9, 10), such as the
2
1 integrin, GPVI, or indirectly via von
Willebrand factor, which binds to both collagen and the vWF receptor on
the platelet surface. Receptor-mediated adhesion to collagen elicits a
cascade of signals within the platelets, which eventually results in
secretion of platelet granula, in platelet aggregation and activation
of platelet integrin
IIb
3 which then
binds to fibrin with high affinity. Insoluble fibrin, which has been
produced by the enzymatic blood clotting cascade and provides a
scaffold, which together with platelets form the blood clot as the
first and essential step in hemostasis. The key role of the
2
1 as the sole integrin collagen receptor
on platelets is drastically manifested in patients with severe bleeding disorders, caused either by a genetic defect or lack of the integrin
2 subunit (11) or by auto-antibodies against the
integrin
2 subunit (12).
IIb
3 from binding to fibrin (13).
However, very little is known about disintegrins that act on the
interaction of
2
1 integrin with collagen,
the initial step of platelet activation and aggregation. From the venom
of the Malayan pit viper (Calloselasma rhodostoma),
rhodocytin/aggretin (14, 15), and, more recently, rhodocetin (16) have
been shown to induce and inhibit, respectively, collagen-elicited
platelet activation and aggretion. However, no direct proof was
provided that rhodocytin/aggretin and rhodocetin are the agonist and
antagonist, respectively, that interact directly and specifically with
2
1 integrin among the different collagen
receptors of blood platelets.
2
1 integrin, we could rule out that
rhodocytin/aggretin binds directly to
2
1
integrin. On the other hand, having established an inhibition assay
with the purified soluble
2
1 integrin
apart from whole platelets, we could isolate an inhibitor of C. rhodostoma venom that inhibits the binding of soluble
2
1 integrin to collagen on the molecular
level. N-terminal sequencing identified this inhibitor to be the lately
published rhodocetin (16). We could demonstrate that rhodocetin binds
directly to the
2
1 integrin. Rhodocetin
efficiently competes with collagen for the
2
1 integrin, even though it does not
contain any collagenous triple helix domain, which has been surmised to
be a prerequisite for high affinity binding to collagen-binding
integrins. Nevertheless, the native conformation that is stabilized by
disulfide bridges is essential for binding to
2
1 integrin. In contrast to the majority
of snake venom disintegrins, rhodocetin binds to
2
1 integrin in an RGD-independent manner.
2
1
integrin is not only the integrin receptor for collagen on platelets
but also abundantly expressed in various tissues (17), suggesting an
important role of
2
1 integrin within the
organism. Having proved rhodocetin to be a very specific
2
1-integrin antagonist, we have started to test rhodocetin as a tool in studying
2
1-related functions on the cellular
level and have demonstrated that rhodocetin can efficiently and
entirely inhibit
2
1 integrin-mediated
adhesion of HT1080 fibrosarcoma cells to collagen.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 Integrin--
The
transmembrane and cytoplasmic domain of the integrin
2
subunit were substituted for a GGSTGGG spacer and the dimerizing motif
of the transcription factor Fos. The cloning strategy started from the
construct pUC-HygMT-
3fos, which was described in a
previous paper (18). Briefly, the cDNA sequence coding for the
3 ectodomain within the pUC-HygMT-
3fos
was replaced by the cDNA sequence coding for the ectodomain of the
integrin
2 subunit. To this end,
pUC-HygMT-
3fos was cleaved by SalI and
dephosporylated by calf intestine phosphatase. The 7.2-kilobase
pair-long vector fragment still contains the Fos-coding sequence of the
original construct pUC-HygMT-
3fos, yet lacking the
complete sequence coding for the integrin
3 ectodomain. The human cDNA coding for the signal sequence and the N-terminal 912 amino acids of the mature
2 ectodmain were excised
from pFneo-
2 construct (19) using SalI and
BglII. The cDNA coding for the C-terminal 131 amino
acids of the
2 ectodomain and the first few amino acids
of the GGSTGGG spacer, the latter one of which contains the
SalI restriction site, were obtained by polymerase chain
reaction using the
2 cDNA of pFneo-
2
as template, and the oligonucleotides
ATGCTGAAATTCACTTAACAAGATCTACC with the BglII site underlined and GCCGCCCGTCGACCCTCCTGTTGGTACTTCGGCTTTCTC
with the SalI site underlined as forward and reverse primer,
respectively. In a triple ligation the SalI-cleaved vector
fragment and the cDNA fragments for both the N- and C-terminal part
of the
2 ectodomain were ligated to the
pUC-HygMT-
2fos construct coding for the soluble
2 ectodomain, which bears at its C terminus the short
spacer sequence GGSTGGG and the dimerizing motif of Fos. The
pUC-HygMT-
1jun construct was generated as described in a
previous paper (18).
2
1 Secreting
Schneider Cell Clone--
Both constructs were transfected in an
equimolar ratio into Drosophila Schneider cells, using
TransFastTM Transfection Reagents (Promega, Madison, WI)
according to the manufacturer's instructions. Transfected cells were
selected under 0.1 mg/ml hygromycin B. After two rounds of subcloning
by limited dilution and after screening for positive clones by a
sandwich ELISA1 described
below, the stable clone
2
1-G1.2 was
established, which after induction of the metallothionine promoters
upstream of both integrin
2 and
1
ectodomain cDNAs secreted soluble
2
1 integrin into the cell supernatant in concentrations of about 40 µg/liter.
2
1 integrin, supernatants of
transfectant clones were tested in a sandwich ELISA 4-5 days after
induction by copper sulfate. For the sandwich ELISA, the mouse
monoclonal anti-integrin
2 antibody JA218 (kindly
provided by Danny Tuckwell, University of Manchester, UK) (20) was
immobilized to the plastic surface of a microtiter plate at 8 µg/ml
in TBS (50 mM Tris/HCl, pH 7.4, 150 mM NaCl )
with MgCl2 (TBS/MgCl2). After blockage of
nonspecific binding sites on the microtiter plate with 1% (w/v) heat
denatured BSA in TBS/MgCl2 (BSA/TBS/MgCl2), the
cell supernatants were added into the coated wells. The antibody JA218
captured the soluble
2
1 integrin, which
was then detected by an rabbit anti-human
1
integrin-antiserum as primary antibody and goat anti-rabbit IgG-antibodies coupled to alkaline phosphatase (Sigma) as secondary antibody, diluted 1:300 and 1:600, respectively, in
BSA/TBS/MgCl2. Before each antibody incubation and the
final enzymatic detection reaction, wells were washed three times with
TBS/MgCl2. As substrate of alkaline phosphatase,
p-nitrophenylphosphate tablets were used according to the
manufacturer's instructions (Sigma). Absorbance was measured at 405 nm
using an ELISA-reader (Dynatech, Burlington, MA).
2
1 Integrin--
In spinner flasks,
2
1 G1.2 cells were grown in Sf900
Medium (Life Technologies, Inc.) containing 0.1 mg/ml hygromycin B and
10% fetal calf serum. Once they had reached a density of about 12 million cells/ml, they were induced by addition of copper sulfate at
0.6 mM. Simultanously, glucose was added to 0.1% (v/w) and glutamine was added to 0.8 mM. Cell supernatant was
harvested 5 days after induction and concentrated by ultrafiltration in a YM30 membrane cartridge (Amicon, Witten, Germany). Protease inhibitors aprotinin, leupeptin, and pepstatin were added at 1 µg/ml.
