EC3, a Novel Heterodimeric Disintegrin from Echis carinatus Venom, Inhibits α4 and α5 Integrins in an RGD-independent Manner*

EC3, a heterodimeric disintegrin (M r = 14,762) isolated from Echis carinatus venom is a potent antagonist of α4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing α4β1 and α4β7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6–30 nm, adhesion of K562 cells (α5β1) to fibronectin with IC50 = 150 nm, and adhesion of αIIbβ3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nm; it did not inhibit adhesion of αvβ3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 μm), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mm), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5–10 mm) in contrast to RGDS peptide (IC50 = 3 mm), inhibiting completely at 10 mm.

Integrins are a family of cell surface proteins that mediate cell-cell interactions and the adhesion of cells to extracellular matrix proteins and other ligands. Integrins are heterodimeric structures composed of noncovalently bound ␣ and ␤ subunits (1,2). In humans there are at least 15 different ␣ subunits and 8 different ␤ subunits, and they can combine to form proteins with diverse ligand specificities and biological activities. The integrins play important roles in many diverse biological processes including platelet aggregation, tissue repair, angiogen-esis, bone destruction, tumor invasion, and inflammatory and immune reactions. Integrin ␣IIb␤3 (glycoprotein IIb/IIIa complex) binds fibrinogen on the platelet surface and mediates platelet aggregation. Integrin ␣v␤3 is predominantly expressed on endothelial cells and plays an important role in angiogenesis. It is also expressed on osteoclasts and participates in bone destruction. Integrin ␣5␤1 is widely distributed on a variety of cells; it plays a critical role in cell adhesion to extracellular matrix as well as in the formation of tissues and organs during embryonic development (3). All three integrins, ␣IIb␤3, ␣v␤3, and ␣5␤1, recognize RGD sequence in the adhesive ligands (1,2).
The ␣4 integrins ␣4␤1 and ␣4␤7 are widely expressed on leukocytes and lymphoid cells and play a major role in inflammation and autoimmune diseases (4). The ␣4␤1 integrin (also called VLA-4, very late antigen-4) mediates cell adhesion to vascular cell adhesion molecule 1 (VCAM-1), 1 an adhesive molecule belonging to the immunoglobulin (Ig) superfamily that is expressed on endothelial cells at sites of inflammation. ␣4␤1 also binds to alternatively spliced variants of fibronectin that contain connecting segment 1 (CS-1). The ␣4␤7 integrin binds to mucosal addressin cell adhesion molecule 1 (MadCAM-1) and to a lesser extent to VCAM-1 and CS-1. Interaction of these integrins with VCAM-1 or MadCAM-1 (which are up-regulated by cytokines) on endothelium mediates leukocyte infiltration, which can lead to tissue and organ destruction (4). Selectins and ␤2 integrins (expressed on neutrophils and monocytes) also contribute to this process. Leukocyte engagement via ␣4 integrins is believed to play a significant role in the progression of many diseases including insulin-dependent diabetes, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, arteriosclerosis, asthma, allergy, and re-stenosis of arteries after surgery and angioplasty (4,5).
Over the last decade a number of investigators have sought naturally occurring or synthetic peptides that may selectively inhibit integrin-ligand interactions. Research on disintegrins, low molecular weight, cysteine-rich, RGD-containing peptides isolated from viper venoms was stimulated by this long term objective. The first disintegrin described in the literature, trigramin, was identified and characterized on the basis of its ability to block platelet aggregation and inhibit fibrinogen binding to ␣IIb␤3 (6). Subsequently a number of laboratories have isolated several other RGD containing viper venom disintegrins of similar size, including kistrin (rhodostomin) (7), applagin (8), and flavoridin (triflavin) (9,10). Two short (49 amino acids) RGD disintegrins, echistatin (11) and eristostatin (12,13), have been isolated from the venoms of Echis carinatus and Eristocophis macmahoni, respectively. A number of NMR studies on kistrin, echistatin, and flavoridin showed that their RGD sequences are located in a mobile loop joining two strands of ␤ sheet protruding from the protein core (reviewed in Ref. 14). The disulfide bonds around the RGD sequence in disintegrins maintain the hairpin loop conformation in each peptide, which is important for their potency and selectivity.
