EC3, a Novel Heterodimeric Disintegrin from Echis carinatus Venom, Inhibits alpha 4 and alpha 5 Integrins in an RGD-independent Manner*

Cezary MarcinkiewiczDagger , Juan J. Calvete§, Mariola M. MarcinkiewiczDagger , Manfred Raida, Senadhi Vijay-KumarDagger , Ziwei Huangparallel , Roy R. Lobb**, and Stefan NiewiarowskiDagger Dagger Dagger

From the Dagger  Department of Physiology, Sol Sherry Thrombosis Research Center, Fels Cancer Research Institute, Temple University, School of Medicine, Philadelphia, Pennsylvania 19140,  Institute of Peptide Research, Hannover, Germany 30559, ** Biogen Inc., Cambridge Massachusetts 02142, § Instituto de Biomedicina, Consejo Superior de Investigaciones Científicas, Valencia, Spain E 46010, and parallel  Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania 19107

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha 4 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 alpha 4beta 1 and alpha 4beta 7 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 (alpha 5beta 1) to fibronectin with IC50 = 150 nM, and adhesion of alpha IIbbeta 3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alpha vbeta 3 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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 alpha  and beta  subunits (1, 2). In humans there are at least 15 different alpha  subunits and 8 different beta  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, angiogenesis, bone destruction, tumor invasion, and inflammatory and immune reactions. Integrin alpha IIbbeta 3 (glycoprotein IIb/IIIa complex) binds fibrinogen on the platelet surface and mediates platelet aggregation. Integrin alpha vbeta 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 alpha 5beta 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, alpha IIbbeta 3, alpha vbeta 3, and alpha 5beta 1, recognize RGD sequence in the adhesive ligands (1, 2).

The alpha 4 integrins alpha 4beta 1 and alpha 4beta 7 are widely expressed on leukocytes and lymphoid cells and play a major role in inflammation and autoimmune diseases (4). The alpha 4beta 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. alpha 4beta 1 also binds to alternatively spliced variants of fibronectin that contain connecting segment 1 (CS-1). The alpha 4beta 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 beta 2 integrins (expressed on neutrophils and monocytes) also contribute to this process. Leukocyte engagement via alpha 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 alpha IIbbeta 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 beta  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 alpha 4beta 1 and alpha 5beta 1 integrins but do not express beta 3 integrins. We isolated and characterized a new protein, referred as EC3, that is selective and a highly potent inhibitor of alpha 4 integrins and shows a low level of interaction with beta 3 integrins. EC3 is the member of a new protein family called heterodimeric disintegrins, which is first reported in this paper.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Monoclonal antibodies (mAb) HP2/1 (anti-alpha 4 subunit of VLA-4) and SAM-1 (anti-alpha 5 subunit of VLA-5) were purchased from Immunotech, Inc. (Westbrook, ME). HP2/4 (anti-alpha 4 subunit of VLA-4) was a gift from Dr. F. Sanchez-Madrid (Madrid, Spain). Because the biological effects of HP2/1 and HP2/4 were identical, only data with HP2/1 are shown. Highly purified human fibrinogen was a gift from Dr. A. Budzynski (Temple University, Philadelphia, PA). Recombinant human VCAM-1 (16) was a gift from Dr. M. Renz (Genentech, San Francisco, CA). Human vitronectin and fibronectin were purchased from Calbiochem and Sigma, respectively. GRGDSP and GRGESP peptides were purchased from Bachem (Torrance, CA). RGDS was purchased from Sigma. Echistatin was isolated from E. carinatus suchoreki venom as described previously (13). Fluorescein isothiocyanate-conjugated goat anti-mouse IgG for flow cytometry was purchased from Jackson Immune Research (West Grove, PA).