Mn2+ ions that increase integrin affinity to ligands were
added to a final concentration of 1 mM. The concentration
of dithiothreitol (DTT) was adjusted to 2 mM, before the
concentrated cell supernatant was loaded onto the collagen I column.
The collagen I column had been generated by covalently coupling bovine
type I collagen to cyanogen bromide-activated Sepharose 4B CL according
to the manufacturer's instruction (Amersham Pharmacia Biotech). The
loaded collagen I column was washed with TBS containing 2 mM MgCl2, 1 mM MnCl2, and 2 mM DTT (wash buffer A). After a stringent wash with
buffer A with a NaCl concentration of 300 mM, the collagen
I column was washed with buffer A, before the soluble
2
1 integrin was eluted with TBS
containing 20 mM EDTA. Immediately after elution,
MgCl2 was added to 30 mM, and the eluate
fraction was neutralized with 2 M Tris/HCl, pH 8.0. The
2
1 containing eluate fractions were concentrated by ultrafiltration.
2
1
containing solution was loaded onto a Mono Q column and eluted with a
linear gradient of 0 to 50% Mono Q buffer B (1 M NaCl in
Mono Q buffer A) within 60 min. The
2
1
containing eluate fractions were concentrated by centrifugational
ultrafiltration using a Centricon 50 tube (Amicon, Witten, Germany).
Protein concentration was determined using the bichinonic acid assay
according to the manufacturer's instructions (Pierce). Purity was
assessed by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie staining.
2
1 Integrin to
Various Extracellular Matrix Molecules--
Bovine type I collagen and
chicken type II collagen was kindly provided by Peter Bruckner
(University of Münster, Germany). Type IV collagen, the type IV
collagen fragment CB3[IV], type V collagen, and murine Laminin-1
(Engelbreth-Holm-Swarm-Laminin) were gratefully obtained from Klaus
Kühn, Rupert Timpl, and Albert Ries (Max-Planck-Institute for
Biochemistry, Martinsried, Germany). Collagens were plated in 0.1 M acetic acid, except for CB3[IV], which like laminin was
coated in TBS/MgCl2 onto the microtiter plate. After the
wells were blocked with a BSA/TBS/MgCl2, the integrin
dissolved in the same solution was allowed to bind to the immobilized
substratum. MnCl2, activating antibody 9EG7 or EDTA were
added as indicated. The activating monoclonal anti
1 integrin antibody 9EG7 (21) was isolated from cell supernatant according to standard protocols. The 9EG7 hybridoma was kindly provided
by Dieter Vestweber (University of Münster, Münster, Germany). After a 2-h incubation at room temperature, nonbound integrin
was washed away with HEPES wash buffer (50 mM HEPES, pH
7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM MnCl2) twice. Then collagen-bound
2
1 integrin was covalently cross-linked
to the substratum with 2.5% glutaraldehyde solution in HEPES wash
buffer for 10 min at room temperature. After washing the plate three
times with TBS/MgCl2, the amount of bound
2
1 was measured in an ELISA-like
procedure with a rabbit anti-human integrin
1 subunit
antiserum as primary antibody and an anti-rabbit IgG-antibody
conjugated to alkaline phosphatase as secondary antibody, diluted 1:300
and 1:600, respectively, in BSA/TBS/MgCl2. Each antibody
incubation, all of which lasted for 1.5 h, was followed by washing
the plate with TBS/MgCl2 three times. For detection,
p-nitrophenylphosphate tablets (Sigma) were used as
substrate for the alkaline phosphatase according to the manufacturer's
recommendations. The yellow reaction product was measured at 405 nm in
an ELISA reader.
2
1 integrin to immobilized type I
collagen. The fractions containing the Low molecular weight Calloselasma inhibitor (LMW-CI) was diluted in 20 mM MES/NaOH, pH 6.5 (Mono S buffer A) and passed through a
Mono S HR5/5 column (Amersham Pharmacia Biotech). The retained proteins
were eluted with a linear gradient of 0-20% Mono S-buffer B (1 M NaCl in Mono S-buffer A) within 60 min. In the third
purification step, the LMW-CI containing solution was adjusted to pH
8.5 by diluting into 20 mM Tris/HCl, pH 8.5 (Mono Q-buffer
A). The LMW-CI was eluted from the Mono Q column using a linear
gradient of 0-50% Mono Q-buffer B (1 M NaCl in Mono
Q-buffer A). The elute fractions containing LMW-CI were concentrated in
a Centricon 10 tube by centrifugal ultrafiltration. To reduce
contaminating proteins any further, a final gel filtration on a TSK
G3000SWXL column (TosoHaas, Stuttgart, Germany) was performed at 0.4 ml/min. N-terminal sequencing by Edman degradation identified LMW-CI to
be identical to rhodocetin (16).
2
1
Binding to Immobilized Monomeric Type I Collagen--
Dissolved in 0.1 M acetic acid at 40 µg/ml, type I collagen was coated as
monomeric molecule onto the plastic surface of a microtiter plate at
4 °C overnight. After washing with TBS/MgCl2, nonspecific binding sites on the plastic surface were blocked with
BSA/TBS/MgCl2 for 2 h at room temperature. Then
soluble
2
1 integrin was added as a 6 µg/ml solution in BSA/TBS/MgCl2 either without any
inhibitor (positive control of 100% binding), in the presence of a
snake venom fraction, or with 10 mM EDTA (nonspecific binding; negative control with 0% binding). To increase the binding signal of
2
1 integrin, both 1 mM MnCl2 and a 3-fold molar surplus of
integrin-activating antibody 9EG7 was added. To prevent any protease
activity of the snake venom that could degrade the
2
1 integrin or the collagen substratum,
resulting in a likewise decrease of binding signals, the following
protease inhibitors were added to final concentrations as follows: 2 µg/ml of each aprotinin, leupeptin, and pepstatin, and 2 mM of each 1,10-phenanthroline and phenylmethylsulfonyl
fluoride. After having bound to the immobilized collagen ligand in
either the presence or the absence of inhibitor for 2 h at room
temperature, nonbound
2
1 integrin was
washed off the plate with HEPES wash buffer. After chemical fixation, the bound integrin was measured in the ELISA-like procedure described above. As blank value, the binding signal obtained in the presence of
EDTA was subtracted from all other values. To calculate relative binding values, the binding signal of
2
1
integrin to type I collagen without any inhibitor was taken as
100%.
