(Received for publication, August 7, 1995; and in revised form, October 5, 1995)
From the
Previous studies have shown that the neurotoxin dendroaspin and
the disintegrin kistrin, which show little overall sequence homology
but similar residues around RGD (PRGDMP), preferentially inhibited
platelet adhesion to fibrinogen. In contrast, the elegantin which has
different amino acids around RGD (ARGDNP) preferentially inhibited
platelet adhesion to fibronectin. To investigate further the role of
amino acids around RGD in disintegrins, we have constructed the genes
of a wild-type and of two mutant dendroaspins with substitutions around
the RGD, namely [Asn]- and
[Ala
,Asn
]dendroaspins. Proteins
were expressed in Escherichia coli as glutathione S-transferase fusion recombinants and purified to homogeneity
by affinity chromatography and reversed phase high performance liquid
chromatography. Platelet aggregation studies revealed that wild-type
dendroaspin showed an IC
value similar to that of native
dendroaspin, with
[Ala
,Asn
]dendroaspin showing an
IC
value similar to that of elegantin. Interestingly, in
platelet adhesion assays, the mutants showed a progressive shift in
inhibitory preference, in particular,
[Ala
,Asn
]dendroaspin showed nearly
identical behavior as elegantin when fibronectin was the immobilized
ligand (IC
= 0.33 µM and 0.6
µM, respectively, compared with 20 µM for
native dendroaspin). Native and recombinant wild-type dendroaspin bound
to a single class of binding site exhibiting a K
= 67 nM; [Asn
]- and
[Ala
,Asn
]dendroaspins, however,
both produced biphasic isotherms with K
values = 87 nM and 361 nM for
[Asn
]dendroaspin and 33 nM and 371
nM for
[Ala
,Asn
]dendroaspin, which are
close to those of elegantin (K
values
= 18 nM and 179 nM). These studies prove that
the amino acids flanking RGD provide an extended locus that regulate
the affinity and selectivity of RGD protein dendroaspin.
Integrins are a family of cell surface receptors that mediate
adhesion of cells to each other or to extracellular matrix substrate (1, 2, 3, 4, 5) . They are
composed of noncovalently associated and
transmembrane
subunits selected from among 16
and 8
subunits that
heterodimerize to produce 20 receptors(6) . Among the
integrins, the platelet membrane
is the best characterized(3, 5) . Upon cell
activation, the
integrin binds
several glycoproteins, predominantly through the Arg-Gly-Asp (RGD)
tripeptide sequence (6, 7, 8) present in
fibrinogen(9) , fibronectin(10) , von Willebrand
factor(11) , vitronectin(12) , and
thrombospondin(13) . The nature of the interactions between
these glycoprotein ligands and their integrin receptors is known to be
complex, and conformation changes occur in both the receptor (14) and the ligand(15) .
Recently, many proteins
from a variety of snake venoms have been identified as potent
inhibitors of platelet aggregation and integrin-dependent cell
adhesion. The majority of these proteins which belong to the
disintegrin family share a high level of sequence homology, are small
(4-8 kDa), cysteine-rich, and contain the sequence RGD (16) or KGD(17) . In addition to the disintegrin
family, a number of non-disintegrin RGD proteins of similar inhibitory
potency, high degree of disulfide bonding, and small size have been
isolated from both the venoms of the Elapidae family of snakes (18, 19) and leech homogenates(20) . All of
these proteins are approximately 1000 times more potent inhibitors of
the interactions of glycoprotein ligands with the integrin receptors
than simple linear RGD peptides, a feature that is attributed to an
optimally favorable conformation of the RGD motif held within the
protein scaffold. The NMR structures of several inhibitors including
kistrin(21, 22, 23) ,
flavoridin(24) ,
echistatin(25, 26, 27, 28) ,
albolabrin(29) , decorsin(30) , and dendroaspin ()(31, 32) have been reported, and the only
common structural feature elucidated so far is the positioning of the
RGD motif at the end of a solvent exposed loop, a characteristic of
prime importance to their inhibitory action.