Cell Lines-- A5 and VNRC3 cells, Chinese hamster ovary (CHO) cells transfected with human alpha IIbbeta 3 and alpha vbeta 3 integrins, respectively (17), were kindly provided by Dr. M. Ginsberg (Scripps Research Institute, La Jolla, CA). CHO cells with deleted alpha 5 integrin (B2 cells) were kindly provided by Dr. R. Juliano (University of North Carolina, Chapel Hill, NC). CHO cells transfected with human alpha 4 or its G190A mutant (18) and B2 cells transfected with human alpha 4 (alpha 4B2) were kindly provided by Dr. Y. Takada (Scripps Research Institute). JY cells expressing alpha 4beta 7 were a gift from Dr. S. Burakoff (Dana-Farber Cancer Institute, Boston MA), and RPMI8866 cells were from Dr. A. Garcia-Padro, Madrid, Spain. K562 cells transfected with alpha 6 and alpha 2 integrin were gifts of Dr. A. Sonnenberg (Netherlands Cancer Institute, Amsterdam, Holland) and M. E. Hemler (Dana Farber, Boston, MA), respectively. Jurkat cells, K562 cells, and nontransfected CHO K1 cells were purchased from ATCC (Manassas, VA).

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 µg of EC3 was incubated in 200 µl 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 µl 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 N2 atmosphere and in the dark). Peptides were separated by reverse-phase of HPLC using a 0.4 × 25-cm Lichrospher RP100 C-18 (5-µm 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.

Peptide Synthesis-- The peptides were prepared by solid phase synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy on a 430A peptide synthesizer (Applied Biosystems, Foster City, CA) and a 9050 Pepsynthesizer Plus (Perseptive Biosystems, Cambidge, MA), as described previously (20).

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 µM 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 × 105/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).

Direct Binding Assay-- Direct binding assay alkaline phosphatase conjugated with VCAM-1Ig was performed using Jurkat (alpha 4beta 1-expressing) and JY (alpha 4beta 7-expressing) cells according to procedures described previously (23).

    RESULTS
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INTRODUCTION
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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: NSVHPXXDPV(K/T)XEPREGEHXISGP. The complete amino acid sequences of EC3A and EC3B were determined by N-terminal 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 cysteine-rich 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 E66), (1-67: R33, V37, G64, E66), and (1-66: N33, I37, D64, D66) are 7412 Da, 7440 Da, and 7341 Da, respectively, corresponding to major and minor ions of reduced EC3 mass spectrum. The major EC3A isoforms might be the one with molecular weight 7412, which yields a mass of 8478 after reduction and ethylpyridylethylation. On the other hand, reduced EC3B isoforms (1-67: T11, K40, S55), (1-67: K11. R40, T55), and (1-67: T11, R40, T55) have calculated masses of 7370 Da, 7439 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).


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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.

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 alpha IIbbeta 3-dependent platelet aggregation and alpha IIbbeta 3-, alpha vbeta 3-, and alpha 5beta 1-dependent cell adhesion (Table I). In contrast, EC3 only weakly inhibited alpha IIbbeta 3-dependent interactions (IC50 = 1 µM for platelet aggregation and IC50 = 500 nM for A5 cell adhesion to fibrinogen) and showed no inhibition of alpha vbeta 3-dependent adhesion up to 10 µM, although inhibition of alpha 5beta 1-dependent adhesion was observed at an IC50 of 150 nM (Table I). When the two disintegrins were evaluated in a panel of alpha 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 alpha 4beta 1 with either VCAM-1 or the CS-1 fragment of fibronectin, whereas echistatin showed no detectable activity at 10 µM (Table I). EC3 inhibited to the same extent adhesion of A2 (CHO alpha 4+alpha 5+) cells and alpha 4B2 (CHO alpha 4+alpha 5-) cells to immobilized VCAM-1, confirming direct inhibition of binding to alpha 4beta 1 integrin. To further extend the data on alpha 4 integrins, the potency of EC3 in assays measuring VCAM-Ig binding directly to either alpha 4beta 1 on Jurkat cells or alpha 4beta 7 on JY cells was evaluated. EC3 potently inhibited alpha 4beta 1 and alpha 4beta 7 binding at concentrations of 28 nM and 6 nM, respectively. Moreover, adhesion of RPMI 8866 cells was inhibited by EC3 with IC50 = 17 nM, whereas echistatin was not inhibitory. Cell adhesion assays and direct binding assays yielded similar results. Neither echistatin nor EC3 inhibited adhesion of alpha 6beta 1-transfected cells to laminin and adhesion of alpha 2beta 1 cells to collagen (Table I). We also studied biological function of both disintegrins in direct binding assay, confirming the specificity of EC3 for alpha 4beta 1 and alpha 4beta 7 integrin.