It is known that disintegrin-like and cysteine-rich domains occur in larger venom proteins containing a metalloproteinase domain and that the RGD sequence in these proteins is substituted with other amino acids (15). We considered the possibility that viper venoms may contain low molecular weight disintegrins with anti-adhesive properties mediated by epitopes other than RGD. We fractionated E. carinatus venom on HPLC reverse-phase column, and we tested each fraction for its ability to bind to Jurkat cells, which express ␣4␤1 and ␣5␤1 integrins but do not express ␤3 integrins. We isolated and characterized a new protein, referred as EC3, that is selective and a highly potent inhibitor of ␣4 integrins and shows a low level of interaction with ␤3 integrins. EC3 is the member of a new protein family called heterodimeric disintegrins, which is first reported in this paper.
Cell Lines-A5 and VNRC3 cells, Chinese hamster ovary (CHO) cells transfected with human ␣IIb␤3 and ␣v␤3 integrins, respectively (17) Purification of EC3-Lyophilized E. carinatus suchoreki venom obtained from Latoxan (Rosans, France) was dissolved in 0.1% trifluoroacetic acid (30 mg/ml). The solution was centrifuged for 5 min at 5000 rpm to remove the insoluble proteins. The pellet was discarded, and the supernatant was applied to a C-18 HPLC column. The column was eluted with an acetonitrile linear gradient of 0 -80% over 45 min. The venom was separated into 17 fractions. EC3 fraction, eluting at approximately 40% of acetonitrile, was collected, lyophilized, and then dissolved in water. This solution was re-injected into the same HPLC column. However, a "flatter" gradient of acetonitrile was applied (0 -60% over 45 min). The main peak, which contained EC3, was collected and lyophilized. Purity of EC3 was tested by SDS-polyacrylamide gel electrophoresis and mass spectrometry. The yield of EC3 was about 4 mg/1 g of crude venom.

Separation of Reduced and Ethylpyridylethylated EC3 Subunits-
Reduction and alkylation of EC3 were performed according to procedures used before for trigramin (6). Briefly, 100 g of EC3 was incubated in 200 l of 0.1 M Tris-HCl, pH 8.5, buffer containing 6 M guanidine hydrochloride, 4 mM EDTA, 3.2 mM dithiothreitol together with 2 l of 4-vinylpyridine for 2 h in the dark at room temperature. Modified subunits epEC3A and epEC3B were isolated by reverse-phase HPLC on a C-18 column with an acetonitrile gradient of 0 -80% over 45 min. In some experiments, EC3 subunits were reduced and carboxymethylated with iodoacetate acid before HPLC separation.
Structural Characterization of EC3A and EC3B-Determination of the molecular mass of native EC3 or reduced and alkylated EC3 subunits was done by electrospray ionization mass spectrometry using a Sciex API-III triple quadrupole instrument. The sequences of native EC3 electroblotted onto a polyvinylidene difluoride membrane (19), and residues 1-40 of epEC3A and epEC3B were determined by N-terminal sequence analysis using an Applied Biosystems Procise instrument. The primary structures of EC3A and EC3B were deduced from Edman degradation of overlapping peptides obtained by digestion with endoproteinase Lys-C (Roche Molecular Biochemicals) (2 mg/ml protein in 100 mM ammonium bicarbonate, pH 8.3, for 18 h at 37°C using an enzyme:substrate ratio of 1:100 (w/w)) and CNBr (10 mg/ml protein and 100 mg/ml CNBr in 70% formic acid for 6 h under N 2 atmosphere and in the dark). Peptides were separated by reverse-phase of HPLC using a 0.4 ϫ 25-cm Lichrospher RP100 C-18 (5-m particle size) column (Merck) eluting at 1 ml/min with acetonitrile gradient. For determination of sulfhydryl groups (free cysteines), native EC3 (2 mg/ml in 100 mM ammonium bicarbonate, pH 8.3, containing 6 M guanidine hydrochloride) was treated for 2 h at room temperature with a 100-fold molar excess of iodoacetamide, dialyzed against distilled water, lyophilized, and subjected to amino acid analysis (after sample hydrolysis with 6 N HCl for 18 h at 110°C) using a Amersham Pharmacia Biotech AlphaPlus amino acid analyzer.
