Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach

ARTICLE IN PRESS

Toxicon 51 (2008) 659–671 www.elsevier.com/locate/toxicon

Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach$ Ana Lu´cia Oliveira-Carvalhoa, Patrı´ cia Ramos Guimara˜esa, Paula Alvarez Abreub, Denis L.S. Dutraa, Ina´cio L.M. Junqueira-de-Azevedoc,d, Carlos Rangel Rodriguesb, Paulo Lee Hoc,d, Helena C. Castrob,, Russolina B. Zingalia, a Rede Proteoˆmica do Rio de Janeiro and Laborato´rio de Hemostase e Venenos (LabHemoVen), Unidade de Espectrometria de Massas e Proteoˆmica, Programa de Biologia Estrutural, Instituto de Bioquı´mica Me´dica-ICB, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro/RJ, Brazil b Laborato´rio de Antibio´ticos, Bioquı´mica e Modelagem Molecular (LABioMol), Departamento de Biologia Celular e Molecular-IB/CEG, Universidade Federal Fluminense, CEP 24001-970, Nitero´i/RJ, Brazil c Centro de Biotecnologia, Instituto Butantan—IBU, CEP 05503-900, Sa˜o Paulo/SP, Brazil d Instituto de Biocieˆncias, Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil

Received 9 September 2007; received in revised form 28 November 2007; accepted 29 November 2007 Available online 14 December 2007

Abstract Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains a and b. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family. r 2007 Elsevier Ltd. All rights reserved. Keywords: Snake venom; Thrombin inhibitor; Structure; Lectin C-type; Phylogenetic; Electrophoresis 2D; Proteomics

1. Introduction

$

Ethical statement: We would like to declare that the manuscript did not utilize experiments with animals or humans. The study was realized with in vitro assays using purified samples. Corresponding authors. E-mail addresses: [email protected] (H.C. Castro), [email protected] (R.B. Zingali). 0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2007.11.026

Snake venoms contain a variety of proteins with diverse pharmacological activities (Lu et al., 2005; Farsky et al., 2005). These proteins belong to multiple protein families, such as phospholipases A2, serine proteinases, metalloproteinases, C-type lectins, C-type lectin-like proteins (CLPs), Kunitz-type

ARTICLE IN PRESS 660

A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

protease inhibitors, and three-finger toxins, which are encoded by multigenes (Ogawa et al., 2005; Daltry et al., 1996). C-type lectins are nonenzymatic proteins found in animals, which bind in a calcium-dependent manner to mono- and oligosaccharides. They are generally multidomain proteins composed of one or more highly conserved carbohydrate-recognition domain consisting of 115–130 amino acid residues (Drickamer, 1988; Drickamer and Taylor,1993). Snake venom CLPs differ by ca. 70% from the C-type lectins sequences and display different biological activities, but no carbohydrate-binding activity (Drickamer, 1999; Morita, 2004; Weis et al.,1991, 1992; Hirabayashi, et al., 1991; Guimara˜esGomes et al., 2004; Abreu et al., 2006). Some CLPscontaining proteins interfere with clotting factors or plasmatic proteins. Russel’s viper (Vipera russeli) (RVV-X), which contains a metalloproteinase and a C-type lectin-like domain, activates blood coagulation factor X (Takeya et al., 1992). Some proteins binds to the Gla domain of factors IX, X, or both such as—factor IX/X-binding protein, factor IXbinding protein (Atoda et al., 1991, 1995), and factor X-binding protein (Atoda et al., 1998; Tani et al., 2002). Botrocetin binds to the von Willebrand factor (vWF) and blocks platelet aggregation and agglutination (Read et al., 1989; Andrews et al., 1989). Bothrojaracin (BJC) interacts with thrombin through the anion-binding exosites I and II, thus inhibiting platelet aggregation, fibrinogen clotting, and other thrombin functions (Zingali. et al., 1993, 2005; Arocas et al., 1997; Monteiro et al., 1999). Apparently BJC-like proteins are conserved in Bothrops sp. venoms, as their presence has been demonstrated in at least five venoms (Castro et al., 1999). Bothroalternin, isolated from Bothrops alternatus venom, was the first BJC-like molecule to be isolated (Castro et al., 1999, 1998). The cDNAs encoding BJC a and b chains have been cloned, and the deduced amino acid sequences show extensive similarity with botrocetin (a ¼ 80% and b ¼ 66%), factor IX/X-binding protein (a ¼ 57% and b ¼ 54%), and alboaggregin-B (a ¼ 54% and b ¼ 56%) (Arocas et al., 1997). B. insularis is an endemic species restricted to the island of Queimada Grande, located 64 km off the coast of the State of Sa˜o Paulo, Brazil (Campbell and Lamar, 1989). Compared with other Brazilian species of Bothrops, the toxicology of B. insularis is still poorly understood. Interestingly, this serpent has a restricted diet, mainly composed of birds, a