2
1 Integrin--
Both native and
inactive rhodocetin were coated onto a microtiter plate at 50 µg/ml
in TBS/MgCl2 at 4 °C overnight. Rhodocetin had been
inactivated by heat denaturation at 95 °C for 20 min in the presence
of 40 mM DTT, followed by blockage of free thiol groups
with 120 mM iodacetamide for 10 min at room temperature. After the microtiter plate was blocked with BSA/TBS/MgCl2,
soluble
2
1 integrin at different
concentrations was incubated with the immobilized rhodocetin. Soluble
2
1 integrin was dissolved in BSA/TBS/MgCl2 containing 1 µg/ml of each aprotinin,
leupeptin, and pepstatin, as well as 0.5 mM
phenylmethylsulfonyl fluoride and 1,10-phenanthroline. After a 2-h
incubation at room temperature, wells were washed twice with HEPES wash
buffer. Bound
2
1 integrin was fixed, and
its amount was determined by ELISA as described above. Nonspecific
binding signals measured as
2
1 binding to the blocking agent BSA were subtracted from the binding values for
2
1 binding to native and denatured
rhodocetin, respectively. The titration curves were linearized, and a
Kd value was determined according to the algorithm
given by Heyn and Weischet (22).
2
1
Binding to Rhodocetin--
Inhibition of
2
1 binding to immobilized rhodocetin by
RGD peptide was performed similarly to the titration experiments. After
the microtiter plate was coated with rhodocetin at 50 µg/ml overnight
at 4 °C and blocked with BSA/TBS/MgCl2 at room
temperature for 2 h, soluble
2
1 at
15 µg/ml was added either in the absence or presence of various
concentrations of the linear GRGDSP peptide (Bachem, Heidelberg,
Germany) for 2 h at room temperature. Then unbound
2
1 integrin was removed by washing with
HEPES wash buffer twice. Bound
2
1
integrin was fixed with 2.5% glutaraldehyde in HEPES wash buffer. Its
amount was determined by ELISA as described above. The binding signals
were corrected for the blank values measured as
2
1 binding to BSA and afterward
normalized to the noninhibited binding of
2
1 to rhodocetin in the absence of GRGDSP peptide (positive control, 100% binding).
helix, parallel, and anti-parallel
strands, random coil) were
calculated with the deconvolution program of CDNN by Gerhard Böhm
(23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 Integrin--
A recombinant soluble
human
2
1 integrin that consists of the
ectodomains of both
2 and
1 integrin
subunits being noncovalently associated by the dimerizing motif of Fos
and Jun, respectively, was secreted by transfected
Drosophila Schneider cells. Affinity purification of the
cell supernatant on a type I collagen column yielded not only the
soluble
2
1 integrin but also a protein of
45 kDa as determined by SDS-PAGE under reducing conditions (Fig.
1, lane 5). Edman degradation
of the latter one revealed its N-terminal sequence as
STEFSEDLLDEDLDLDIDE and, thus, identified the 45-kDa protein as
Drosophila BM40 (GenBankTM accession number
AJ1333736). Interestingly, BM40 was abundantly expressed by Schneider
cells. Like the soluble
2
1 integrin, it
bound to type I collagen column in a divalent
cation-dependent manner. About 10 times more BM40 than
soluble
2
1 integrin was eluted from the
type I collagen column by EDTA. However, binding of BM40 to type I
collagen did not interfere with
2
1
integrin binding to its collagen ligand. Being a less acidic protein
than BM40, the soluble
2
1 integrin was
further purified by anion exchange chromatography on a Mono Q column,
from which the soluble
2
1 integrin was
eluted at lower ion strength than the highly acidic BM40. Yields of
soluble
2
1 integrin ranged from 30 to 40 µg/liter of cell supernatant.
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Fig. 1.
SDS-PAGE of soluble human
2
1
integrin (lanes 1 and 4, without and
with prior reduction, respectively) and Drosophila
BM40 (lanes 2 and 5, without
and with prior reduction, respectively) in a 7.5-15% polyacrylamide
gradient gel. Broad range molecular mass markers (Bio-Rad) were
used as standard proteins (lane 3) with their molecular
masses indicated on the far right side. Proteins in the gel
were stained with Coomassie dye. Protein bands under both nonreduced
and reduced conditions are labeled on the left and
right side, respectively, of the gel.
2
1 Integrin--
In SDS-PAGE, the
soluble
2
1 integrin heterodimer was
separated into the Fos zipper containing
2-ectodomain,
2-Fos, and the Jun zipper containing
1-ectodomain,
1-Jun, which run at 150 and
95 kDa, respectively, under nonreducing conditions and at 140 and 100 kDa, respectively, after reduction (Fig. 1, lanes 1 and
4). The identity of the
2 band was proven by
N-terminal sequencing. Edman degradation revealed the sequence
YNVGLPEAKI in agreement with the mature human integrin
2
subunit (19), demonstrating that the human
2 subunit was
correctly processed proteolytically within the insect cells. Like the
wild-type form on human cells, the human
1-Jun chain
expressed by the insect cells was N-terminally blocked and thus
inaccessible to Edman degradation. However, it was identified in
Western blot by a polyclonal antiserum against the human integrin
1 subunit (data not shown). Unlike other integrin
subunits, the
2-ectodomain is not proteolytically processed into a heavy and light chain. Neither was the human soluble
2
1 integrin cleaved in the heterologous
expression system of the Drosophila Schneider cells. Having
very similar isoelectric points, the
2
1
integrin and BSA could not efficiently be separated by anion exchange
chromatography leading to a slight contamination of BSA in the
2
1 integrin preparation.
2
1 integrin was able to bind
to collagen types I, II, IV, and V and to laminin-1
(Engelbreth-Holm-Swarm-Laminin) (Fig. 2).
The highest binding signals were observed to type I and II collagen,
which is in good agreement with results of wild-type
2
1 integrin (25). Like the wild-type
form, the soluble
2
1 integrin gave a
smaller binding signal on the basal membrane collagen, type IV
collagen, and likewise to its triple helical fragment CB3[IV], which
comprise the binding sites for both
1
1
and
2
1 integrin (25). A significantly
lower binding signal was measured to type V collagen, which together
with type I collagen forms the collagen fibrils of the connective
tissue. As a ligand without any collagenous triple helix, laminin-1 was
bound by the soluble
2
1 integrin, albeit
with a much lower binding signal than the collagenous ligands. The
latter finding corroborated studies of wild-type
2
1 integrin binding to laminin-1 (26).
Identical to cell membrane-anchored wild-type
2
1 integrin, soluble
2
1 required divalent cations to recognize
its ligands. Therefore, EDTA abolished
2
1
binding (Fig. 2). The soluble
2
1 integrin seemed to be regulated by extracellular factors in a manner similar to
that of the the wild-type
2
1 integrin on
the cell surface, because integrin-activating Mn2+ ions and
the activating monoclonal antibody 9EG7 increase the binding signal of
soluble
2
1 integrin to its ligands (Fig.
2). Taken together, the soluble
2
1
integrin showed ligand binding properties similar to the
membrane-anchored wild-type
2
1 integrin. However, no detergent was needed to extract the soluble
2
1 integrin or to keep it in solution.
Furthermore, unlike the detergent-extracted wild-type
2
1 integrin, soluble
2
1 integrin remained active even after a
longer storage period of several months.
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Fig. 2.
