(Received for publication, January 28, 1997, and in revised form, May 28, 1997)
From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom
Integrin ligands almost invariably employ a
variant of either the RGD or LDV motif as a key element of their
receptor recognition site. These short acidic peptide sequences
collaborate with specific nonhomologous flanking residues and spatially
separate "synergy" sequences to determine receptor binding
specificity. Although the consensus sequences for RGD and LDV motifs
are quite different, their common use suggests that they might share a
critical role in receptor-ligand engagement. To date, the effects of
interconversion of the two motifs within a natural protein framework
have not been tested; however, in this study, we have converted the
natural RGD site found in the snake venom disintegrin kistrin into an LDV motif and examined the effects of the change on the specificity of
integrin recognition and on disintegrin potency. While an assessment of
receptor binding using cell adhesion and purified integrin solid-phase
assays demonstrated recognition of recombinant RGD kistrin by V
3
and
5
1, a series of LDV kistrin chimeras did not bind to these
integrins, but instead were recognized specifically by
4
1. The
minimal change to elicit this distinct switch in receptor specificity
was found to involve alteration of only three residues within kistrin.
Alanine scanning mutagenesis was used to provide further information on
the functional contribution of the three residues. More important, the
LDV kistrin chimeras also retained much of the characteristic potency
of RGD kistrin, indicating that the kistrin scaffold is optimized for
presentation of both RGD and LDV sequences. These findings provide
evidence for similarities in motif pharmacophore and reinforce the
hypothesis that RGD and LDV sites have an equivalent functional role in
receptor binding. They also demonstrate the potential for other
disintegrin-containing proteins, perhaps from the ADAM family, to
employ LDV sequences for integrin binding.
Cell-surface adhesion receptors mediate the cell-cell and
cell-matrix interactions that are fundamental to normal cell behavior and tissue organization. The integrins, a superfamily of structurally related heterodimers, represent a major class of adhesion
receptors (for reviews, see Refs. 1-4).
Many studies of the molecular basis of integrin-ligand binding have now
been performed, and frequently, integrins have been found to recognize
short peptide motifs within their ligands. The RGD motif, which
contains a critical aspartate residue, was first identified in the
central cell-binding domain of fibronectin (5-7), but has subsequently
been shown to be functional in other molecules, including vitronectin
and von Willebrand factor. The molecular mechanisms involved in the
recognition of the RGD sequence are complex since it binds to a number
of different integrins, and receptor specificity varies between
different matrix molecules. A second peptide motif, LDV, present within
the alternatively spliced CS1 sequence of the IIICS region of
fibronectin, also contains a crucial aspartate and is primarily
recognized by integrin 4
1 (8-10). The integrin-binding members
of the immunoglobulin superfamily possess motifs that are homologous to
LDV, including the tetrapeptide sequence IDSP in domains 1 and 4 of
VCAM-1 (11-14) and glutamate-containing sequences in the
membrane-distal domains of intercellular adhesion molecules (15-17). A
third aspartate-dependent motif, which contains QAGDV as
the minimal active sequence, is found near the C terminus of the
chain of human fibrinogen and is recognized by platelet integrin
IIb
3 (18).
The active-site motifs of integrin ligands can be reproduced synthetically in the form of peptides, and these reagents have been instrumental in providing information on the specificities of receptor-ligand binding and for the development of therapeutic agents for diseases involving aberrant adhesion (19). In addition, the insertion of peptide motifs into nonadhesive protein scaffolds has previously been reported to generate adhesive activity. The RGD sequence has been incorporated into a long exposed loop within human lysozyme by site-directed mutagenesis, and the resultant mutant protein was able to promote cell adhesion (20). Insertion of RGD into the finger-like structure of hirudin produced a molecule capable of inhibiting platelet aggregation (21). Both the RGD- and LDV-containing CS1 sequences have been successfully grafted into staphylococcal protein A, with the creation of the cell adhesive activities of both motifs (22, 23).
Although short sequence motifs such as RGD and LDV are critical for integrin recognition, they do not account for the entire binding event, as synergistic sequences have been identified that collaborate with them (24, 25), and nonfunctional RGD and LDV sequences are also present in many molecules. The affinity and selectivity of linear peptides can be improved by the introduction of a conformational constraint, for example, by cyclization (7, 26). Thus, differences in conformation, flanking residues, and the use of synergy sequences may all contribute to the binding specificities of different integrin-ligand combinations.