condition imposed by physical isolation. Therefore, B. insularis venom may constitute a valuable protein source for studying the structure–activity relationship of proteins involved in the envenomation process. Recently, different authors have explored venom proteomics in order to better understand this intriguing source of new compounds (Guercio et al., 2006; Bazza et al., 2005). So far, the study of lectin molecules has been restricted to finding a or b chains in 2D gels or Mudpit approaches (Guercio et al., 2006; Bazza et al., 2005). Our group has evaluated the activity of BJC-like molecules in some of the most common Bothrops venom (Castro et al., 1999). Thus, in an effort to identify and understand the structure–activity relationship of these snake venom thrombin-binding proteins, we used proteomics technologies to initially identify the expressed protein from B. insularis and then to purify and characterize. In this work we also present the homology models for BJC and this new thrombin inhibitor based on their similarity with some C-type lectin family member, also comparing them to other CLPs to identify important structure features. 2. Materials and methods 2.1. Materials B. insularis venom was gently provided by the Butantan Institute. Sodium dodecyl sulfate (SDS), azocasein, trichloroacetic acid, ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fuoride (PMSF), D-Phe-Pro-Arg- chloromethylketone (PPACK), anti-rabbit IgG with alkaline phosphatase conjugate, p-nitrophenyl phosphate, and benzamidine were from Sigma (St. Louis, MO). Bovine a-thrombin was from Roche (Basel, Switzerland), human prothrombin was purified as described previously (Bezeaud et al., 1985). Egg lecithin was from Merck (Darmstadt, Germany). Phenol red was from Reagen (Rio de Janeiro, Brazil). Electrophoresis reagents are from GEHealth Care. 2.2. Methods 2.2.1. 2D electrophoresis B. insularis venom (175 or 1800 mg) was dissolved in rehydratation solution (Urea 8 M, chaps 2%, IPG buffer 0.5%, bromophenol blue 0.002%) in the absence of dithiotreitol (DTT). IEF was carried out

ARTICLE IN PRESS A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

in strips (7 or 18 cm, pH 4–7 linear gradient) in the IPG-Phor unit as described by the manufacturer (GE-Healh Care). Precast IPG strip was focused in the gradient steps of 12 h. The temperature was maintained at 20 1C. The IPG strips were then incubated in SDS-polyacrylamide gel and electrophoresis (PAGE) equilibration buffer without DTT for 30 min with gentle shaking. After equilibration, they were directly applied onto 15% polyacrylamide gel and electrophoresis was performed as described by the manufacturer. A Dalt six GE-Healh Care apparatus was used for 18 cm strips, electrophoresis was conducted at 25 W per gel. The gels were fixed and then stained with Comassie Blue G 250 (SigmaAldrich). Direct scanning and image analysis was performed on a ImageScanner with ImageMaster 2D Platinum software. 2.2.2. In gel digestion Each gel spot was sliced and washed with 25 mM ammonium bicarbonate/acetonitrile 1:1 (v/v) three times. After washing, they were covered in with 100% acetonitrile that was removed after a few seconds and replaced by 10 mM DTT in 25 mM ammonium bicarbonate for 45 min at 56 1C. Then it was incubated with 55 mM iodoacetamide in 25 mM ammonium bicarbonate for 45 min at room temperature in the dark. A digestion buffer containing 10 mg/mL trypsin (Promega, modified sequencing grade) in 25 mM ammonium bicarbonate was added to the gel, incubated for 45 min, and then replaced by 50 mM ammonium bicarbonate without trypsin. The samples were incubated at 37 1C overnight and the remaining supernatant was removed and saved. The peptides were extracted from the gel pieces by incubating it in 5% trifluoracetic acid/acetonitrile 1:1 (v/v) for 30 min, when the remaining supernatant was removed and stored. The extraction step was repeated twice and all fractions were pooled and lyophilized to a final volume 10 mL. 2.2.3. Mass spectrometry analysis and database searching Database searching was done using the Mascot search engine (Matrix Science, UK). The data were searched against a venom gland transcriptome databank (Junqueira-de-Azevedo and Ho, 2002) with a mass accuracy of 15 ppm for the parent ion (MS) and 0.2 Da for the fragment ions (MS/MS). Tryptic peptides were admitted with a maximum of one missed cleavage. Carbamidomethylation of cysteine was considered a fixed modification,

661

whereas oxidation of methionine residues was considered as variable modification. An initial list of proteins were generated based on which further analysis was performed. A ‘‘positive list’’ was generated by considering only proteins containing at least one unique peptide with a Mascot score above 20 (p-value 435) in the dataset. 2.2.4. Purification of bothroinsularin Pooled B. insularis crude venom (250 mg) from the Institute Butantan (Sa˜o Paulo, SP, Brazil) was applied to a Sephacryl S-200 column in Tris-saline buffer pH 8.8 and monitored at Abs280 nm. The fraction presenting the inhibitory profile against thrombin-induced platelet aggregation was identified and incubated with a PPACK-a-thrombinsepharose affinity column in 20 mM Tris–Cl, pH 7.5 overnight at 4 1C. The retained material was eluted using 0.001 N HCl, 0.5 M de NaCl, pH 2.0, and immediately neutralized using 1 M Tris-base. The retained material was further applied onto a Superdex G-75 column equilibrated in 20 mM Tris–Cl, pH 7.5, containig 0.5 M NaCl coupled to a FPLC system (fow rate of 1.0 mL/min) and the fractions were further analyzed with anti-BJC serum in reducing and non-reducing conditions. This pool (4 mL) was extensively dialyzed against 2 mM Tris–HCl, pH 7.5, tested for inhibitory activity on thrombin-induced platelet aggregation, and analyzed with anti-BJC serum using western blots. 2.2.5. Platelet aggregation Washed rabbit platelets were prepared from blood containing 5 mM EDTA. Platelets were isolated by centrifugation and washed with calcium-free Tyrode’s buffer and platelet aggregation induced by thrombin (2 nM) was performed in an aggregometer as described elsewhere (Castro et al., 1998). 2.2.6. Fibrinogen clotting This activity was induced by thrombin measured in 10 mM HEPES, 100 mM NaCl, 0.1% PEG 6000, pH 7.4 buffer using a Thermomax Microplate Reader (Molecular Devices, Menlo Park, CA) as described elsewhere (Francischetti et al., 1999). Briefly, thrombin (2 nM) was incubated for 5 min at 37 1C at various concentrations of bothroinsularin (BIN) and the reaction was started by addition of human fibrinogen (2 mg/mL, final concentration). The initial rate of fibrinogen clotting was determined by the increase in Abs405 at 6 s intervals.