Binding of soluble
2
1
integrin to bovine type I collagen (bCol-1), chicken
type II collagen (chCol-2), human type IV collagen
(hCol-4) and its triple helical cyanogen
bromidederived fragment (CB3[IV]), human type V
collagen (hCol-5), and murine
Engelbreth-Holm-Swarm-Laminin (mLam-1). The
collagen molecules were immobilized onto the microtiter plate in 0.1 M acetic acid, except for CB3, which like laminin is coated
in TBS containing Mg2+ ions. Binding of soluble
2
1 integrin (12 µg/ml) was tested in
the presence of 1 mM Mn2+ ions (lightly
hatched bars), in the presence of 1 mM
Mn2+ and a 3-fold molar surplus of activating antibody 9EG7
(densely hatched bars), or in the presence of 10 mM EDTA (open bars). Duplicate measurements were
performed for every binding condition. Mean values and standard
deviations of a representative experiment are shown.
2
1 Integrin to Immobilized Type I
Collagen--
The strong binding signal of soluble
2
1 integrin to immobilized type I
collagen (Fig. 2) was diminished and completely inhibited by the crude
snake venom of C. rhodostoma in a dose-dependent manner with an IC50 value of about 50 µg/ml (data not
shown). Like other snake venoms, C. rhodostoma venom
contains several proteases that could be detected by zymogram developed
with gelatin. Rhodostoxin (kistomin and major hemorrhagin), a
metalloprotease (27, 28), and ancrod, a serine protease (29), could be
detected in the zymogram among other proteolytic activities (data not
shown). To rule out the possibility that any snake venom protease
diminishes the
2
1 binding signal to
immobilized collagen, protease inhibitors directed against all four
classes of proteases, such as aprotinin, leupeptin,
phenylmethylsulfonyl fluoride, pepstatin, and 1,10-phenanthroline, were
added to the venom protein fraction when applied in the inhibition ELISA to test its capability to inhibit binding of soluble
2
1 to immobilized type I collagen by a
nonproteolytic interaction.
2
1 Integrin to Type I
Collagen--
Rhodocytin or aggretin are the two names of a 29-kDa
protein of C. rhodostoma venom, which induces activation and
aggregation of thrombocytes (15, 30). It was isolated from the snake
venom according to Shin and Morita (31). In SDS-PAGE (Fig.
3, lane 1), the purified
rhodocytin/aggretin showed a molecular mass of about 29 kDa under
nonreducing conditions. Being a disulfide cross-linked heterodimer, it
was cleaved under reducing conditions into two subunits of 19 and 15 kDa (Fig. 3, lane 4). The N-terminal sequences of both
subunits, GLEDDFGWSPYDQ[H/(Q)]2 and
DPSGWSSYEG[H/(G)](H)YK, proved their
identities as
and
chains, respectively, of rhodocytin/aggretin
(14, 31). To test the postulated interaction of soluble
2
1 integrin with rhodocytin/aggretin, the
latter one was immobilized on a microtiter plate, and the binding of
soluble
2
1 was tested. Whereas the soluble
2
1 binds to immobilized monomeric
type I collagen in a divalent cation-dependent manner, no
binding to immobilized rhodocytin/aggretin was observed (Fig.
4A). A similar result was obtained when wild-type
2
1 integrin,
which had been purified from platelets (kindly provided by Albert Ries
and Rupert Timpl, Max-Planck-Institute for Biochemistry, Martinsried,
Germany), was used (data not shown).
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Fig. 3.
SDS-PAGE of rhodocytin/aggretin (lanes
1 and 4, without and with prior reduction,
respectively) and LMM-CI/rhodocetin (lanes 2 and
5, without and with prior reduction, respectively) in
a 12-18% polyacrylamide gradient gel. Broad range molecular mass
marker (Bio-Rad) was used as standard proteins (lane 3) with
their molecular masses indicated on the far right side.
Proteins in the gel were stained with Coomassie dye. Protein bands
under nonreduced and reduced conditions are labeled on the
left and right side, respectively, of the
gel.
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Fig. 4.
Rhodocytin/aggretin from C. rhodostoma snake venom is not recognized by soluble
2
1
integrin (A) and does not inhibit binding of
2
1
integrin to type I collagen (B). In A,
bovine type I collagen at 20 µg/ml in 0.1 M acetic acid
and rhodocytin/aggretin at 50 µg/ml in TBS/MgCl2 were
coated onto a microtiter plate. After blockage with heat denatured BSA,
soluble
2
1 integrin was added for 2 h at room temperature. After the wells had been washed twice, the bound
2
1 integrin was chemically fixed to the
immobilized substratum and its amount measured by ELISA. In
B, bovine type I collagen was immobilized on the microtiter
plate at 40 µg/ml in 0.1 M acetic acid. After blockage
with heat denatured BSA, the soluble
2
1
integrin was allowed to bind to the immobilized collagen in both
absence and presence of various concentrations of soluble
rhodocytin/aggretin for 2 h at room temperature. After the wells
had been washed twice, the collagen-bound
2
1 integrin was chemically fixed, and its
amount was determined by ELISA. Blank values measured in BSA-coated
wells were subtracted from the measured absorbance values. In
B, the absorbance values were normalized to the noninhibited
binding value, taken as 100%. Each value was measured in duplicate.
Standard deviations are indicated.
2
1 to soluble rhodocytin/aggretin was
tested by measuring the capability of the snake venom component to
inhibit
2
1 integrin binding to
immobilized type I collagen. However, rhodocytin/aggretin does not
prevent
2
1 integrin from binding to
collagen (Fig. 4B). Both the binding test and the inhibition test rule out any direct interaction between rhodocytin/aggretin and
soluble
2
1 integrin on the molecular level.
2
1 Integrin
with Collagen--
Although rhodocytin/aggretin did not inhibit
2
1 binding to collagen (Fig. 4), the
whole snake venom hampered binding of soluble
2
1 integrin to immobilized type I
collagen. Taking advantage of the inhibition ELISA, the constituent of
C. rhodostoma venom that is responsible for the inhibition
of
2
1 integrin binding to type I collagen
was searched. In the first purification step, the venom proteins were
separated according to their molecular masses by gel filtration on a
Superose 6 column (Fig. 5A).
When the eluate fractions were screened for their capability to inhibit
2
1 binding to immobilized type I
collagen, two peaks of inhibitory activity could be identified (Fig.
5B). Because of their different molecular masses, they were
referred to as high molecular weight and low molecular weight
Calloselasma inhibitor. Purification and identification of
the LMW-CI activity was further pursued. Ion exchange chromatography
both on Mono S and Mono Q could clearly separate rhodocytin/aggretin
from the
2
1 integrin inhibitory activity
of LMW-CI. The Mono S column retained LMW-CI at pH 6.5 up to a ionic
strength of 105 mM NaCl, whereas rhodocytin/aggretin barely
bound to Mono S at pH 6.5 and was washed off the column at very low
ionic strength. In the opposite elution order, LMW-CI was eluted from
the Mono Q column at pH 8.5 at low ionic strength of about 100 mM NaCl, whereas rhodocytin/aggregetin remained bound to
the Mono Q resin at NaCl concentrations of up to 300 mM
NaCl. In conclusion, the isoelectric point of LMW-CI must be higher than the one of rhodocytin/aggretin, although the isoelectric points of
both proteins must be in a pH range of 6.0-8.5. As final purification
step of LMW-CI, another gel filtration chromatography on a TSK
G3000SWXL was performed, resulting in a highly purified band at 27 kDa
in SDS-PAGE. Furthermore, coprecipitation experiments with
2
1 integrin showed that the 27-kDa
protein binds to the
2
1 integrin,
suggesting that the 27-kDa protein is the LMW-CI (data not shown).