Recent studies have
implied a role for the amino acids around the tripeptide RGD in
regulating the ligand binding specificity shown by snake venom
proteins. Scarborough et al.(33) examined a range of
disintegrins and observed that those containing RGDW were very
effective at inhibiting the interactions of fibrinogen to purified
but not of vitronectin and
fibronectin to purified
and
, respectively, whereas the converse
was true for disintegrins containing the sequence RGDNP. Other regions
of amino acid sequence divergence may also be
contributory(33) . We have reported that dendroaspin, a short
chain neurotoxin analogue containing the RGD sequence, and the
disintegrin kistrin, which show little overall sequence homology but
have similar amino acids flanking the RGD sequence (PRGDMP), are both
potent inhibitors of platelet adhesion to fibrinogen but poor
antagonists of the binding of platelets to immobilized
fibronectin(34) . In contrast, elegantin, which has 65%
sequence homology to kistrin but markedly different amino acids around
RGD (ARGDNP), preferentially inhibited platelet adhesion to fibronectin
as opposed to fibrinogen and binds to an allosterically distinct site
on
complex. These studies
suggested that the amino acids around the RGD determine the affinity
and selectivity of these RGD proteins. In addition to RGD domains, a
number of recent studies have suggested that amino acids at the
carboxyl terminus of these proteins may affect their interactions with
integrins. Deletion of the PRNP sequence from echistatin has been
reported to reduce its ability to inhibit platelet aggregation,
implying a reduction in the binding affinity(16) . Furthermore,
the complete carboxyl-terminal peptide (PRNPHKGPAT) of echistatin not
only competed with the binding of echistatin to the
complex but also enhanced the
binding of fibronectin and vitronectin to the purified
, indicating that this non-RGD
component of the protein was able to alter the integrin affinity for
glycoprotein ligands(35) . However, the mechanism by which
amino acids at the carboxyl terminus of these proteins interact with
their receptors and their binding characteristics are not yet
understood.
In this study we examined the role of amino acids
flanking the RGD sequence by expressing the neurotoxin variant
dendroaspin in Escherichia coli and using site-directed
mutagenesis. Dendroaspin, unlike echistatin, does not have any
appreciable sequence at its carboxyl-terminal after Cys making it an excellent model to study the functional role of the
nature of the amino acids flanking the sequence RGD. We show that
recombinant dendroaspin has inhibitory properties identical with native
dendroaspin indicating that the expressed protein has the correct
folding and disulfide bonding and that substituting Met
Asn (PRGDMP
PRGDNP) or Met
Asn
and Pro
Ala (PRGDMP
ARGDNP) dramatically
altered the preferential inhibitory properties and binding
characteristics of the protein to the
complex to that of the disintegrin elegantin containing the
sequence ARGDNP. These studies prove that the amino acid flanking
sequence RGD provide an extended locus that determines the preferential
selectivity of dendroaspin.
The functional characterization of the
recombinant wild-type and of the mutant dendroaspins purified from cell
lysates of E. coli was determined by platelet aggregation and
adhesion assays. In order to verify that the expression system
generated was satisfactory, we first compared the functional properties
of recombinant dendroaspin with that of native dendroaspin purified
from snake venom. As shown in Table 2, recombinant dendroaspin
showed platelet aggregation inhibition as potent as native dendroaspin
and displayed similar inhibitory activity toward ADP-induced platelet
aggregation both in platelet-rich plasma and washed platelets. This
indicated that the protein folded correctly and formed the correct
disulfide bondings. The mutant [Asn]dendroaspin
showed an IC
value similar to that of recombinant
dendroaspin, while the mutant with two substitutions,
[Ala
,Asn
]dendroaspin, showed an
IC
value similar to that observed with elegantin (Table 2).
We previously observed that measurement of
ADP-activated platelet adhesion to immobilized glycoproteins highlights
selective inhibitory preferences for RGD snake venom proteins that are
less easily discernible using the platelet aggregation
assay(34, 39) . In such experiments, we have shown
that dendroaspin and kistrin are potent inhibitors of platelet adhesion
to fibrinogen, whereas elegantin preferentially inhibits platelet
adhesion to fibronectin(34) . Fig. 1and Table 3(showing IC values) illustrate the results
obtained with the wild-type recombinant and mutant dendroaspins in
comparison with the inhibitory properties of native dendroaspin and
elegantin. Wild-type dendroaspin showed an identical inhibitory profile
toward the inhibition of platelet adhesion to fibrinogen compared to
native dendroaspin. Interestingly, the mutants
[Asn
]dendroaspin and
[Ala
,Asn
]dendroaspin exhibited a
progressive decrease in their ability to inhibit platelet adhesion to
fibrinogen in both maximal inhibitory levels (using a maximum of 5
µM protein) and IC
values. Indeed,
appropriate substitutions at both flanking positions, i.e. [Ala
,Asn
]dendroaspin, showed
an 8-fold lower IC
that approached the value obtained with
elegantin. Studies using fibronectin as the immobilized ligand showed
an even more striking change with respect to antagonistic preference.