ARTICLE IN PRESS 662

A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

2.2.7. Non-denaturing gel electrophoresis Complex formation between BJC and various proteins (thrombin, prothrombin) was analyzed by PAGE in non-denaturing conditions. After preincubation for 2 min in 20 mM Tris–HCl (pH 7.5) with or without 5 mM CaCl2, the mixtures were applied to a 12% acrylamide gel in 365 mM Tris–HCl (pH 8.8). The migration buffer consisted of Tris–glycine-buffer saline, pH 8.8.

2.2.8. Protein sequencing The N-terminal sequence of BIN (both subunits simultaneously, without previous separation of a and b chains) was performed by automated Edman degradation in a Porton Integrated Microssequencing System (Model PI 2090). Phenylthiohydantoin derivatives of amino acids were identified by on-line reverse-phase HPLC.

2.2.9. Rabbit antiserum Rabbit antiserum was obtained by s.c. injection of 150 mg (10 microspots) of highly purified BJC emulsified with 0.5 mL 50% v/v Freund’s complete adjuvant followed by boosters given by the same route with the same antigen and emulsified with Freund’s incomplete adjuvant. Serum titers were monitored by double immunodifusion (Ouchterlony and Nilsson, 1978). Preimmune rabbit serum was used for all immunological assays as controls.

2.2.10. SDS-PAGE and western blots Venom samples were analyzed by SDS-PAGE according to Laemmli (1970), with the Bio-Rad system according to the recommendations of the manufacturer. Proteins were stained with the Coomassie coloration method. Western blots were performed by electro-transferring proteins (200 mA, 2 h) from gels (15%) onto 0.45 mm Immobilon-P transfer membranes. The membranes were blocked with Tris-buffered-saline solution containing 5% milk (TBS-milk) and then incubated with the primary antibody anti-BJC serum. After washing with TBS-milk containing 0.1% tween-20, the membranes were exposed to alkaline phosphataselabeled anti-rabbit immunoglobulins. The reaction was developed according to the manufacturer’s recommendation (Sigma St. Louis, Mo) using chromogenic substrates (nitro-blue tetrazolium and bromo-chloro-indol-phosphate).

2.2.11. Computer analysis and modeling The primary structure alignment of the new thrombin inhibitor from B. insularis (BIN, BJC was performed using a multiple-sequence alignment program: Clustal-W (Thompson et al., 1994), available at http://www.ebi.ac.uk/clustalw. BIN and BJC homology modeling were performed using Swiss Model and Swiss–PDB Viewer programs (Guex and Peitsch, 1997; Schwede et al., 2003) available at http://swissmodel.expasy.org/ and http:// www.expasy.org/spdbv/, respectively. The crystal structures of botrocetin from Bothrops jararaca (PDB entry code 1FVU) and factor IX/factor X-binding protein from Trimeresurus flavoviridis venom (PDB entry code 1IXX) were used as templates. Blocks of structurally conserved regions were identified and the structure alignment of the C-type lectin sequences was generated. Coordinates for all residues were transferred to both BIN and BJC sequences monomers, and loops were constructed in a single round. Several cycles of constrained energy minimization regularized the structures and their geometrical parameters. In subsequent runs, the dimers were constructed according to the templates looking for steric hindrances. The BIN and BJC dimeric models were minimized using Gromos96 parameter set available at Swiss-PDB Viewer program. The energy minimization of the models was performed in vacuo without reaction field and cutoff of 10,000 A˚. After evaluation of the energy of each structure and the repair of distorted geometries through energy minimization, the models were validated as described elsewhere (Castro et al., 2001) and deposited in Protein Data Bank with PDB entry codes 2IMV and 2IMY, respectively. 3. Results and discussion 3.1. Analysis of crude venom The transcriptome analysis of B. insularis venom glands showed at least 16 different sequences homologous to C-type lectins (Junqueira-de-Azevedo and Ho, 2002) denoted mostly as lectins or lectin-like molecules. Data are in agreement with our previous result that indicated the presence of BJC-like molecules in other Bothrops venoms (Castro et al., 1999). We further analyzed B. insularis venom using both non-reduced 2D gel followed by western blot revealed with anti-BJC antibodies, in order to verify the presence of proteins from

ARTICLE IN PRESS A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

663

Fig. 1. 2D analysis of B. insularis crude venom. (A) and (B) Two 2D gel electrophoresis of 175 mg of crude venom were carried out (focalization with 7 cm strips pH 4–7 and 15% SDS PAGE) as described in Section 2. One of them was stained with Comassie blue (A) and the other one was used for the Western blot (B) developed with anti-bothrojaracin serum (1:500) as described. (C) Venom (1800 mg) was applied to 4–7 IPG strips followed by electrophoresis on 15% acrylamide gels. Gel was stained with Coomassie Blue R. Rectangles indicate gel regions that were chosen for spots comparison and identification by MALDI/MS and/or MS/MS (Table 1), and are amplified in (D). All separation was conducted in the absence of DTT.