A conformationally constrained, functional RGD motif is also found
within many disintegrins, a family of naturally occurring polypeptide
integrin antagonists present in snake venoms (for reviews, see Refs.
27-29). Disintegrins inhibit platelet aggregation by blocking the
adhesive function of integrin IIb
3 (30), but are also able to
disrupt the adhesive functions of other RGD-dependent integrins and are therefore relatively nonspecific (31-33). It is
notable that disintegrins are up to 1000-fold more potent than linear
RGD-containing peptides (34), probably because the RGD sequence exists
in a favorable conformation at the apex of a long loop across the
surface of the molecule (35).
The disintegrin molecule may be considered as a model scaffold for the presentation of an integrin recognition motif, and in this study, we have examined the effects of engineering the replacement of the RGD sequence in the disintegrin kistrin with LDV to assess whether it is possible to retain potency in an integrin antagonist while altering specificity. We have reported in preliminary form the design of synthetic kistrin cDNA and the expression of the molecule as a recombinant protein in which specific residues or segments can be mutated to create chimeric kistrins (36).
Rat
mAb1 13 (IgG), recognizing
the human integrin 1 subunit, and rat mAb 16 and mAb 11 (IgG),
recognizing the human integrin
5 subunit, were gifts from S. K. Akiyama (NIDR, National Institutes of Health, Bethesda, MD) (37). Mouse
mAb HP2/1 (IgG), recognizing the human integrin
4 subunit, was
purchased from Serotec Ltd. (Oxford, United Kingdom). LM609 mAb
(ascites), recognizing human integrin
V
3, was a gift from D. A. Cheresh (Scripps Research Institute, La Jolla, CA), and mAb 17E6
(IgG), recognizing the human integrin
V subunit, was from S. Goodman
(E. Merck, Darmstadt, Germany). Mouse anti-human mAb P1E6 (ascites),
recognizing the integrin
2 subunit, was obtained from GIBCO
(Paisley, Scotland). All anti-integrin antibodies used were
function-blocking, with the exception of mAb 11. An 80-kDa fragment of
plasma fibronectin containing the central cell-binding domain was
generated as described (38). H/120, a recombinant fragment of
fibronectin containing the HepII/IIICS region, was produced as
described (39). Integrin
4
1 was isolated from the MOLT-4 human T
lymphoblastic cell line as described (39). Integrin
5
1 was
purified from human placenta as described (40).
Kistrin
cDNA was generated from complementary oligonucleotides designed
from the protein sequence using Escherichia coli codon usage
data. BamHI and EcoRI restriction enzyme sites
were incorporated at the ends of the cDNA sequence to allow cloning
into the phagemid vector pUC118. Internal BspMII,
NarI, BglII, and KspI restriction sites were also incorporated to allow exchange of different segments of
the molecule (Fig. 1). The complementary
phosphorylated oligonucleotides were annealed and ligated into the
dephosphorylated vector pUC118. Competent E. coli DH5F
cells were transformed with the ligation product, and the kistrin
cDNA sequence was verified before subcloning into the pGEX2T
expression vector (Pharmacia Biotech, Milton Keynes, UK) using the same
restriction sites. DH5
F
cells were transformed, and glutathione
S-transferase-kistrin fusion proteins were induced and
isolated as described (41). Briefly, a 40-ml overnight culture of
transformed DH5
F
cells was diluted 1:10 with fresh LB medium containing 50 µg/ml ampicillin and cultured for 1 h at 37 °C. Isopropyl-
-D-thiogalactopyranoside was added to 0.1 M and cultured for a further 4 h. Cells were then
centrifuged, resuspended in divalent cation-free Dulbecco's
phosphate-buffered saline, and lysed by sonication. The extract was
cleared by centrifugation and applied to a glutathione-agarose affinity
column (Sigma, Poole, UK) pre-equilibrated with 150 mM NaCl
and 10 mM Tris-HCl, pH 7.5. The column was washed with 150 mM NaCl and 10 mM Tris-HCl, pH 7.5, and the
fusion protein was eluted with 5 mM reduced glutathione and
50 mM Tris-HCl, pH 7.5. The glutathione
S-transferase carrier was removed by cleavage with human
thrombin (Sigma) for 3 h at room temperature using an
enzyme/substrate ratio of 1:500 (w/w). A second glutathione-agarose
column was used to separate kistrin from GST, with the kistrin
appearing in the column flow-through fraction. Protein concentrations
were measured using the BCA assay (Pierce, Chester, UK), and peak
fractions were stored at
70 °C.