Adhesion Studies-Adhesion of cultured cells labeled with 5-chloromethylfluorescein diacetate was performed as described previously (21). Briefly, ligands EC3, fibrinogen, vitronectin, fibronectin, or VCAM-1 were immobilized on 96-well microtiter plates (Falcon, Pittsburgh, PA) in phosphate-buffered saline overnight at 4°C. Wells were blocked with 1% bovine serum albumin in Hanks' balanced salt solution. Cells were labeled by incubation with 12.5 M 5-chloromethylfluorescein diacetate in Hanks' balanced salt solution buffer containing 1% bovine serum albumin at 37°C for 15 min. Unbound label was removed by washing with the same buffer. Labeled cells (1 ϫ 10 5 /sample) were added to the well in the presence or absence of inhibitors and incubated at 37°C for 30 min. Unbound cells were removed by washing the wells, and bound cells were lysed by the addition of 0.5% Triton X-100. In parallel, a standard curve was prepared in the same plate using known concentrations of labeled cells. The plates were read using a Cytofluor 2350 fluorescence plate reader (Millipore, Bedford, MA) with a 485-nm excitation filter and a 530-nm emission filter.
Flow Cytometry Analysis-Samples for flow cytometry analysis were prepared as described (22) and analyzed in a Coulter Epics flow cytometer (Miami, FL).

Amino Acid Sequence and Subunit Composition of EC3-
Analysis of the nonreduced EC3 band excised from the Immobilon-P membrane revealed a single amino acid sequence: NS-VHPXXDPV(K/T)XEPREGEHXISGP. The complete amino acid sequences of EC3A and EC3B were determined by Nterminal sequence analysis of reverse phase HPLC-isolated peptides derived by degradation of each subunit with endoproteinase Lys-C and CNBr. Both EC3A and EC3B are cysteinerich proteins of 67 amino acids. They display amino acid sequence heterogeneity at several positions, indicating the existence of isoforms. The isotope-averaged molecular masses calculated for the reduced EC3A isoforms (1-67: N33 I37 G64 Da, and 7412 Da, respectively. The major EC3B isoform, i.e. the one that would have a molecular mass of 7950 after reduction and carboxymethylation, is the 7370-Da isoform. The possibility of a number of dimers involving combinations of various EC3A and EC3B isoforms should be considered. However, we propose that EC3A-EC3B heterodimers may represent the major species because homodimers would not yield separated subunits displaying the distinct biological activities demonstrated for the HPLC-purified EC3A and EC3B fractions, and a mixture of EC3A and EC3B homodimers would display a more complex HPLC separation profile. EC3A and EC3B showed a high degree of sequence similarity to each other and to eristostatin, echistatin, flavoridin, and kistrin, including the alignment of conserved cysteines identified in each subunit. The EC3A amino acid sequence had high homology with the disintegrin domain of Le3, a metalloproteinase-disintegrin identified in Vipera lebetina (24) (Fig. 1). Surprisingly, neither EC3 subunit contained an RGD sequence. The hairpin loop sequence of echistatin, KRARGDDMDDY, was substituted in EC3A and EC3B with KRAVGDDVDDY and KRAMLDGLNDY, respectively (Fig. 1).