BJC-like group. Fig. 1A shows the 2D profile (pH 4–7, MW 100–10 kDa) obtained with 175 mg of crude venom that show one group of proteins near the 60 kDa and two patches near 30 kDa. Analysis by western blot revealed two clusters of proteins in the 30 kDa range, which indicated the presence of related molecules (Fig. 1B) as previously shown for other CLPs from Bothrops sp. venom (Castro et al., 2003). With the objective of identifying these proteins, we have performed a larger 2D gel with 1.8 mg of crude venom in the absence of reducing

agent. The gel was better resolved displaying sharp spots (Fig. 1C). A total of 204 spots were observed after gel staining in these conditions, indicating that an analysis of oligomeric proteins bound by disulfide bridges is possible in 2D gels. Since BJC is an acidic protein we decide to better analyze the first patch of proteins showed in the inset of Fig. 1D. The spots were trypsinized after reduction and alkylation, and analyzed by mass spectrometry using a MALDI-TOF-TOF spectrometer. Five

ARTICLE IN PRESS A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

664

spots matched venom lectin and lectin-like proteins using the MASCOT search program and a data bank specific for B. insularis EST sequences or the NCBI Data Bank (Table 1), which included (a) spot 439 that matched botrocetin (one peptide from a chain), the most homologous protein to BJC that cross-reacts with BJC-antibody (Castro et al., 2003); (b) spots 346 and 347 that matched a C-type lectin molecule correlated with GPIb Bp (with one peptide each from a chain); and (c) spots 348 and 340 in which peptides from both chains matched with clusters BINS009 and BINS900, corresponding to molecules similar to BJC b and a chains, respectively (Table 1). These clones were previously classified only as C-type-lectin chains (Junqueira-de-Azevedo and Ho, 2002). It is important to emphasize that although the coverture was not very high, peptides that were obtained by MS/MS are non-redundant and exclusively found in the matched proteins. Generally, a proteomic approach used for the analysis of snake venom led to the identification of chains without considering the association between subunits. Recently, Calvete et al. (2007) described the proteomic analysis of Bitis gabonica gabonica where this venom was first separated by HPLC and analyzed by SDS-PAGE with and without reduction. This approach allowed the identification of various multimeric proteins, including some C-type lectins that shared one common chain. We have focused this first analysis on BJC-like molecules, but we believe that a systematic analysis of 2D gels without DTT will yield more information about the venom complexity, and provide new informa-

tion on C-type lectin family and other multimeric proteins. 3.2. Purification and characterization of B. insularis thrombin inhibitor In order to characterize the BJC-like protein from B. insularis, we attempted to purify this molecule using a previously established protocol (Castro et al., 1998). Thus, B. insularis venom was submitted to a three purification steps that included a Sephacryl S-200, a thrombin-affinity chromatography, and a Superdex G-75 columns as described in Section 2 (Fig. 2). Peak II from the last step exhibited only one band (28KDa) in SDS-PAGE under non-reducing conditions and was recognized by BJC antibodies presenting two bands under reduced conditions, which suggested a heterodimer structure. N-terminal sequencing of this protein confirmed its previous identification as a BJC-like molecule (data not shown). The purified protein named herein as BIN was able to bind to thrombin and prothrombin similar to other BJC-like proteins (Fig. 3A and B). BIN inhibitory activity against thrombin activities was determined. The IC50 of thrombin-induced platelet aggregation and fibrinocoagulation was shown to be 62 and 35 nM, respectively (Fig. 3C and D). This activity was at least 10-fold lower than that observed for other thrombin inhibitors purified so far (i.e. BJC IC50 ¼ 2.5 nM and bothroalternin IC50 ¼ 7.0 nM, respectively for platelet aggregation) (Zingali. et al., 1993; Castro et al., 1998). Since only seven amino acids are not conserved in BIN and

Table 1 Identification of proteins from B. insularis venom by MALDI-TOF-TOF Mass Spectrometry, (MS/MS) of selected peptide ions from in-gel digested protein bands Spot

Sequence coverage (%)

Peptides matched

ID

Protein

439 348

10 12

gi|264834 BINS0900S

Botrocetin A Bothrojaracin A

BINS0009C

Bothrojaracin B

BINS0165C

Coagulation factor IX/Xbinding protein A Coagulation factor IX/Xbinding protein A Bothrojaracin A Bothrojaracin B

346

4

NIQSSDLYAWIGLR DCPSDWSPYGQYCYK IQSSDLYAWIGLR DCPPDWSSYEGSCYR DSFVWTGLSDVWK FEWSDGSDLSYK ELNVWIGMR

347

4

ELNVWIGMR

BINS0165C

340

4 6

MNWADAER DSFVWTGLSDVWK

BINS0900S BINS0009C

15

ARTICLE IN PRESS A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

665

Fig. 2. Purification profile of bothroinsularin. (A) B. insularis crude venom (250 mg) was applied onto a Sephacryl S-200 column and elution was monitored at 280 nm. The inhibitory fraction in thrombin-induced platelet aggregation is shown by the dashed line (- - -). These fractions were pooled, treated with PMSF and EDTA, and incubated with a PPACK-Thrombin column (B) as described in Section 2. The retained material was immediately neutralized after elution and fractionated by SDS-PAGE 12% (inset). (C)The retained material was further applied onto a Superdex G-75 in FPLC system and the 28 kDa band (peak II) eluted was analyzed with antibothrojaracin serum in reducing and non-reducing conditions (inset).

BJC primary sequences (Gly10Glu, Gln11Gly, Tyr12His, Ser62Thr, Leu94Val in a-chain, and Gly74Glu, Arg76Ser in b-chain), data suggested that these amino acids are important for their biological activity. In order to determine the structure–activity relationship, we proceeded with the evaluation of the 3D structures of BIN and BJC.