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Fig. 5.
Purification of LMM-CI from C. rhodostoma venom using gel filtration on a Superose 6 HR30/30
column (A). The eluate fractions were tested in the
inhibition ELISA for their capability to inhibit binding of soluble
2
1 to immobilized type I collagen
(B). The inhibitory activity of the eluate fractions is
shown as relative inhibition, which is defined as difference between
noninhibited and inhibited binding signal normalized to the
noninhibited binding signal. Note that two inhibitory peaks are
separated on the size exclusion column that differ in their molecular
masses. They are named high molecular mass Calloselasma
inhibitor (HMW-CI) and low molecular mass
Calloselasma inhibitor (LMW-CI),
respectively.
2
1 integrin to immobilized type I
collagen in a dose-dependent manner (Fig.
6), thus proving that, in contrast to
rhodocytin/aggretin, the effect of rhodocetin on whole platelets (16)
can indeed be imitated on a molecular scale, i.e. on the
interaction of isolated
2
1 integrin to
collagen. With increasing concentrations, LMW-CI/rhodocetin decreased
the binding signal of the collagen receptor to its ligand and
eventually abolished it entirely. From Fig. 6, an IC50
value of about 30 nM could be determined.
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Fig. 6.
LMM-CI/rhodocetin inhibits binding of
soluble
2
1
integrin to immobilized type I collagen in a dose-dependent
manner. Monomeric bovine type I collagen was coated onto a
microtiter plate at 40 µg/ml in 0.1 M acetic acid. After
blockage of the wells with heat denatured BSA, soluble
2
1 integrin at 6 µg/ml was allowed to
bind to immobilized type I collagen in both absence and presence of
various concentrations of LMM-CI for 2 h at room temperature.
After the wells had been washed twice, the collagen-bound
2
1 integrin was detected by ELISA.
Nonspecific binding signal as measured in the presence of 10 mM EDTA was subtracted from all values. The binding signals
were then normalized to the noninhibited binding signal. Each value was
measured in duplicate. Relative standard deviations are shown.
2
1 Integrin--
Addition of various
protease inhibitors to the inhibition ELISA ruled out the possibility
that the decrease of
2
1 binding to
collagen was caused by proteolytic digestion of either binding partner
by a snake venom protease. Therefore, a direct, yet nonenzymatic binding interaction of LMW-CI/rhodocetin with either
2
1 integrin or with the integrin-binding
site on type I collagen must be responsible for its inhibitory effect.
To test a direct interaction of rhodocetin with the soluble
2
1 integrin, rhodocetin was immobilized
onto a microtiter plate, and binding of soluble
2
1 was measured. As shown in Fig.
7, the soluble
2
1 integrin directly bound to rhodocetin,
thereby qualifying it to be a disintegrin. The binding signal could be
increased slightly by addition of 1 mM MnCl2
and the integrin-activating antibody 9EG7. However, in contrast to other integrin ligands, binding of
2
1 to
rhodocetin did not require any divalent cations, because addition of
EDTA did not abolish
2
1 binding to
rhodocetin. A binding signal similar to the one of soluble
2
1 integrin was obtained when
detergent-extracted wild-type
2
1 integrin
from human platelets was applied (data not shown). Another soluble
integrin, the laminin-5 receptor
3
1 integrin (18), did not bind to immobilized, native rhodocetin, although
it showed binding activity to laminin-5 (Fig. 7). The soluble
3
1 integrin had been produced in our lab
by insect cells similarly to the soluble
2
1 integrin (18). Even more striking, another widespread collagen receptor,
1
1
integrin, which had been isolated from human placenta according to Kern
et al. (25) and was tested biologically active by its
binding to type I and IV collagen, entirely fails to bind to rhodocetin
(Fig. 7), proving the specificity of LMW-CI/rhodocetin to recognize
2
1 integrin selectively.
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Fig. 7.
LMW-CI is a disintegrin which interacts
directly and specifically with
2
1
integrin. LMW-CI at 30 µg/ml, monomeric bovine type I collagen
(bCol-1) at 30 µg/ml, monomeric human type IV collagen at
30 µg/ml (hCol-4), and human laminin-5 (hLam-5)
at 10 µg/ml were coated onto the microtiter plate in
TBS/Mg2+ ions, except for the collagen, which were
dissolved in 0.1 M acetic acid. After blocking with heat
denatured BSA, the wells were incubated with
octylglucoside-solubilized
1
1
integrin (open, filled, and gray
bars), with soluble
2
1 integrin
(thinly, thickly, and densely striped
bars), or with soluble
3
1 integrin
(thinly, thickly, and densely hatched
bars) for 2 h at room temperature either in the presence of 1 mM Mn2+ ions (open, thinly
striped, and hatched bars, respectively), or in the
presence of both 1 mM Mn2+ and a 3-fold molar
surplus of integrin-activating 9EG7 antibody (filled,
thickly striped, and hatched bars, respectively)
or in the presence of 10 mM EDTA (gray,
densely striped, and hatched bars, respectively).
Wild-type
1
1 had been extracted and
isolated from human placental tissue according to Kern et
al. (25). Both soluble human
2
1 and
3
1 integrin had been recombinantly
expressed in Drosophila Schneider cells and isolated as
described "Experimental Procedures" and according to Eble et
al. (18), respectively. After the wells had been washed twice,
substratum-bound integrin was chemically fixed, and its amount was
determined by ELISA. The binding signals onto heat denatured BSA were
taken as blanks. Each value was measured in duplicate, and standard
deviations are shown. Binding of
1
1 and
2
1 integrin to laminin-5 and binding of
3
1 integrin to type I and IV collagen
were not determined (n.d.).
2
1 integrin depended on its disulfide
bridges, which stabilize both its quartenary and tertiary structure.
Preincubation of LMW-CI at DTT concentrations higher than 0.016 mM without any thermal denaturation resulted in a strong
decrease of
2
1 binding (Fig.
8A). However, when scrutinized
by SGS-PAGE (Fig. 8B), the partially reduced
LMW-CI/rhodocetin run as stable heterodimer even up to 10 mM DTT. Amazingly, rhodocetin does not possess any intercatenary disulfide bridges (16), but its subunits stayed together
even under the harsh denaturating condition of the SDS-PAGE sample
buffer containing 2% SDS. Reduction of the intracatenary disulfide
bridges at DTT concentrations higher than 10 mM made the
rhodocetin heterodimer dissociate. Although lacking an intercatenary disulfide bridge, quaternary structure of rhodocetin is very stable and
depends on the tertiary structure of both subunits, which is stabilized
by intracatenary disulfide bridges. As the binding signal of soluble
2
1 integrin gradually decreased with
increasing DTT concentrations higher than 0.016 mM and is
entirely lost at 10 mM DTT, it can be envisioned that the
intracatenary disulfide bridges are of paramount importance in
maintaining the native tertiary structure of rhodocetin, which is
essential for
2
1 integrin binding.