Biological Activities of EC3-The biological activities of EC3 and the RGD-containing disintegrin echistatin were compared in a panel of integrin assays (Table I). As expected, echistatin at concentrations of 20 -130 nM potently inhibited ␣IIb␤3-dependent platelet aggregation and ␣IIb␤3-, ␣v␤3-, and ␣5␤1-dependent cell adhesion (Table I). In contrast, EC3 only weakly inhibited ␣IIb␤3-dependent interactions (IC 50 ϭ 1 M for platelet aggregation and IC 50 ϭ 500 nM for A5 cell adhesion to fibrinogen) and showed no inhibition of ␣v␤3-dependent adhesion up to 10 M, although inhibition of ␣5␤1-dependent adhesion was observed at an IC 50 of 150 nM (Table I). When the two disintegrins were evaluated in a panel of ␣4 integrin-mediated cell adhesion assays, the specificities were reversed. At concentrations of 25-100 nM, EC3 was a highly potent inhibitor of the interaction of both anchorage-dependent and -independent cells expressing ␣4␤1 with either VCAM-1 or the CS-1 fragment of fibronectin, whereas echistatin showed no detectable activity at 10 M (Table I). EC3 inhibited to the same extent adhesion of A2 (CHO ␣4ϩ␣5ϩ) cells and ␣4B2 (CHO ␣4ϩ␣5Ϫ) cells to immobilized VCAM-1, confirming direct inhibition of binding to ␣4␤1 integrin. To further extend the data on ␣4 integrins, the potency of EC3 in assays measuring VCAM-Ig binding directly to either ␣4␤1 on Jurkat cells or ␣4␤7 on JY cells was evaluated. EC3 potently inhibited ␣4␤1 and ␣4␤7 binding at concentrations of 28 nM and 6 nM, respectively. Moreover, adhesion of RPMI 8866 cells was inhibited by EC3 with IC 50 ϭ 17 nM, whereas echistatin was not inhibitory. Cell adhesion assays and direct binding assays yielded similar results. Neither echistatin nor EC3 inhibited adhesion of ␣6␤1transfected cells to laminin and adhesion of ␣2␤1 cells to collagen (Table I). We also studied biological function of both disintegrins in direct binding assay, confirming the specificity of EC3 for ␣4␤1 and ␣4␤7 integrin.
We also evaluated the biological activity of the EC3A and EC3B subunits after reduction and ethylpyridylethylation. Although residual activity of both subunits was significant, it was decreased by approximately 200-fold. It has been previously reported that reduction and ethylpyridylethylation of flavoridin and albolabrin decreased their ability to inhibit ADP-induced platelet aggregation by approximately 40-fold (25). epEC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC 50 ϭ 6 M), whereas epEC3A was inactive in this system. However, epEC3A and epEC3B both inhibited adhesion of K562 cells to fibronectin (IC 50 ϭ 30 M and 6 M, respectively) (Fig. 2). This experiment suggests that the specificity of EC3 for ␣4 integrins likely resides in the MLD sequence in the EC3B subunit, whereas the ability if EC3 to inhibit ␣5␤1 likely resides in both subunits. Obviously, the MLDG sequence in EC3B is replacing the RGDX motif in monomeric disintegrins. Both RGDX and MLDG motifs appear to represent integrin binding sites. Fig. 3 shows that MLDG peptide inhibited adhesion of Jurkat cells to immobilized VCAM-1 in a dose-dependent manner approaching saturation at 5-10 mM. Adhesion of K562 cells to immobilized fibronectin showed a similar pattern of inhibition by RGDS. On the other hand RGDS did not cause any significant inhibition of Jurkat cell adhesion to immobilized VCAM-1. Inhibition of K562 to fibronectin by MLDG was only partial at 10 mM. It should be noted that the inhibitory effect on Jurkat cell adhesion to VCAM-1 was increased when longer MLDG-containing peptides were used. For instance CKRAMLDGLNDYC inhibited Jurkat cell adhesion with IC 50 of 800 M, whereas the peptide CRAMLDGLNDYCTGKSSD caused 50% inhibition at 50 M (not shown).