3.3. Structural and theoretical study of bothroinsularin (BIN) and bothrojaracin (BJC) Initially we performed the sequence alignment of BIN, BJC, and other proteins from C-type lectin family that showed striking conservation exemplified by the six cysteine residues forming intrachain disulfide bridges (Cys2–Cys13, Cys30–Cys127 and Cys102–Cys119 in a-chain, and Cys2–Cys13, Cys30–Cys123 and Cys100–Cys115 in b-chain) (Fig. 4). Interestingly, Cys79 in a-chain and Cys75 in b-chain formed the interchain disulfide bridge in both BIN and BJC, similar to other CLPs, but differently from true lectins such as B. insularis lectin, where Cys88 is involved in forming the interchain disulfide link (Guimara˜es-Gomes et al., 2004; Schwede et al., 2003) (Fig. 4). The primary structure analysis of BIN revealed the highest sequence similarity with BJC (a ¼ 94% and b ¼ 98%), followed by botrocetin (a ¼ 79% and b ¼ 69%), GPIb-bp (a ¼ 68% and b ¼ 62%), factor IX/X-binding protein

(a ¼ 61% and b ¼ 56%), and B. insularis lectin (a ¼ 30% and b ¼ 31%). Experimentally BIN and BJC activities are not calcium-dependent (not shown) although both present a calcium-binding site (Ser41, Gln43, Glu47, and Glu128) in b-chain and an incomplete calcium-binding site in a-chain subunit, where Glu128 is replaced by Lys128 (Fig. 4). Therefore, these proteins are more similar to bitiscetin and Echis IX/X-bp, which are also calcium-independent proteins (Maita et al., 2003; Atoda et al., 2002; Hirotsu et al., 2001), than to Tf-IX/X, whose crystal structure revealed Ca2+-binding sites in both a and b subunits, and botrocetin that has only one metal ion-binding site in b subunit for Mg2+ (Maita et al., 2003; Sen et al., 2001; Mizuno et al., 1999) . These metal-binding sites are reported to be important for ligand recognition and in the stabilization of native structure for some proteins but not for all C-type family members such as BIN and BJC (Atoda et al., 2002; Hirotsu et al., 2001). Construction of BIN and BJC homology models revealed that each subunit of these proteins has a compact globular unit and an extended long loop as described for other C-type lectin-like family members (Fig. 5). BIN and BJC dimeric structures are mainly maintained by hydrophobic interactions between the extended loop of the a-chain and the globular domain of the b-chain and vice versa, reinforcing the conserved folding mechanism of this

ARTICLE IN PRESS 666

A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

Fig. 3. Bothroinsularin biological activities. (A) and (B) Non-denaturing gel electrophoresis was conducted after incubation of bothroinsularin (BIN) at 37 1C for 2 min with thrombin (THRO) (A) and prothrombin (PRO) (B) non-denaturing PAGE (12% acrylamide) was carried out. Samples were applied after incubation in a ratio molar 1:1. Results are from one representative experiment out of three. The complexes bothoinsularin–thrombin and bothroinsularin–prothrombin are indicated by an arrow. (C) and (D) Inhibitory activity of bothroinsularin upon thrombin (2 nM) activities. (C) Platelet aggregation was performed as described in Section 2. Inset: The arrow indicates the increase in light transmittance that accompanies platelet aggregation registered by the lumi-aggregometer: (1) control without bothroinsularin, (2) BIN 20 nM, (3) BIN 100 nM, (4) BIN 200 nM; (D) fibrinocoagulation was performed as described in Section 2. Inset: % of inhibitory activity.

family (Sen et al., 2001; Monteiro et al., 2003) (not shown). Importantly, BIN and BJC maintained almost identical secondary and three-dimensional (3-D) structures revealing a low root mean square deviation (RMSD ¼ 0.07) on the backbone chain structure alignment (Fig. 5). These results suggested that the differences on BIN and BJC thrombin inhibitory profiles are probably due to some variation in their surface and electrostatic features instead of just the 3-D structure (Fig. 5). In fact,

BIN and BJC homology models reinforced this interpretation as the substitution of all seven amino acids including those of BJC polar and hydrophilic residues (Glu10, Glu74, His12) that are replaced in BIN for neutral or hydrophobic residues (Gly10, Gly74, Tyr12) produced a slightly different electrostatic potential map that could justify their difference in platelet aggregation and fibrinocoagulation potencies (Fig. 5). BIN and BJC show a predominantly negative region that may favor the

ARTICLE IN PRESS A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

667

Fig. 4. Sequence alignment of six C-type lectins a and b subunits using CLUSTAL W (http://www.ebi.ac.uk/clustalw). Identical amino acids in the C-type lectin-like proteins are labeled with *, the conserved amino acids are +, semiconserved amino acids are , and the conserved cysteines are underlined. Amino acids identical to bothroinsularin are shown in gray. Abbreviations are bothrojaracin a (Bjca) (gi:62131098) and b (Bjcb) (gi:62131100) chains and botrocetin a (gi:399125) (Botca) and b (Botcb) (gi:399126) chains from Bothrops jararaca venom, factor IX/factor X-binding protein a (Tf IX/Xa) (gi:233489) and b (Tf IX/Xb) (gi:233490) chains from Trimeresurus flavoviridis, platelet glycoprotein Ib-binding protein a (GPIb-bpa) (gi:1839441) and b (GPIb-bpb) (gi:1839442) chains from B. jararaca venom, and B. insularis lectin (BiL) (gi:41353970).