Furthermore, the tertiary structure of its subunits as evidenced by its
integrin binding function seems to be even more sensitive to
denaturation than its quaternary structure, i.e.
dissociation into its two subunits.
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Fig. 8.
The native tertiary structure of
LMW-CI/rhodocetin, as evidenced by its inhibitory activity toward
2
1
integrin (A), is destroyed at much lower concentration
of reducing agents than its quartenary structure, i.e.
association of its two subunits, as detected by SDS-PAGE under
nonreducing conditions (B). A, LMW-CI
was incubated for an hour with increasing concentrations of DTT at
37 °C. After inactivation of DTT with a surplus of iodacetamide,
LMW-CI was coated onto a microtiter plate at 40 µg/ml. Soluble
2
1 integrin at 6 µg/ml was allowed to
bind to the pretreated LMW-CI. After being chemically fixed, bound
integrin was detected by ELISA. Binding signals were corrected for the
nonspecific binding signal on heat denatured BSA and normalized to
LMW-CI, which had been incubated without DTT and treated with
iodacetamide. Values were measured in duplicate. Standard deviations
are shown. Note that inibitory activity drops at DTT concentration
higher than 0.016 mM, and is completely lost at 10 mM. B, DTT and iodacetamide-treated LMW-CI,
which was used to coat the microtiter plate, was separated in a
12-18% polyacrylamide gradient gel under nonreducing conditions. Note
that nonreduced LMW-CI heterodimer vanishes at DTT concentrations
higher than 2 mM with a concomitant pronounced appearance
of the two LMW-CI subunits.
2
1 integrin, both native and denatured
rhodocetin were immobilized onto a microtiter plate and titrated with
soluble
2
1 integrin (Fig. 9). Treatment of rhodocetin with 40 mM DTT in addition to thermal denaturation entirely
abolished its binding activity to the integrin, again demonstrating
that the specific interaction of rhodocetin with
2
1 integrin requires the
disulfide-stabilized native conformation of rhodocetin. For binding of
soluble
2
1 integrin to native rhodocetin,
saturation was achieved at
2
1
concentrations of about 100 nM. From such titration curves,
an apparent Kd value of LMW-CI/rhodocetin binding to
2
1 integrin was calculated to be 10.3 nM.
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Fig. 9.
Titration of native (closed
symbols) and inactivated (open symbols)
LMW-CI/rhodocetin with soluble
2
1
integrin. Inactivation of LMW-CI was achieved by reduction of
disulfide bridges with 40 mM DTT and heat denaturation at
95 °C for 20 min. Reduced thiol groups were then blocked with 120 mM iodacetamide. Both native and inactivated LMW-CI was
coated onto the microtiter plate at 40 µg/ml and titrated with the
indicated concentrations of soluble
2
1.
Wells coated with heat denatured BSA were taken as blanks. The bound
2
1 integrin was chemically fixed, and its
amount was determined by ELISA. The blank values were subtracted from
the binding signals. Each value was measured in duplicate. Standard
deviations are indicated.
2
1 integrin are mostly collagenous
molecules. Laminin-1, which lacks any collagenous structure, is bound
by
2
1 integrin with much lower affinity.
Although LMW-CI/rhodocetin competed with the high affinity binding of
2
1 to type I collagen, the CD spectrum of LMW-CI (data not shown) did not reveal any collagenous triple helical
structure within the disintegrin. Although lacking a collagen domain,
rhodocetin possesses a distinct native structure, because deconvulation
of the CD spectrum recorderd at 20 °C disclosed a high content of
59.5%
-sheet and a minor amount of 10.3%
-helical secondary
structure for rhodocetin. Heat denaturation abrogated any secondary
structural signals in the CD spectrum, leaving a spectrum typical of
random coil.
2
1 integrin
with immobilized LMW-CI/rhodocetin (Fig.
10). Even at concentrations of 4 mM, which represented an 800,000-fold molar surplus to the
soluble
2
1 integrin in the inhibition
experiment, the GRGDSP peptide did not affect the
2
1 disintegrin interaction, thus showing
that LMW-CI/rhodocetin belongs to the small group of RGD-independent disintegrins.
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Fig. 10.
Binding of the disintegrin LMW-CI/rhodocetin
to
2
1
integrin does not depend on an RGD peptide sequence. LMW-CI was
coated onto a mircrotiter plate at 40 µg/ml. After being blocked with
heat denatured BSA, wells were incubated with soluble
2
1 integrin for 2 h in the absence
and presence of the linear peptide GRGDSP at concentrations indicated
in the plot. After wells had been washed twice, bound receptor was
chemically fixed, and its amount was determined by ELISA.
2
1 binding to heat denatured BSA was
taken as blank and subtracted from the binding values. Binding signals
were normalized to the noninhibited binding signal measured in the
absence of peptide. Values were determined in duplicate. Standard
deviations are indicated.
2
1-mediated
Adhesion of Fibroblasts--
Whether LMW-CI/rhodocetin can be used
in vivo, e.g. to inhibit
2
1-mediated cell adhesion and migration
or to influence other cellular reactions triggered by the
2
1-collagen interaction, the effect of
the isolated rhodocetin on adhesion of HT1080 cells onto immobilized
type I collagen was examined. HT1080 is a human fibrosarcoma cell line
that abundantly expresses
2
1 integrin on
its surface and that adheres to immobilized type I collagen mainly via
its
2
1 integrin (32). When HT1080 cells
were plated onto monomeric type I collagen in the presence of soluble
rhodocetin, cell adhesion declined with increasing concentrations of
the snake venom disintegrin and was eventually abolished completely
(Fig. 11). At a cell density of 500,000 cells/ml, plated onto 0.2 mg/ml type I collagen, an IC50
value for LMW-CI/rhodocetin of about 2 µg/ml = 60 nM
was measured, showing that, even at low concentrations, this
disintegrin is able to specifically affect
2
1-collagen interaction on a cellular
level.
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Fig. 11.
LMW-CI/rhodocetin efficiently and entirely
inhibits
2
1
integrin-mediated cell adhesion of HT1080 fibrosarcoma cells to type I
collagen. Monomeric type I collagen was coated at 0.2 µg/ml in
0.1 M acetic acid. After the wells were washed and blocked
with heat denatured BSA, HT1080 cells were plated onto the collagen
substratum at a density of 50,000 cells/well for 35 min in the absence
and presence of various concentrations of LMW-CI as indicated. Adhered
cells were stained with crystal violet, which was solubilized from the
cells after the wells had been destained. Absorbance was measured at
560 nm. Integrin-specific cell adhesion was completely abolished in the
presence of 10 mM EDTA. This blank value was subtracted
from the binding signals. The adhesion signals were normalized to the
noninhibited adhesion of HT1080 cells. Each value was measured in
triplicate. Standard deviations are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 integrin, one of the collagen
receptors on the surface of blood platelets. However, from the same
snake venom, we have isolated and characterized LMW-CI, which is
identical to rhodocetin, a recently published inhibitor of
collagen-induced platelet aggregation (16). We show that
LMW-CI/rhodocetin is a disintegrin that specifically and avidly binds
to
2
1 integrin. Independently of Wang
et al. (16), we have purified LMW-CI/rhodocetin as a
component of C. rhodostoma venom, which inhibits the binding of
2
1 integrin to immobilized collagen on
the molecular level. We used recombinantly expressed and purified,
soluble
2
1 integrin in an inhibition
ELISA to screen the snake venom for components that specifically and
nonproteolytically inhibit the interaction of
2
1 integrin with collagen. Although
commonly used, the method of using whole platelets to test for integrin
agonists and antagonists may be biased by the presence of various other
collagen receptors on the platelet surface. Furthermore, here we
describe our detailed studies on the interaction of the novel
disintegrin LMW-CI/rhodocetin with
2
1 integrin.