                              
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Table I
Comparison of the inhibitory effects of echistatin and EC3 on various integrins
The data represent the mean from three independent experiments. Fg, fibrinogen; Fn, fibronectin; Vn, vitronectin; Lm, laminin; Coll, collagen; ADP-PA, ADP-induced platelet aggregation; CA, cell adhesion; DBA, direct binding assay; ND, not determined. *, assays performed at Biogen; all other assays were performed at Temple University.

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 (IC50 = 6 µM), whereas epEC3A was inactive in this system. However, epEC3A and epEC3B both inhibited adhesion of K562 cells to fibronectin (IC50 = 30 µM and 6 µM, respectively) (Fig. 2). This experiment suggests that the specificity of EC3 for alpha 4 integrins likely resides in the MLD sequence in the EC3B subunit, whereas the ability if EC3 to inhibit alpha 5beta 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 IC50 of 800 µM, whereas the peptide CRAMLDGLNDYCTGKSSD caused 50% inhibition at 50 µM (not shown).


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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.


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Fig. 3.   Effect of MLDG and RGDS peptides on the adhesion of Jurkat cells to immobilized VCAM-1 (A) and on the K562 cells adhesion to immobilized fibronectin (B). The experiment was performed as described in the legend to Fig. 2. The inhibitory effects of MLDG peptide (open circles) and RGDS peptide (closed circles) are shown. Error bars indicate S.D. from three independent experiments.

Further experiments showed that EC3 competes with mAb HP2/1 for binding to alpha 4 integrin. HP 2/1 at a concentration of 1 µg/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. 4B shows EC3-mediated inhibition of HP2/1 binding to alpha 4B2 (CHO alpha 4+alpha 5-) cells. The inactive G190A alpha 4 mutant did not interact with EC3 (data not shown). In addition, the ability to directly inhibit binding to alpha 5beta 1 was confirmed using mAb SAM-1, which blocked adhesion of K562 cells to immobilized EC3 (data not shown).


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Fig. 4.   Competition of EC3 with RGD peptides and mAb HP2/1. A, effect of GRGDSP, GRGESP, and HP 2/1 mAb on the adhesion of Jurkat cells to immobilized EC3. An adhesion study was performed using 5-chloromethylfluorescein diacetate-labeled Jurkat cells in the absence or presence of 1 mM GRGDSP, 1 mM GRGESP, or 10 µg/ml HP 2/1. Error bars represent S.D. from three independent experiments. B, effect of EC3 on the binding of HP2/1 mAb to alpha 5-deficient CHO cells transfected with alpha 4 integrin. Cells were incubated with 10 µg/ml HP 2/1 in the absence () or presence () of 60 nM EC3 for 30 min at room temperature. After washing, 10 µg/ml of fluorescein isothiocyanate-conjugated goat anti-mouse IgG was added, and the samples were incubated for another 30 min at room temperature. The samples were fixed by the addition of 1% paraformaldehyde before measurement of fluorescence intensity by flow cytometry. The control binding of mouse IgG is shown in the unfilled trace.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha 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 alpha 5beta 1 binding resides on both subunits. However, only EC3B was active in the inhibition of alpha 4beta 1/VCAM-1 interactions. This observation suggests, that MLD is the active sequence in EC3 mediating its anti-alpha 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 beta 1 and beta 3 integrins (27), was not significantly active in this system. The MLDG peptide partially inhibits adhesion of alpha 5beta 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-alpha 4 and anti-alpha 5 inhibitory antibodies are in agreement with this suggestion.