interaction with the positive regions of thrombin (exosite 1 and 2). They also present some positively charged areas that may help in the orientation of this binding event as thrombin present negative regions near from the anion-binding exosites (ABEI and ABE-II) (Castro and Rodrigues, 2006). Therefore, as the center of the BIN molecule is slightly more apolar and its positive patches were more exposed than BJC, these features may be affecting the interaction of this molecule with thrombin in some way (Fig. 5). Our theoretical study suggested that the position 74 in b-chain is an

important residue that apparently modulates the electrostatic profile of these molecules. According to our theoretical mutation study, the substituted b-chain 74 position is the one that most contribute for the electrostatic difference observed between BJC and BIN compared with to other positions (i.e. 10 and 12) (not shown). Therefore, this is an interesting position for evaluation using site-directed mutagenesis on BIN and BJC for further structure–activity relationship studies. Compared with other CLPs (i.e. coagulation factor IX-BP, Flavocetin, and Bitiscetin), the overall

ARTICLE IN PRESS 668

A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

Fig. 5. Comparison of bothroinsularin model with other C-type lectin family members. (A) Structural alignment of bothroinsularin (BIN) and bothrojaracin (BJC) homology models in ribbon (RMS ¼ 0.08) with the four regions containing the non-conserved positions in solid spheres (BIN ¼ pink and BJC ¼ light pink) in the full model and in stick in the zoom view (1–4). (B) Electrostatic potential maps of BIN and BJC homology models (front view—larger, back view—smaller). (C) Representation of the dimeric structure—a-chain in yellow and b-chain in light gray (first line), the secondary structures (second line), and the electrostatic potential map (front view-third line and back view-fourth line) of the C-type lectin family members.The binding sites described in the literature for botrocetin, factor IX/X-binding protein, and bitiscetin are within the yellow box.

3-D folding of BIN and BJC models was also very similar (Fig. 5). However, despite the similar folding, the structure alignment of BIN model with factor IX/X-binding protein (Tf-IX/X) and botrocetin revealed a high RMSD (5.15 and 6.61 A˚, respectively) for the dimeric structure. These high RMSD values are mainly due to differences in b subunit, which alone presented the highest RMSD (7.25 and 8.91 A˚, respectively) compared with the a-chain fold (1.35 and 3.10 A˚, respectively). These results indicate that these structural differences may in part account for the biological activities observed for these molecules (Sen et al., 2001).

Comparison of BIN and BJC a and b chains with other CLPs revealed differences where the 81–97 loop of b chain folds more distantly from the a-chain globular unit than the other CLPs (Fig. 5). Despite of that BIN and BJC 80s-loop extends to the adjoining subunit to form a loop swapping dimer similar to Tf IX/X-BP and botrocetin and other CLPs. Since true lectins such as B. insularis lectin present this loop folded back to their globular domain (Guimara˜es-Gomes et al., 2004; Mizuno et al., 1999). Literature suggests that CLPs loop swapping leads to loss of carbohydrate-binding activity, whereas the concave surface created by

ARTICLE IN PRESS A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

the dimerization is predicted to function as a coagulation factor-binding site (Maita et al., 2003; Monteiro et al., 2003). Recently it has been proposed that the concave surfaces of many CLPs form the binding site for a variety of highly diverse ligands (Hirotsu et al., 2001), and interestingly, the electrostatic potential maps for these proteins differ in this region (Fig. 5). The analysis of BIN electrostatic potential map revealed significant differences from other CLPs, including Tf-IX/X. The positively charged patch (Arg 114, 116, 120) near to the concave surface (Fig. 5) of Tf-IX/X is apparently required for interaction with the g-carboxyl glutamic acid (Gla) residues as seen in factors IX/X-BP and IX-BP (Sen et al., 2001). Interestingly, these residues are replaced by Arg114, Ile116, and Thr120 in BIN, and Trp114, Ile116, and Thr120 in botrocetin. According to the literature, 20 acidic residues (6 in a-chain and 14 in b-chain) form the botrocetin negatively charged cleft supposedly involved in binding to the vWF A1 domain positive region (Fig. 5) (Sen et al., 2001; Matsushita et al., 2000). Similarly, bitiscetin also present a negative region that binds to the vWF A1 domain. However, a bitiscetin large positive surface (i.e. Lys 17, 20, 23, 30, 60 and 116, and Arg 62) that probably binds to the vWF A3 domain negative patch is also observed (Hirotsu et al., 2001). Interestingly, BIN and BJC homology models also pointed to the concave center of these molecules as a feasible region to be directly or indirectly involved in binding thrombin. Despite the similar fold, the CLPs specific substitutions in the primary structure involved in changing charge distribution are probably responsible for their different biological activity (i.e. BIN, Tf IX/X-BP, and botrocetin) or their degree of activity (i.e. BIN and BJC). Even though the exact binding sites of BIN or BJC to thrombin were not indicated, these homology models that are now available on Protein Data bank are important tools for further site-directed mutation studies.

Acknowledgments We thank the Instituto Butantan (Sa˜o Paulo, Brazil) for providing the snake venom. We also thank Mr Hugo R.O.B. Filho and Dione M. Silva for their technical assistance, Dr. Franklin Rumjanek for critical reading of the manuscript and Dr. Debora Foguel for the use of HPLC columns.