Production of Mutant Kistrins
The segment of cDNA
encoding the kistrin RGD loop was removed by digestion with
BglII and KspI restriction endonucleases and replaced with a double-stranded insert encoding a mutant sequence. The
insert was generated from two complementary oligonucleotides that were
annealed, phosphorylated, and ligated into the dephosphorylated vector.
Competent DH5F
cells were transformed with the ligation product,
and the correct sequence was verified before expression of the mutant
kistrins as described above.
The purity of the recombinant kistrins was assessed by discontinuous SDS-polyacrylamide gel electrophoresis using a Tris/Tricine buffer system (42). The fidelity of bacterial translation and thrombin cleavage were validated by N-terminal sequencing and mass spectroscopy.
N-terminal SequencingPrior to analysis, recombinant kistrin was dialyzed extensively at 4 °C against 0.1% (v/v) trifluoroacetic acid. Approximately 1-nmol samples were sequenced by automated Edman degradation and high pressure liquid chromatography using a Model 476A Protein Sequencer (Applied Biosystems, Warrington, Cheshire, UK). The sequence obtained for recombinant kistrin was GSGKEXDXS(G/S)PENPXXD, which is identical to that of authentic kistrin with the exception of the ambiguous position, which is actually Ser, and the initial Gly and Ser, which are derived from the GST fusion partner following thrombin cleavage. The four unidentified residues (X) are cysteines that are destroyed during sequencing.
Mass SpectrometryRecombinant kistrins were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry using a VG-TofSpec E spectrometer (Fisons Instruments, Manchester, UK). A mass of 7469 obtained for recombinant RGD kistrin was within 0.1% of the calculated mass of 7474.5 based on the amino acid sequence. For ILDV kistrin, the value of 7451 obtained was also within 0.1% of the calculated mass of 7474.5.
Solid-phase Receptor-Ligand Binding AssayAssays were
performed using a method based on that of Charo et al. (43).
The 80-kDa fragment of fibronectin (500 µg/ml in PBS) or the H/120
recombinant fragment of fibronectin (450 µg/ml) was biotinylated by
mixing with an equal mass of
sulfo-N-hydroxysuccinimidobiotin (Pierce). After rotary
mixing for 40 min at room temperature, excess biotin was removed from
the mixture by dialyzing against several changes of 150 mM
NaCl and 25 mM Tris-HCl, pH 7.4. A 96-well enzyme-linked
immunosorbent assay plate (Immulon-3, Dynatech, Billingshurst, UK) was
incubated overnight at room temperature with 100-µl aliquots of
purified integrins diluted with PBS to a concentration of ~5 µg/ml.
The wells were then blocked with 200 µl of 5% (w/v) BSA, 150 mM NaCl, 0.05% (w/v) NaN3, and 25 mM Tris-HCl, pH 7.4, for 2 h at room temperature and
washed three times with 200 µl of 150 mM NaCl, 5 mM MnCl2, and 25 mM Tris-HCl, pH
7.4, containing 1 mg/ml BSA (buffer A). 100-µl aliquots of biotinylated H/120 (~0.2 µg/ml), diluted with buffer A, with or without recombinant kistrins, were then added to the wells and incubated at 30 °C for 3 h. Unbound biotinylated ligand was
removed, and the wells were washed three times with buffer A. Bound
ligand was quantitated by addition of 100 µl of ExtrAvidin-peroxidase conjugate (Sigma) diluted 1:200 in buffer A for 10 min at room temperature, following which the wells were washed four times with
buffer A. The color was developed by addition of
2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma), and the
absorbance was measured at 405 nm. Nonspecific binding was determined
from the level of ligand binding to BSA alone, and these values were
subtracted from those obtained with integrin-coated wells. Each point
represented the mean ± S.D. of four replicate wells.