2
1 integrin, was made feasible
by the recombinant production of a soluble
2
1 integrin and its purification in sufficient amounts. Soluble
2
1 integrin
was generated by replacing the transmembrane and cytoplasmic domain of
both integrin subunits
2 and
1 with the
dimerizing motifs of the transcription factor Fos and Jun,
respectively. Lately, a similar attempt to produce soluble
3
1 had been successful (18). However,
yields of soluble
2
1 integrins were
generally lower than with soluble
3
1
integrin in compliance with the comparatively lower expression of
membrane-bound
2
1 on transfected
mammalian cells, such as erythroleukemic K562 cells.4 Although the human
integrin ectodomains were heterologously expressed by
Drosophila cells, both sunbunits were correctly processed
proteolytically, because the leader sequences were cleaved off to give
the N termini of both mature human subunits. Whereas the N-terminal
amino acid sequence of mature human
2 subunit was
accessible to Edman degradation, the
1 subunit was
N-terminally blocked. However, loss of signal sequence of the
1 subunit and its subsequent N-terminal blockage reaction to a pyroglutamate residue also occurs in the homologously expressed human
1 subunit, indicating a correct
processing of the soluble human
1 integrin subunit by
the insect cells. Furthermore, the ectodomain heterodimer of the
collagen receptor
2
1 integrin binds
avidly to monomeric type I collagen, suggesting that the integrin is
not only correctly processed but also correctly folded. The binding
signals of soluble
2
1 to different types
of collagen and laminin-1 demonstrated a ligand preference similar to
the wild-type
2
1 integrin, with
decreasing binding signals in the order of type I, type II, type IV,
and type V collagen and laminin-1 (25, 26). Binding affinities of
soluble
2
1 integrin could be increased
with Mn2+ ions and an activating antibody 9EG7, an
observation that resembles the activity regulation of wild-type
2
1 integrin.
2
1
integrin in comparison with the membrane-bound wild-type
2
1 integrin, which was isolated by
extracting blood platelets with the mild detergent
octylglucoside, is its stability and the comparatively high
yield. Whereas the detergent-extracted wild-type
2
1 integrin lost activity within days
after preparation,2 the soluble
2
1 integrin remained stable for several
weeks. However, we have not found any explanation for this observation yet.
2
1 integrin at hand, we could address the
question of which component of the hemorrhagic snake venom of C. rhodostoma is responsible for inhibiting the interaction of
2
1 integrin with type I collagen. This
interaction is of major importance for platelet reactions to collagen.
Collagen becomes accessible to platelets after damage of the blood
vessels or tissue injury. It initiates the activation of platelets (8,
30, 33), which results in degranulation and an increase in number
and/or affinity of other cell adhesion molecules on the platelet, such
as the major platelet integrin
IIb
3 (3),
which eventually leads to platelet aggregation and blood clotting.
RGD-containing disintegrins, such as rhodostomin (kistrin) from
C. rhodostoma (27, 34), inhibit the interaction of the
RGD-dependent
IIb
3 integrin
with fibrin, thereby impairing a later step in the blood clotting
mechanism. Moreover, snake venoms contain several proteases, such as
the metalloprotease rhodostoxin (kistomin and major hemorrhagin)
(27, 28), and the serine protease ancrod (29) from C. rhodostoma, which cleave fibrinogen/fibrin or prothrombin, thus
again interfering in the blood clotting cascade and resulting in
bleeding and hemorrhages.
2
1 integrin is of paramount
importance in hemostasis (9, 10, 17). Previous studies with the
hemorrhagic snake venom of C. rhodostoma were performed on
whole platelets. However, platelets contain various collagen receptors
on their surface with different characteristics, e.g.
2
1 integrin (GPIa/IIa) and GPVI (9, 10,
17). Whereas GPVI mainly recognizes type I collagen molecules, which
are bundled into collagen fibrils, in a divalent cation-independent
manner,
2
1 integrin mainly binds to
monomeric type I collagen molecules in the presence of divalent cations
(35-37). The importance of
2
1 integrin
in normal hemostasis is corroborated by severe bleeding disorders in
patients, who either lack the
2
1 integrin
receptor on their platelets (11) or who have developed inhibiting
autoantibodies against the
2 integrin subunit (12).
2
1 integrin-collagen interaction,
inasmuch as snake venoms by themselves contain a whole battery of
various agents interfering with platelet activation and blood clotting.
Based on such studies with whole platelets, rhodocytin/aggretin have
been published to be an activator of
2
1
integrin-mediated platelet aggregation (15, 31). However, we have
isolated rhodocytin/aggretin, proved its identity by N-terminal sequencing, and could not see any interaction of rhodocytin with
2
1 integrin. Nor could we observe any
influence of rhodocytin on the integrin-collagen interaction.
Therefore, a direct interaction of rhodocytin/aggretin with the soluble
2
1 integrin ectodomain on the protein
level can be ruled out. Alternatively, its effects on platelets
aggregation may be caused by protein-carbohydrate interactions.
Wild-type
2
1 integrin on the platelets
surface may differ from recombinantly expressed soluble
2
1 integrin in their carbohydrate side
chains, because Drosophila Schneider cells are unable to
process the N-linked carbohydrate side chains of proteins
from high mannose type into complex type carbohydrate antennary
structures (18). Because rhodocytin/aggretin belongs to the family of
C-type lectins bearing homology to the carbohydrate recognition domains
of Ca2+-dependent lectins (31), it can be
surmised that rhodocytin cross-links several
2
1 integrins on the platelet surface via their carbohydrate side chains, thereby imitating the recruitment of
integrins into focal contact-like structures, which eventually leads to
platelet activation and aggregation. However, because both
recombinantly expressed soluble
2
1
integrin and wild-type
2
1 integrin
extracted from platelets that are likely to bear high-mannose type and
complex-type N-linked carbohydrate side chains,
respectively, fail to interact with rhodocytin/aggretin in our binding
tests, even in the presence of Ca2+ ions, a mechanism
involving a direct interaction of rhodocytin/aggretin with
2
1 integrin to explain platelet
activation and aggregation by rhodocytin can be considered very unlikely.