Further experiments showed that EC3 competes with mAb HP2/1 for binding to ␣4 integrin. HP 2/1 at a concentration of 1 g/sample blocked adhesion of Jurkat cells to immobilized EC3, whereas at the concentration of 1 mM, neither the hexapeptide GRGDSP nor a control peptide GRGESP had any effect (Fig. 4A). Competition between EC3 and HP2/1 was also confirmed using fluorescence-activated cell sorter analysis. Fig.   FIG. 1. Comparison of amino acid sequences of EC3A and EC3B with other disintegrins. Eristostatin (13) and echistatin (12) represent short disintegrins; kistrin (7) and flavoridin (9) represent medium size disintegrins. Le3 is a metalloproteinase from V. lebetina venom with a disintegrin domain (24). The cysteines are boxed. EC3A and EC3B now have Swiss-Prot entry codes P81630 and P81631.
4B shows EC3-mediated inhibition of HP2/1 binding to ␣4B2 (CHO ␣4ϩ␣5Ϫ) cells. The inactive G190A ␣4 mutant did not interact with EC3 (data not shown). In addition, the ability to directly inhibit binding to ␣5␤1 was confirmed using mAb SAM-1, which blocked adhesion of K562 cells to immobilized EC3 (data not shown). DISCUSSION The experimental data described in this paper identify a novel, heterodimeric disintegrin in the venom of E. carinatus. This disintegrin, named EC3, is a potent and relatively selective antagonist of ␣4 integrins, which inhibits their interaction with ligands in an RGD-independent manner. EC3 is composed of two covalently linked subunits A and B, which show a high degree of homology (including alignment of conserved cysteines) with other viper venom disintegrins. It is likely that the integrin binding sites of EC3 are located in two loops encompassing 13 amino acids (Cys-38 to Cys-50), corresponding to hairpin loops extending from Cys-20 to Cys-32 in echistatin and from Cys-45 to Cys-57 in kistrin and flavoridin. It is well known that the hairpin loops in disintegrins are maintained in appropriate conformation by S-S bridges (14,15,26), and the same appears to be true regarding EC3. The biological activity of this protein is decreased by 2 orders of magnitude after reduction and alkylation (Fig. 3). In contrast to all other viper venom disintegrins, EC3 contains neither RGD nor KGD sequences. In fact, the RGD motif is substituted with VGD in EC3A and with MLD in EC3B. The activity of EC3 with regard to inhibition of ␣5␤1 binding resides on both subunits. How-

FIG. 2. Effect of reduced and ethylpyridylethylated EC3A and EC3B on adhesion of Jurkat cells to immobilized VCAM-1 (A) and K562 cells to immobilized fibronectin (B).
Recombinant VCAM-1 (0.5 mg/well) or fibronectin (0.5 mg/well) were immobilized overnight at 4°C on a 96-well plate in phosphate-buffered saline buffer. After blocking, the 5-chloromethylfluorescein diacetate-labeled cells were added to each well in the presence or absence EC3 subunits. The adhesion was performed as described under "Experimental Procedures." Open circles and closed circles indicate different concentrations of EC3A and EC3B, respectively. Error bars indicate S.D. from three independent experiments. ever, only EC3B was active in the inhibition of ␣4␤1/VCAM-1 interactions. This observation suggests, that MLD is the active sequence in EC3 mediating its anti-␣4 effects. The experiment with synthetic peptides (Fig. 3) confirmed this expectation. MLDG linear peptide blocked adhesion of Jurkat cells to VCAM-1. In contrast, RGDS peptide, which is a very well known inhibitor of several ␤1 and ␤3 integrins (27), was not significantly active in this system. The MLDG peptide partially inhibits adhesion of ␣5␤1-expressing cells to fibronectin (Fig.  3). This is consistent with the dual inhibitory effect of EC3 and of EC3B subunit containing MLDG (Fig. 2). The inhibitory effects of anti-␣4 and anti-␣5 inhibitory antibodies are in agreement with this suggestion.