The A375-SM human melanoma cell line (provided by I. J. Fidler, M. D. Anderson Hospital and University of Texas, Houston, TX) was maintained as described (44) in Eagle's minimal essential medium supplemented with 10% (v/v) fetal bovine serum, minimal essential medium vitamins, nonessential amino acids, 2 mM L-glutamine, and 1 mM sodium pyruvate. HT-1080 human fibrosarcoma cells were obtained from the European Collection of Animal Cell Cultures (Porton Down, UK) and cultured in Dulbecco's modified Eagle's medium containing 0.11 g/liter sodium pyruvate, 10% (v/v) fetal bovine serum, and 2 mM L-glutamine. Assays were performed as described by Humphries et al. (45) using 96-well tissue culture plates (Costar, High Wycombe, Bucks, UK). Ligands were diluted in Dulbecco's phosphate-buffered saline (PBS), and 100-µl aliquots were used to coat the wells for 1 h at room temperature. The ligand solution was then removed, and sites that could support nonspecific cell adhesion were blocked with 100 µl of 10 mg/ml heat-denatured BSA (45). After incubation for 30 min at room temperature, the BSA was removed, and 50-µl aliquots of inhibitors in PBS were added. Cells were detached using 0.05% (w/v) trypsin and 0.02% (w/v) EDTA and then washed and resuspended to 2 × 105 cells/ml in Dulbecco's minimal essential medium. Cell suspensions were allowed to recover at 37 °C for 10 min before addition of 50-µl aliquots to the wells. Following incubation at 37 °C for 90 min in a humidified atmosphere of 6% CO2, cells were fixed with 5% (w/v) glutaraldehyde in PBS. Using phase-contrast microscopy, the percentage of cells with a flattened, phase-dark morphology was estimated. Each point was obtained by counting 3 × 100 cells in random fields. No cell spreading was observed on wells coated only with heat-denatured BSA.
The disintegrin kistrin was selected for use as a scaffold for the
insertion of the LDV motif because the sequence of the RGD loop shows
some homology to the LDV-containing CS1 sequence in fibronectin. In
both sequences, the critical aspartate is immediately followed by a
residue with a hydrophobic side chain, and a proline residue is present
on either side of the motif: kistrin RGD loop sequence,
RIPRGDMPDDR; and CS1 sequence,
PEILDVPSTV. Synthetic kistrin cDNA was
generated from overlapping oligonucleotides and expressed as a
recombinant protein in E. coli. Samples of recombinant kistrin (r-kistrin) from different stages of purification were analyzed
by SDS-polyacrylamide gel electrophoresis (Fig.
2). The r-kistrin was resolved as a
slightly diffuse band of ~7.5 kDa under both reducing and nonreducing
conditions, which corresponds with its expected molecular mass. The
band at 26 kDa was the major contaminant, but was removed by repeating
the glutathione-agarose affinity chromatography and therefore appeared
to be GST (data not shown).
Following the expression of kistrin as a recombinant protein, the
activity of r-kistrin was compared with that of native kistrin isolated
from venom. The ability of both molecules to inhibit the spreading of
A375-SM cells on the RGD-containing 80-kDa fragment of fibronectin,
which is recognized by integrins 5
1 and
V
3, was examined
(Fig. 3). Half-maximal inhibition of cell
spreading was obtained at 2 µM for native kistrin and at
7 µM for the recombinant molecule. When compared with the
published value of 100 µM for the linear peptide GRGDS
(34), these data demonstrate that both native and recombinant kistrins
are potent integrin antagonists; they also suggest that the recombinant
molecules are largely correctly folded. The biological activity of
disintegrins appears to be dependent on correct disulfide bond-mediated
folding, as suggested by the dramatic loss of activity following
reduction and alkylation of the disulfide bonds, but nevertheless,
E. coli appears to be a suitable host for their recombinant
expression (31, 46).