Both recombinant and native dendroaspins were relatively poor
inhibitors of activated platelet adhesion to fibronectin displaying
only 40% inhibition at 15 µM. However, the singly
substituted Met
Asn of dendroaspin has a similar
IC
for platelet adhesion to both fibrinogen and
fibronectin, whereas the doubly substituted (Pro
Ala and Met
Asn) mutant shows an approximately
4-fold preference for inhibition of binding to fibronectin. In
particular, the latter showed a maximal extent of inhibition and
IC
values that were markedly similar with those displayed
by elegantin. Thus, substituting Pro
Ala and
Met
Asn in the residues immediately flanking the
RGD in dendroaspin altered the inhibitory preferences of dendroaspin to
that of elegantin. The presence of asparagine adjacent to aspartic acid
would be particularly important in inhibiting the interactions of
fibronectin with its receptor. This study strongly supports our
previous studies(34) .
Figure 1:
Inhibition of platelet adhesion to
immobilized glycoproteins by RGD-containing proteins. Washed platelet
suspensions were incubated with various concentrations of native
dendroaspin (), recombinant wild-type dendroaspin (
),
[Asn
]dendroaspin (
),
[Ala
,Asn
]dendroaspin (
), or
elegantin (
) for 3 min prior to application to microtiter wells
coated with either 10 µg/ml fibrinogen (A) or fibronectin (B). The number of adherent platelets were determined by
measurement of endogenous acid phosphatase as described
previously(34) . Results are expressed as percent inhibition
relative to the number of adherent platelets observed in the absence of
inhibitors. All points were performed in quadruplicate, and the mean
± S.E. are shown in Table 3(n =
2-4).
Binding of I-labeled
recombinant and mutated dendroaspins and of
I-elegantin
to activated platelets was studied to determine whether the alterations
in functional properties of the mutated dendroaspins were reflected in
their binding characteristics. All four
I-labeled
proteins bound to ADP-activated platelets in a saturable and
cation-dependent manner (Fig. 2, insets). Scatchard
analysis of the data using the Kinetic, EBDA, Ligand, and Lowry version
4 software programs (BIOSOFT, Cambridge, UK) indicated that recombinant
dendroaspin bound to a single class of binding site exhibiting a K
= 67 nM (Fig. 2A and Table 3) with a B
equal to
approximately 29,100 sites per platelet. The
[Asn
]- and
[Ala
,Asn
]dendroaspin, however, both
produced biphasic isotherms again using the Kinetic, EBDA, Ligand, and
Lowry version 4 software with K
values = 87
nM and 361 nM for
[Asn
]dendroaspin (Fig. 2B and Table 3) and 33 nM and 371 nM for
[Ala
,Asn
]dendroaspin (Fig. 2C and Table 3). In agreement with the
results obtained with the adhesion experiments, the mutant dendroaspins
showed a progressive shift in their binding characteristics toward
those of elegantin (K
values = 18
nM and 179 nM) as shown in Fig. 2D and Table 3. Considering that
[Ala
,Asn
]dendroaspin and elegantin
share little sequence homology ( Table 1and Table 2) and
have structures unrelated, except for the (A)RGD(N) domain, the close
similarity of the dissociation constants is striking.
Figure 2:
Scatchard analysis of binding of I-labeled-proteins to ADP treated platelets. Scatchard
analysis was performed with the RADLIG (radioligand) software version 4
of Kinetic, EBDA, Ligand, and Lowry (BIOSOFT, Cambridge, UK). Varying
concentrations of
I-labeled recombinant wild-type
dendroaspin (A; one-site fit, R
=
0.990),
I-labeled [Asn
]dendroaspin (B; two-site fit, R
= 0.999 and
0.999, respectively),
I-labeled
[Ala
,Asn
]dendroaspin (C;
two-site fit, R
= 0.996 and 0.983,
respectively), and
I-labeled elegantin (D;
two-site fit, R
= 0.990 and 0.997,
respectively) were incubated with ADP-treated, washed platelets (3
10
/ml) for 30 min at room temperature in a volume
of 350 µl. Bound and free levels of
I-labeled
RGD-containing proteins were determined by loading 300 µl of the
platelet suspension onto a cushion of 25% (w/v) sucrose, 1% bovine
serum albumin and centrifuged for 10 min at 12000
g.