EC3 is a new naturally occurring ligand for ␣4 integrins. The LD motif from its B subunit is also present in other ligands of ␣4 integrins. An ILDV sequence was found in alternatively spliced connective segment I of fibronectin (28,29), and KLDAPT is present in the fibronectin type III5 repeat (30). The LDT sequence occurring in MadCAM (31) appears to be important for its ability to bind to ␣4␤7. Recently Tselepis et al. (32) produced a number of mutants of recombinant kistrin and demonstrated that ILDV kistrin (kistrin in which PRGD sequence was substituted with ILDV) inhibited binding of the LDV-containing fibronectin fragment to immobilized ␣4␤1, with an IC 50 close to 0.1 M. It is difficult to compare activities of EC3 with LDV-kistrin and synthetic peptides because preparations have been tested in different assay systems; however, in our hands LDV-kistrin was some 50-fold less active than EC3 in the direct binding assay. 2 Until now, no MLDG motif has been identified and functionally characterized. Most investigators have achieved better inhibitory effects for tested ␣4 inhibitors in the presence of Mn 2ϩ . However EC3 has almost the same activity in the presence or absence of Mn 2ϩ (data not shown).
EC3 inhibits quite selectively adhesion of ␣4␤1-expressing cells to immobilized VCAM-1. Its effect on ␣5␤1 and on ␣IIb␤3 appears to be lower by 1 and 2 orders of magnitude, respectively. The effect of EC3 on ␣4␤1 does not appear to be related to the inhibition of ␣5␤1 integrin, because this disintegrin inhibited to the same extent the adhesion to VCAM-1 of CHO cells transfected with ␣4 and of ␣5-deficient CHO cells transfected with ␣4. EC3 also inhibits adhesion of ␣4␤7-expressing cells to MadCAM. Because mAb HP2/1 competes with EC3 for binding to ␣4, it appears that EC3 may bind to the N-terminal domain of ␣4, where the epitope of this antibody also resides (18,36,37).
It should be noted that EC3 weakly inhibited ADP-induced platelet aggregation and the binding of CHO cells transfected with ␣IIb␤3 to fibrinogen, although it had no significant effect on ␣v␤3-mediated adhesion. This is in agreement with other observations that the RGD motif in disintegrins is not absolutely required for expression of platelet aggregation inhibitory activity. For instance, Jia et al. (38) expressed in insect cells the disintegrin/cysteine-rich domain of atrolysin A from Crotalus atrox and demonstrated that the recombinant protein inhibited collagen and ADP-induced platelet aggregation. This recombinant protein contained RSEC instead of the RGD motif.
Trikha et al. (39) and Clark et al. (40) isolated a homodimeric, RGD-containing protein, contortrostatin, from the venom of Agkistrodon contortrix contortrix. The amino acid sequences of this protein, which appears to be a disintegrin, have not been reported. As determined by mass spectrometry, molecular mass of nonreduced contortrostatin is 13,505 Da, and the molecular mass of reduced and pyridylethylated contortrostatin is 8,000 Da. This bivalent protein is a potent inhibitor of platelet aggregation, and in contrast to monomeric disintegrins, it induces tyrosine phosphorylation of platelet proteins. In addition, contortrostatin is a potent inhibitor of ␤1 integrin-mediated melanoma cell adhesion in vitro and lung colonization in vivo. Most recently we isolated three other dimeric disintegrins, EMF10 from E. macmahoni and CC5 and CC8 from Cerastes cerastes venom, which all seem to be heterodimeric disintegrins with an molecular mass of 14 -15 kDa. 3 We are in the process of determining the amino acid sequences and function of these novel disintegrins. Further studies are required to establish how the bivalent structure of dimeric disintegrins affects the biological properties of the individual subunits.
In conclusion, we describe a novel dimeric disintegrin, EC3, that is a potent inhibitor of ␣4 integrin binding to VCAM-1 and moderately inhibits ␣5␤1 integrins. We propose that the activity of EC3 is associated with the MLDG sequence in the putative hairpin loop of this disintegrin.