669

This research was supported in part by the International Foundation of Science (Stockholm, Sweden) grant to R.B.Z. (contract F/3156-1). Additional support was provided by Coordenac- a˜o de Aperfeic- oamento de Pessoal de Nı´ vel Superior (CAPES); Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico (CNPq), Fundac- a˜o de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundac- a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Fundac- a˜o Butantan. References Abreu, P.A., Albuquerque, M.G., Rodrigues, C.R., Castro, H.C., 2006. Structure–function inferences based on molecular modeling, sequence-based methods and biological data analysis of snake venom lectins. Toxicon 48 (6), 690–701. Andrews, R.K., Booth, W.J., Gorman, J.J.P., Castaldi, P.A., Berndt, M.C., 1989. Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex. Biochemistry 28 (21), 8317–8326. Arocas, V., Castro, H.C., Zingali, R.B., Guillin, M.C., JandrotPerrus, M., Bon, C., Wisner, A., 1997. Molecular cloning and expression of bothrojaracin, a potent thrombin inhibitor from snake venom. Eur. J. Biochem. 248 (2), 550–557. Atoda, H., Hyuga, M., Morita, T., 1991. The primary structure of coagulation factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis. Homology with asialoglycoprotein receptors, proteoglycan core protein, tetranectin, and lymphocyte Fc epsilon receptor for immunoglobulin E. J. Biol. Chem. 266 (23), 14903–14911. Atoda, H., Ishikawa, M., Yoshihara, E., Sekiya, F., Morita, T., 1995. Blood coagulation factor IX-binding protein from the venom of Trimeresurus flavoviridis: purification and characterization. J. Biochem. 118 (5), 965–973. Atoda, H., Ishikawa, M., Mizuno, H., Morita, T., 1998. Coagulation factor X-binding protein from Deinagkistrodon acutus venom is a Gla domain-binding protein. Biochemistry 37, 17361–17370. Atoda, H., Kaneko, H., Mizuno, H., Morita, T., 2002. Calciumbinding analysis and molecular modeling reveal echis coagulation factor IX/factor X-binding protein has the Cabinding properties and Ca ion-independent folding of other C-type lectin-like proteins. FEBS Lett. 531 (2), 229–234. Bazza, A., Marrakchi, N., Ayeb, M.E., Sanz, L., Calvette, J.J., 2005. Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina. Proteomics 5 (16), 4223–4235. Bezeaud, A., Denninger, M.H., Guillin, M.C., 1985. Interaction of human alpha-thrombin and gamma-thrombin with antithrombin III, protein C and thrombomodulin. Eur. J. Biochem. 153 (3), 491–496. Calvete, J.J., Marcinkiewicz, C., Sanz, L., 2007. Snake venomics of Bitis gabonica gabonica. Protein family composition, subunit organization of venom toxins, and characterization