Both the radioactivity in the platelet pellets and supernatants were
determined. Insets, saturation isotherms of
I-labeled RGD-containing proteins binding to ADP-treated
washed platelets. The curves show nonspecific (
),
specific (
), and total (bound + free) binding (
).
The values are representive of three similar experiments with all
points performed in duplicate. (S.E. were less than
10%.)
To confirm
that both binding sites occupied by the two dendroaspin mutants on
ADP-treated platelets were present on the
integrin complex, the effects of
two inhibitory antibodies on radioligand binding were monitored.
PM6/248, a monoclonal antibody with specificity for the native
complex(36) , effectively
inhibited in a dose-related manner the binding of all these
I-labeled recombinant dendroaspins by 80-100% (Fig. 3). In contrast, an anti-
antibody was comparatively ineffective, confirming that the
binding parameters observed were specifically associated with the
complex.
Figure 3:
Inhibition of I-labeled
proteins to ADP-treated platelets by antibodies. Platelet suspensions
were incubated with various concentrations of the
specific monoclonal antibody
PM6/248 (filled symbols) or a polyclonal antibody raised
against the integrin
(open
symbols) for 30 min before addition of
I-labeled
recombinant dendroaspin (
,
),
I-labeled
[Asn
]dendroaspin (
,
),
I-labeled
[Ala
,Asn
]dendroaspin (
,
), and
I-labeled elegantin (
,
) at 30
nM for monoclonal antibody PM6/248 and 170 nM for the
polyclonal antibody raised against the integrin
and ADP. Platelet suspensions were
then incubated at room temperature for an additional 60 min. Levels of
bound
I-labeled proteins were determined as described in
the legend to Fig. 2. The results are expressed as percent
inhibition.
Further evidence for
the close similarity in the binding of
[Ala,Asn
]- and
[Asn
]dendroaspin and of elegantin to the
complex was obtained by examining
the association kinetics of three ligands (Fig. 4). Native and
recombinant dendroaspin show simple and rapid binding, reaching
equilibrium by 5 min. However, elegantin, [Asn
]-
and [Ala
,Asn
]dendroaspin showed
complex association kinetics with approximately 3- to 4-fold higher
binding before equilibrium than at equilibrium. The reasons for this
complex association pattern are not known at present, but are not due
to internalization of the ligand as the binding was fully reversible
(data not shown). That [Asn
]- and
[Ala
,Asn
]dendroaspin, but not
native dendroaspin, behaved in this manner points to this property
being solely due to the presence of the ARGDN sequence, and whether
other ARGDN-containing disintegrins, e.g. viridin, jararacin,
cotiarin (Table 1), behave in a similar manner remains to be
examined.
Figure 4:
Association kinetics of I-labeled proteins to ADP-treated platelets.
I-labeled [Asn
]dendroaspin
(
),
I-labeled
[Ala
,Asn
]dendroaspin (
),
I-labeled dendroaspin (
),
I-labeled
recombinant dendroaspin (
), and
I-labeled elegantin
(
) (all at 40 nM) were incubated with ADP-treated
platelets for various lengths of time before determination of the bound
and free ligand concentrations as described previously. Results are
expressed as percent ligand bound relative to bound levels at
equilibrium. All data shown are either a representive experiment or a
compilation of two independent determinations with points performed in
duplicate.
These studies report that the amino acids around the RGD motif regulate the affinity and selectivity of the RGD protein dendroaspin and support our earlier studies (34) and those of Scarborough et al.(33) . Further details of the mechanisms of integrin/ligand interactions will benefit greatly from the analysis of both wild-type and mutant dendroaspins by x-ray crystallography or NMR spectroscopy. Until the receptor-ligand complexes are available for such structural studies, the further structure/function evaluation of snake venom adhesive ligands may allow us to engineer potent antagonists that show not only ligand specificity but also receptor specificity.