Having established the anti-adhesive activity of recombinant kistrin, the synthetic cDNA was used as a template for the creation of chimeras with different integrin recognition motifs. In the initial chimera targeting the RGD loop, the entire 11-residue loop sequence was removed by excision of the segment of cDNA encoding this region and replaced with a segment encoding an 11-amino acid sequence from the C-terminal end of the CS1 peptide of fibronectin. A second chimera in which the RGDM tetrapeptide sequence from the loop was replaced by ILDV was also generated, the minimal change needed to convert RGD to LDV. In further mutagenesis, the contribution of individual residues to the activity of the molecule was investigated by replacement with alanine. The active-site aspartate was retained in the same position as in the native kistrin sequence in all mutants. The amino acid sequences of the RGD loop were as follows: r-kistrin, CRIPRGDMPDDRC; CS1 kistrin, CGPEILDVPSTVC; ILDV kistrin, CRIPILDVPDDRC; ALDV kistrin, CRIPALDVPDDRC; IADV kistrin, CRIPIADVPDDRC; ILAV kistrin, CRIPILAVPDDRC; and AAAV kistrin, CRIPAAAVPDDRC.
Promotion of Cell Spreading by Recombinant Kistrin Fusion ProteinsTo compare the abilities of mutant kistrins to promote
cell spreading, they were purified as fusion proteins retaining the GST
carrier and immobilized on the surface of 96-well plates. A high
maximal level (>95%) of HT-1080 cell spreading was observed on
GST-r-kistrin containing the native sequence, with half-maximal spreading at a coating concentration of 1.1 µg/ml. HT-1080 cell spreading was not supported by any of the mutants (Fig.
4 and data not shown).
Since A375-SM cells express integrin 4
1 and recognize the ILDV
motif within the CS1 sequence of fibronectin, spreading assays were
performed to determine whether these cells were able to recognize the
ILDV motif in kistrin chimeras (Fig. 5).
Dose-dependent promotion of spreading was observed, but
activity varied between the different mutant kistrins. GST-r-kistrin
supported up to 98% spreading, with half-maximal spreading observed at
a coating concentration of 0.7 µg/ml. Half-maximal spreading was
supported at 7.4 µg/ml by the ILDV kistrin chimera and at 19 µg/ml
by the ALDV kistrin mutant. Cell spreading on either the IADV kistrin
or CS1 kistrin fusion proteins reached a maximum of 20% at ~200
µg/ml (data not shown). No cell spreading was observed on GST alone
or on the two mutants AAAV kistrin and ILAV kistrin. The results of the cell spreading assay were mirrored by A375-SM cell attachment assays
(data not shown).
Effect of Anti-integrin Antibodies on Cell Spreading on Fusion Proteins
To investigate which receptors were used by the HT-1080
and A375-SM cells to spread on the kistrin chimeras, the effects of function-blocking anti-integrin antibodies were tested. The effects of
anti-integrin antibodies on the spreading of HT-1080 cells on RGD
kistrin are shown in Fig. 6. Spreading
was partially inhibited by function-blocking anti-5 (33%),
anti-
V (43%), anti-
1 (46%), and anti-
V
3 (40%)
antibodies, but not by anti-
4 or anti-
2 antibodies. Substantial
inhibition was observed when anti-
1 and anti-
V
3 (97%)
antibodies or when anti-
5 and anti-
V (95%) antibodies were used
together. A combination of anti-
5 and anti-
V
3 antibodies gave
~77% inhibition of spreading. These data suggest that the receptors
used by HT-1080 cells to recognize r-kistrin are integrins
V
3,
5
1, and possibly
V
1.
Spreading of A375-SM cells on RGD kistrin (Fig.
7A) was substantially
inhibited by anti-V (85%) and anti-
V
3 (73%) antibodies and
was slightly reduced by anti-
5 (14%) and anti-
1 (14%)
antibodies. These data suggest that A375-SM cells employ
V
3 as
the primary receptor for r-kistrin, but that
5
1 may also have a
minor contribution. A375-SM cell spreading on ILDV kistrin (Fig.
7B), CS1 kistrin (Fig. 7C), ALDV kistrin (Fig.
7D), and IADV kistrin (Fig. 7E) was almost
completely blocked by anti-
1 and anti-
4 antibodies, suggesting
that the melanoma cells were using integrin
4
1 only for spreading
on these chimeras.