ARTICLE IN PRESS 670

A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671

of dimeric disintegrins bitisgabonin-1 and bitisgabonin-2. J. Proteome Res. 6 (1), 326–336. Campbell, J.A., Lamar,W.W., 1989. The Venomous Reptiles of Latin America, Ithaca, London, 425p. Castro, H.C., Rodrigues, C.R., 2006. Current status of snake venom thrombin-like enzymes. Toxin Rev. 25 (3), 291–318. Castro, H.C., Dutra, D.L.S., Oliveira-Carvalho, A.L., Zingali, R.B., 1998. Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus. Toxicon 36 (12), 1903–1912. Castro, H.C., Fernandes, M., Zingali, R.B., 1999. Identification of bothrojaracin-like proteins in snake venoms from Bothrops species and Lachesis muta. Toxicon 37 (10), 1403–1416. Castro, H.C., Silva, D.M., Craik, C., Zingali, R.B., 2001. Structural features of a snake venom thrombin-like enzyme: thrombin and trypsin on a single catalytic platform? Biochim. Biophys. Acta 1547 (2), 183–195. Castro, H.C., Lemos, M.G., Bon, C., Zingali, R.B., 2003. Comparative evaluation of immunological and structural similarities of snake venom C-type lectin proteins. Toxicon 41 (4), 525–528. Daltry, J.C., Wuster, W., Thorpe, R.S., 1996. Diet and snake venom evolution. Nature 379 (6565), 537–540. Drickamer, K., 1988. Two distinct classes of carbohydraterecognition domains in animal lectins. J. Biol. Chem. 263 (20), 9557–9560. Drickamer, K., 1999. C-type lectin-like domains. Curr. Opin. Struct. Biol. 9 (5), 585–590. Drickamer, K., Taylor, M.E., 1993. Biology of animal lectins. Rev. Cell Biol. 9, 237–264. Farsky, S.H., Antunes, E., Mello, S.B., 2005. Pro and antiinflammatory properties of toxins from animal venoms. Curr. Drug Targets Inflamm. Allergy 4 (3), 401–411. Francischetti, I.M., Valenzuela, J.G., Ribeiro, J.M., 1999. Anophelin: kinetics and mechanism of thrombin inhibition. Biochemistry 38 (50), 16678–16685. Guercio, R.A., Shevchenko, A., Shevchenko, A., Lopez-Lozano, J.L., Paba, J., Souza, M.V., Ricart, C.A., 2006. Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox. Proteome Sci. 4, 11. Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modeling. Electrophoresis 18 (15), 2714–2723. Guimara˜es-Gomes, V., Oliveira-Carvalho, A.L., Junqueira-deAzevedo, I.L., Dutra, D.L.S., Pujol-Luz, M., Castro, H.C., Ho, P.L., Zingali, R.B., 2004. Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom. Arch. Biochem. Biophys. 432 (1), 1–11. Hirabayashi, J., Kusunoki, T., Kasai, K., 1991. Complete primary structure of a galactose-specific lectin from the venom of the rattlesnake Crotalus atrox. Homologies with Ca2(+)- dependent-type lectins. J. Biol. Chem. 266 (4), 2320–2326. Hirotsu, S., Mizuno, H., Fukuda, K., Qi, M.C., Matsui, T., Hamako, J., Morita, T., Titani, K., 2001. Crystal structure of bitiscetin, a von Willebrand factor-dependent platelet aggregation inducer. Biochemistry 40 (45), 13592–13597. Junqueira-de-Azevedo, I.L.M., Ho, P.L., 2002. A survey of gene expression and diversity in the venom glands of the pitviper snake Bothrops insularis through the generation of expressed sequence tags (ESTs). Gene 299 (1–2), 279–291.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (5259), 680–685. Lu, Q., Clemetson, J.M., Clemetson, K.J., 2005. Snake venoms and hemostasis. Thromb. Haemost. 3, 1791–1799. Maita, N., Nishio, K., Nishimoto, E., Matsui, T., Shikamoto, Y., Morita, T., Sadler, J.E., Mizuno, H., 2003. Crystal structure of von Willebrand factor A1 domain complexed with snake venom, bitiscetin insight into glycoprotein Iba binding mechanism induced by snake venom proteins. J. Biol. Chem. 278 (39), 37777–37781. Matsushita, T., Meyer, D., Sadler, J.E., 2000. Localization of von Willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis. J. Biol. Chem. 275 (15), 11044–11049. Mizuno, H., Fujimoto, Z., Koizumi, M., Kano, H., Atoda, H., Morita, T., 1999. Crystal structure of coagulation factor IXbinding protein from Habu snake venom at 2.6 A: implication of central loop swapping based on deletion in the linker region. J. Mol. Biol. 289 (1), 103–112. Monteiro, R.Q., Raposo, J.G., Wisner, A., Guimaraes, J.A., Bon, C., Zingali, R.B., 1999. Allosteric changes of thrombin catalytic site induced by interaction of bothrojaracin with anion-binding exosites I and II. Biochem. Biophys. Res. Commun. 262 (3), 819–822. Monteiro, R.Q., Foguel, D., Castro, H.C., Zingali, R.B., 2003. Subunit dissociation, unfolding, and inactivation of bothrojaracin, a C-type lectin-like protein from snake venom. Biochemistry 42, 509–515. Morita, T., 2004. C-type lectin-related proteins from snake venoms. Drug Targets Cardiovasc. Haematol. Disord. 4 (4), 357–373. Ogawa, T., Chijiwa, T., Oda-Ueda, N., Ohno, M., 2005. Amino acid sequence of a basic aspartate-49-phospholipase A2 from Trimeresurus flavoviridis venom and phylogenetic analysis of Crotalinae venom phospholipases A2. Toxicon 45 (2), 1–14. Ouchterlony, O., Nilsson, L.A., 1978. Immunodiffusion and immunoelectrophoresis. In: Weir, D.M. (Ed.), Handbook of Experimental Immunology. Blackwell Scientific, Oxford, pp. 99.1–99.44. Read, M.S., Smith, S.V., Lamb, M.A., Brinkhous, K.M., 1989. Role of botrocetin in platelet agglutination: formation of an activated complex of botrocetin and von Willebrand factor. Blood 74 (3), 1031–1035. Schwede, T., Kopp, J., Guex, N., Peitsch, M.C., 2003. SWISSMODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31 (13), 3381–3385. Sen, U., Vasudevan, S., Subbarao, G., McClintock, R.A., Celikel, R., Ruggeri, Z.M., Varughese, K.I., 2001. Crystal structure of the von Willebrand factor modulator botrocetin. Biochemistry 40 (2), 345–352. Takeya, H., Nishida, S., Miyata, T., Kawada, S., Saisaka, Y., Morita, T., Iwanaga, S., 1992. Coagulation factor X activating enzyme from Russell’s viper venom (RVV-X). A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains. J. Biol. Chem. 267 (20), 14109–14117. Tani, A., Ogawa, T., Nose, T., Nikandrov, N.N., Deshimaru, M., Chijiwa, T., Chang, C.C., Fukumaki, M., Ohno, M., 2002. Characterization, primary structure and molecular evolution of anticoagulant protein from Agkistrodon actus venom. Toxicon 40 (6), 803–813.

ARTICLE IN PRESS A.L. Oliveira-Carvalho et al. / Toxicon 51 (2008) 659–671 Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22 (22), 4673–4680. Weis, W.I., Crichlow, G.V., Murthy, H.M., Hendickson, W.A., Drickamer, J., 1991. Physical characterization and crystallization of the carbohydrate-recognition domain of a mannose-binding protein from rat. J. Biol. Chem. 266 (31), 20678–20686.

671

Weis, W.I., Drickamer, K., Hendrickson, W.A., 1992. Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature 360 (127–134). Zingali, R.B., Jandrot-Perrus, M., Guillin, M.C., Bon, C., 1993. Bothrojaracin: a potent two-site-directed thrombin inhibitor. Biochemistry 32 (28), 10794–10802. Zingali, R.B., Ferreira, M.S., Assafim, M., Frattani, F.S., Monteiro, R.Q., 2005. Bothrojaracin, a Bothrops jararaca snake venomderived (pro)thrombin inhibitor, as an anti-thrombotic molecule. Thromb. Pathophysiol. Haemostasis 34 (4–5), 160–163.