EC3 is a new naturally occurring ligand for alpha 4 integrins. The LD motif from its B subunit is also present in other ligands of alpha 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 alpha 4beta 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 alpha 4beta 1, with an IC50 close to 0.1 µM. 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 alpha 4 inhibitors in the presence of Mn2+. However EC3 has almost the same activity in the presence or absence of Mn2+ (data not shown).

Because EC3A, EC3B, kistrin, and flavoridin show identical alignment of cysteines (Fig. 1) and because the pattern of S-S bonds in kistrin and flavoridin is well established (26, 33-35), it is possible to deduce a hypothetical structure of EC3. Assuming that both subunits of EC3 may have the S-S pattern of kistrin/flavoridin, we propose that Cys-7 and Cys-12 may be involved in two intermolecular bridges. The intramolecular disulfides are likely formed between Cys-6-Cys-29, Cys-20---Cys-26, Cys-25---Cys-50, and Cys-38---Cys-57. On the other hand, if one uses the S-S bonding pattern of albolabrin (25), then Cys-6 and Cys-7 will form intermolecular bridges, and the intramolecular S-S bonds would correspond to Cys-12---Cys-26, Cys-20---Cys-50, Cys-25---Cys-29, and Cys-38---Cys-57. Clearly, further structural studies are needed to establish the S-S pattern of EC3.

EC3 inhibits quite selectively adhesion of alpha 4beta 1-expressing cells to immobilized VCAM-1. Its effect on alpha 5beta 1 and on alpha IIbbeta 3 appears to be lower by 1 and 2 orders of magnitude, respectively. The effect of EC3 on alpha 4beta 1 does not appear to be related to the inhibition of alpha 5beta 1 integrin, because this disintegrin inhibited to the same extent the adhesion to VCAM-1 of CHO cells transfected with alpha 4 and of alpha 5-deficient CHO cells transfected with alpha 4. EC3 also inhibits adhesion of alpha 4beta 7-expressing cells to MadCAM. Because mAb HP2/1 competes with EC3 for binding to alpha 4, it appears that EC3 may bind to the N-terminal domain of alpha 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 alpha IIbbeta 3 to fibrinogen, although it had no significant effect on alpha vbeta 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 beta 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 alpha 4 integrin binding to VCAM-1 and moderately inhibits alpha 5beta 1 integrins. We propose that the activity of EC3 is associated with the MLDG sequence in the putative hairpin loop of this disintegrin.

    ACKNOWLEDGEMENTS

We thank Dr. M. Renz for the generous gift of recombinant VCAM-1, Dr. Y. Takada for providing CHO cells transfected with alpha 4 integrin and its mutants, Drs. M. Ginsberg and J. Loftus for cells transfected with alpha IIbbeta 3 and alpha vbeta 3 integrins, Dr. L. Rosenthal for the critical review of the manuscript, and Diane Leone, Andrew Sprague, and William Yangt for help with assays.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant RO3 DE11844, grants from the American Diabetes Association and Barra Foundation, Ardmore Pennsylvania (to S. N.), an Initial Investigatorship American Heart Association (to C. M.), and a grant-in-aid from the American Heart Association, Southeastern Pennsylvania Chapter (to S. N).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.

Dagger Dagger To whom all correspondence should be addressed: Dept. of Physiology, Temple University School of Medicine, 3400 North Broad St., Philadelphia, PA 19140. Tel.: 215-707-4408; Fax: 215-707-4003; E-mail: stni{at}astro.ocis.templ.edu.

2 Lobb, R., Humpries, M., Tselpis, V., unpublished observation.

3 C. Marcinkiewicz, J. J. Calvete, M. M. Marcinkiewicz, M. Raida, S. Vijay-Kumar, R. R. Lobb, and S. Niewiarowski, unpublished data.

    ABBREVIATIONS

The abbreviations used are: VCAM-1, vascular cell adhesion molecule 1; ep, ethylpyridylethylated; MadCAM-1, mucosal addressin cell adhesion molecule 1; VLA-4, very late antigen-4; HPLC, high performance liquid chromatography; CHO, Chinese hamster ovary cells; CS-1, connecting segment 1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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