Inhibition of Integrin-Ligand Binding by Recombinant Kistrins in Solid-phase Assay
To measure the potency of recombinant kistrins
and to exclude the possibility that the mutant kistrins were exerting
their effects indirectly, for example, via another cell-surface
receptor, the interactions of the mutant kistrins with purified
integrins in solid-phase assays were examined. RGD kistrin was found to be a potent inhibitor of the binding of the RGD-containing 80-kDa fibronectin fragment to immobilized 5
1 (Fig.
8), with half-maximal inhibition being
observed at 0.01 µM. None of the mutant chimeras showed
any inhibitory activity when tested at concentrations of up to 1.2 µM (Fig. 8 and data not shown).
ILDV kistrin was the most potent inhibitor of the binding of the
LDV-containing H/120 fibronectin fragment to immobilized 4
1 (with
half-maximal inhibition at ~0.1 µM). The relative
activities of the alanine replacement mutants followed the same pattern
as for the promotion of melanoma cell spreading and attachment, with half-maximal inhibition observed at ~0.12 µM for ALDV
kistrin and at 0.79 µM for IADV kistrin (Fig.
9A). Half-maximal inhibition was not achieved with either AAAV kistrin or ILAV kistrin at
concentrations up to 3.6 µM. The ability of the chimeras
to block the function of purified
4
1 therefore correlated with
the ability to promote
4
1-dependent cell
spreading.
RGD kistrin and CS1 kistrin were less potent antagonists of 4
1
than ILDV kistrin (Fig. 9B). Half-maximal inhibition was not
achieved at concentrations up to 3.5 µM for CS1 kistrin,
but was observed at 2.7 µM for RGD kistrin. The different
potencies of the recombinant kistrins in Fig. 9 are likely to be a
reflection of their relative affinities for the integrin receptors, but
it is also conceivable that this might be attributed to selective inhibition of different integrin activation states. Although
Mn2+ was included in all of the solid-phase assays to
maximize integrin activation, a proportion of the integrin population
may nevertheless have remained in an inactive conformation.
The aims of this study were to express kistrin (and kistrin
chimeras) in recombinant form and to examine for the first time the
effects of interconverting integrin recognition motifs within a natural
framework on receptor binding potency and specificity. Our major
findings are as follows. (i) Recombinant and native kistrins exhibit
similar potency and specificity, confirming previous studies that have
reported successful bacterial expression of disintegrins. (ii) A
chimeric kistrin containing a minimal RGD to LDV motif change exhibits
a distinct switch in receptor specificity: RGD kistrin recognizes
5
1 and
V
3, whereas ILDV kistrin binds specifically to
4
1. (iii) The ILDV kistrin chimera retains much of the potency of
the parent RGD kistrin, suggesting that the structural features
required for a potent RGD activity also apply to the LDV motif. This
provides further evidence that the RGD and LDV motifs may be
functionally equivalent. (iv) Alanine replacement mutants within
the LDV motif have provided information on the relative contributions
of individual flanking residues to integrin binding activity.
The receptor binding specificity of recombinant kistrins was assessed
both by solid-phase assays and by inhibition of cell spreading by
anti-integrin antibodies. Spreading of both HT-1080 and A375-SM cells
on r-kistrin was inhibited by anti-V
3, anti-
V, anti-
1, and
anti-
5 antibodies, and r-kistrin was able to bind to purified
5
1 in solid-phase assays. These data suggest that r-kistrin is
recognized by integrin
V
3 and with a lower affinity by
5
1
and also possibly by
V
1. Previous studies have suggested that the
hydrophobic methionine residue immediately C-terminal to RGD in kistrin
is responsible for a reduced affinity for
5
1 and
V
3
compared with
IIb
3 (47, 48); however,
V
3 seems to be
tolerant of a more diverse flanking region than
5
1 (49), which
may explain the significant contribution of
V
3 to the adhesion of
both A375-SM and HT-1080 cells to r-kistrin.
The ILDV kistrin chimera was unable to bind to purified 5
1, but
bound to
4
1, and cell spreading on ILDV kistrin was inhibited only by anti-
4 and anti-
1 antibodies. These results suggest that
ILDV kistrin is recognized by
4
1, but not by
V
3 or
5
1, indicating that alteration of only three residues is
sufficient to achieve a major change in specificity. The results of the
alanine replacement mutagenesis reflect the dependence on the aspartate residue for the activity of ILDV kistrin and the relative importance of
the isoleucine and leucine residues, the requirement for the leucine
being greater than for isoleucine.