2
1 integrin instead of
detergent-extracted wild-type
2
1 integrin even allows us to work not only in a cell-free but also in a
detergent-free test system. Therefore, we could identify the
disintegrin LMW-CI from C. rhodostoma venom, which
specifically binds to
2
1 integrin in an
RGD-independent manner, thereby inhibiting the interaction of
2
1 integrin with immobilized, monomeric
type I collagen. N-terminal sequencing of LMW-CI revealed its identity
with rhodocetin (16). We called this inhibitor low molecular weight
Calloselasma inhibitor to distinguish it from another
2
1 integrin inhibiting activity of the
C. rhodostoma venom that was found in an earlier eluate
fraction, i.e. higher molecular mass fraction, of a size exclusion column. However, we have not yet characterized the latter one, which we referred to as high molecular weight
Calloselasma inhibitor. Although binding with high affinity
and specificity, the LMW-CI/rhodocetin does not need any divalent
cations to be bound by the integrin. Its native three-dimensional
structure, which is stabilized by disulfide bridges, is essential for
2
1 bindung. Unlike the other high
affinity collagen ligands of
2
1 integrin,
LMW-CI/rhodocetin lacks a collagenous triple helical conformation.
Nevertheless, it binds to
2
1 integrin
avidly and even competes with collagen efficiently. Because we had
included protease inhibitors into the test assay and proved absence of protease activities in the LMW-CI/rhodocetin preparation by zymogram, the inhibitory effect of LMW-CI/rhodocetin is not caused by proteolytic activity.
2
1 integrin, we found that the
native tertiary structure of rhodocetin, which is required for integrin
binding, is lost at much lower concentrations of reducing agents than
the quartenary structure, detected as dissociation of the two subunits
in SDS-PAGE under nonreducing conditions.
2
1 integrin
and recombinantly expressed, soluble
2
1
integrin bind equally well to immobilized LMW-CI/rhodocetin, although
the two integrins may vary in their glycosylation pattern of
N-linked carbohydrate side chains, suggesting that the
interaction of the novel disintegrin LMW-CI/rhodocetin bases on a
protein-protein interaction. This direct interaction then causes the
inhibition of collagen binding to
2
1 integrin.
1
1 integrin, the other collagen-binding
integrin with a widespread tissue distribution. Nor does this
disintegrin interact with
3
1 integrin.
Therefore, LMW-CI/rhodocetin differs from other, mainly RGD-dependent disintegrins in its unique specificity toward
2
1 integrin. Interestingly,
LMW-CI/rhodocetin does not require divalent cations to bind to
2
1 integrin, because, in contrast to
other integrin ligands, deprivation of divalent cations by EDTA does not abolish
2
1 binding to
LMW-CI/rhodocetin. This suggests a binding mechanism distinct from the
integrin binding mechanism to collagen. Nevertheless, LMW-CI can
completely abolish
2
1 integrin binding to
collagen. Either LMW-CI binds at a site within
2
1 integrin distinct from the ligand
binding site that leads to a conformational change and to an allosteric
inactivation of the integrin, or LMW-CI binds to a site within
2
1 integrin, which is overlapping or even
identical to the collagen binding site, thereby inhibiting collagen
binding sterically. Future structural studies will help to answer this question.
2
1 integrin, LMW-CI indispensibly needs
its native conformation, which is stabilized by disulfide bridges.
Further structural insights into LMW-CI were gained by CD spectroscopy.
The CD spectrum of LMW-CI is in good agreement with the CD spectrum
provided by in Refs. 16. It clearly demonstrated that LMW-CI/rhodocetin
does not bear any structural resemblance to a collagenous triple helix, which is typical of high affinity ligands of
2
1 integrin. Still, LMW-CI/rhodocetin
avidly binds to
2
1 integrin and
efficiently competes with type I collagen.
2
1 integrin and preventing it from
binding to its collagen ligand, we eventually returned to whole cells
to study the
2
1-related functions in the
cellular context.
2
1 integrin not only is
a pivotal trigger in hemostasis, but its widespread distribution on
other cell types also suggests a much broader biological role in the
organism (17, 38). The presence of
2
1
integrin on endothelial cells of newly grown blood capillaries suggests
a potential role in angiogenesis (39). Fibroblasts also bear
2
1 integrin and use it to exert mechanical forces to a surrounding collagen gel, which in
vivo takes place in connective tissue to maintain the shape of
tissues and organs, during wound contraction and scar formation (40). Furthermore, ligand occupancy of
2
1
integrin on fibroblasts and epithelial tumor cells elicits expression
of various matrix metalloproteases (MMPs) (40), such as interstitial
collagenase (MMP-1) (41, 42), stromelysin-1 (MMP-3) (43), collagenase-3 (MMP13), and membrane-bound metallomatrixproteinase-1 (MT1-MMP, MMP14)
(44). The latter one itself proteolytically activates gelatinase A
(MMP-2) (40).
2
1 integrin-triggered
secretion of MMPs is a key point in tumor invasion and metastasis,
because these proteases degrade extracellular matrix proteins, among
them basal membrane proteins, thus opening the path for invading tumor cells. To manipulate such
2
1-triggered
effects, LMW-CI/rhodocetin may be a valuable tool because of its unique
specificity and high affinity toward
2
1
integrin. Another advantage of LMW-CI/rhodocetin is its high solubility
under physiological conditions compared with the poor solubility of
collagen, which because of its high tendency to aggregate and
precipitate cannot be applied as soluble inhibitor. Furthermore,
because of its independence of divalent cations, LMW-CI is likely to
bind in vivo as effectively as in the cell-free test,
whereas
2
1 integrin binds less avidly to collagen in vivo because of the presence of
1-integrins attenuating Ca2+ ions under
physiological conditions. With HT1080 fibrosarcoma cells, which adhere
to type I collagen mainly via
2
1
integrin, we demonstrated that LMW-CI indeed inhibits cell adhesion to
type I collagen as the initial step of integrin-mediated cell
migration, gene activation, and anchorage-dependent growth.
LMW-CI/rhodocetin efficiently and completely inhibits
2
1-mediated cell adhesion to type I
collagen, proving its suitability as specific
2
1 integrin inhibitor in vivo.
Thus, LMW-CI may be a useful agent to study and influence
2
1 integrin-triggered cell function, like
cell adhesion, cell migration, or secretion of MMPs. Therefore, it may
help not only in treating thrombosis but also in treatments aimed to
prevent tumor invasion and metastasis.
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ACKNOWLEDGEMENTS |
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Type IV collagen and its fragment CB3[IV], as well as type V collagen and laminin-1 were kindly provided by Klaus Kühn, Rupert Timpl, and Albert Ries (Max-Planck-Institute for Biochemistry, Martinsried, Germany). Type II collagen was gratefully obtained by Peter Bruckner (Institute for Physiological Chemistry, Münster, Germany). We also appreciated the monoclonal antibody JA218, a kind gift of Danny S. Tuckwell (School of Biological Sciences, Manchester, UK).
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Eb177/3-1 (to J. A. E.).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: Inst. für Physiologische Chemie und Pathobiochemie, Universität Münster, Waldeyerstr. 15, 48149 Münster, Germany. Tel.: 49-251-835-2289; Fax: 49-251-835-5596; E-mail: eble@uni-muenster.de.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009338200
4 J. A. Eble, unpublished observations.
2 Brackets indicate two possible amino acids that could not be clearly identified in the sequencing cycle. Parentheses indicate a less likely amino acid when the Edman degradation cycle did not give a clear identification but an option of two or more possible amino acids.
3 See footnote 2. A hyphen indicates that no amino acid could be detected in the respective cycle of Edman degradation.
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ABBREVIATIONS |
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The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; MMP, matrix metalloprotease.
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