Although the ILDV kistrin chimera is potent, the CS1 kistrin chimera is
considerably less active, although both share the ILDVP pentapeptide
sequence at the same position in the loop. The differences in sequence
between the two lie in the flanking sequences RIP, immediately
N-terminal, and DDR, immediately C-terminal to ILDVP, and it is likely
that they determine the potency of the kistrin molecule. Further
mutagenesis will reveal the contribution of these sequences to the
activity of ILDV kistrin. It has been suggested that the RGD tripeptide
is chiefly responsible for the activity of kistrin (50), but there is
also evidence that the surrounding amino acids contribute to the
affinity and selectivity of disintegrins (51-53). It may also be
possible that the residues in the loop contribute differently to the
binding of 4
1 to ILDV kistrin than to the binding interactions
between
V
3 and r-kistrin. For example, the proline residues lying
N- and C-terminal to the RGD motif in kistrin are not present in the
known RGD loop sequences of most other disintegrins, and alanine
replacement mutagenesis suggests that they are not critical to the
potency of kistrin (50), but may contribute to the activity of ILDV
kistrin.
The kistrin RGD loop is conformationally flexible but more constrained
than a linear peptide, possibly contributing to the potency of the
molecule. The concentration of the linear CS1 peptide required to give
half-maximal inhibition of H/120 binding to 4
1 in a solid-phase
assay has been quoted as 4 µg/ml (1.5 µM) (39) and is
therefore significantly more active than the CS1 kistrin chimera. The
11-residue sequence inserted into the kistrin RGD loop may be
unfavorably constrained, resulting in low affinity interactions with
4
1. Clearly, specific residues or sequences must be present
within the loop to produce a potent integrin inhibitor.
Our finding that it is possible to exchange RGD and LDV motifs (by
exchanging only three residues) in an integrin ligand and alter
specificity but retain activity suggests a functional equivalence between the two motifs. Earlier evidence for this was obtained from
cross-inhibition studies in which short linear RGD peptides were shown
to inhibit 4
1-LDV interactions competitively (34), and certain
cyclic RGD peptides have been produced that were also able to block
4
1 (54, 55). This may explain our finding that r-kistrin was able
to inhibit the adhesive activity of purified
4
1 in the
solid-phase assay. The QAGDV motif is also thought to be functionally
equivalent to RGD since QAGDV- and RGD-containing peptides share common
or mutually exclusive binding sites on
IIb
3 (56).
The aspartate residue critical to the activity of all three motifs may be central to a common integrin-binding mechanism used by these sequences. Integrin-ligand binding and receptor activation are dependent on divalent cations, and ligand-binding sites within integrins coincide with putative cation-binding sites (57, 58). It has been proposed that a divalent cation could be coordinated simultaneously by the ligand motif and the active site within the integrin, with the acidic aspartate side chain providing a cation-coordinating group (59, 60). Subtle differences in motif conformation between ligands and the use of synergy sequences may provide the basis for receptor specificity. In the future, it may be possible to exploit kistrin (or other disintegrin) scaffolds to display different integrin recognition sequences and to generate potent inhibitors that can be used in vivo as probes of adhesive function or for structural analysis.
Finally, it is conceivable that other disintegrins might naturally express an LDV motif. A gene family known as ADAM (for proteins containing a disintegrin and metalloproteinase domain) encodes a family of membrane-anchored proteins in mammals that may play roles in cell-cell interactions (61, 62). Whereas almost all snake venom disintegrins contain an RGD active site, the only ADAM protein that contains RGD in its disintegrin domain that has been identified to date is the molecule metargidin (63). Other ADAM proteins lack this sequence and instead have a motif that includes highly conserved cysteine and aspartate residues and that bears some homology to an LDV motif. As yet, the integrin binding specificity of ADAM proteins and the role of this active site sequence are poorly understood, but it is conceivable that they may be natural LDV forms of disintegrins.
We are grateful to Paul Mould for providing
the 80-kDa fragment of fibronectin and integrin 5
1, to Kath Clark
for integrin
4
1, to Cath Fernandez for the N-terminal sequencing,
and to Janet Askari and Sue Craig for H/120.