Cytotoxic activities of [Ser49]phospholipase A2 from the venom of the saw-scaled vipers Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus

Toxicon 71 (2013) 96–104

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Cytotoxic activities of [Ser49]phospholipase A2 from the venom of the saw-scaled vipers Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus J. Michael Conlon a, *, Samir Attoub b, Hama Arafat b, Milena Mechkarska a, Nicholas R. Casewell c, d, Robert A. Harrison c, Juan J. Calvete e a

Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain 17666, United Arab Emirates Department of Pharmacology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates c Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, UK d Molecular Ecology and Evolution Group, School of Biological Sciences, Bangor University, Bangor, UK e Laboratorio de Venómica y Proteinómica Estructural Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 March 2013 Received in revised form 19 May 2013 Accepted 23 May 2013 Available online 4 June 2013

Fractionation by reversed-phase HPLC of venom from four species of saw-scaled viper: Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus led to identification in each sample of an abundant protein with cytotoxic activity against human non-small cell lung adenocarcinoma A549 cells. The active component in each case was identified by MALDI-TOF mass fingerprinting of tryptic digests as [Ser49]phospholipase A2 ([Ser49]PLA2). An isoform of [Ser49]PLA2 containing the single Ala18 / Val substitution and a partially characterized [Asp49]PLA2 were also present in the E. coloratus venom. LC50 values against A549 cells for the purified [Ser49]PLA2 proteins from the four species are in the range 2.9–8.5 mM. This range is not significantly different from the range of LC50 values against human umbilical vein endothelial HUVEC cells (2.5–12.2 mM) indicating that the [Ser49]PLA2 proteins show no differential anti-tumor activity. The LC50 value for [Ser49] PLA2 from E. ocellatus against human erythrocytes is >100 mM and the MIC values against Escherichia coli and Staphylococcus aureus are >100 mM. It is suggested that the [Ser49]PLA2 proteins play a major role in producing local tissue necrosis and hemorrhage at the site of envenomation. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Echis Phospholipase A2 Cytotoxicity Anti-cancer activity Adenocarcinoma A549 cells Human umbilical vein endothelial cells

1. Introduction The saw-scaled vipers (Viperidae: Echis) are widely distributed in Northern Africa, the Middle East, Western Asia and the Indian subcontinent and are responsible for

* Corresponding author. Tel.: þ971 791 3 7137484; fax: þ971 791 3 7672033. E-mail address: [email protected] (J.M. Conlon). 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.05.017

more deaths by snakebite than snakes from any other genus (Warrell, 1995). Envenomation generally results in localized edema, hemorrhage and necrosis at the bite site and with systemic hemorrhage, disseminated intravascular coagulation and fibrinolysis with up to 20% mortality rates (Warrell et al., 1977). The taxonomy of Echis is controversial and has been subject to several revisions [reviewed in Pook et al., 2009]. Twelve species and 20 subspecies were recognized by Cherlin (1990) using morphological criteria but the proposal to divide the genus into subgenera has not

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been generally accepted (David and Ineich, 1999). Analysis of nucleotide sequences of mitochondrial gene fragments demonstrates that populations of Echis may be divided into four main clades: the Echis carinatus, E. coloratus, E. ocellatus and E. pyramidum species groups (Pook et al., 2009). The E. pyramidum and E. coloratus groups are regarded as sister taxa and some evidence suggests the E. ocellatus group forms a sister group to that clade, with the E. carinatus group the most basal lineage (Barlow et al., 2009; Casewell et al., 2011). Venoms also represent a huge and largely unexplored reservoir of bioactive components, and may be regarded as “oceans of opportunity” for the pharmaceutical industry (Escoubas and King, 2009; Calvete, 2009). The composition of the venom of the ocellated carpet viper E. ocellatus from Nigeria has been studied in detail using a combined proteomics and transcriptomics approach (Wagstaff et al., 2009). This involved fractionation by reversed-phase HPLC and characterization of the major components by MALDI-TOF mass fingerprinting and MS/MS amino acid sequencing together with analysis of approximately1000 EST sequences from a venom gland cDNA library. Around 35 distinct proteins were identified belonging to 8 different toxin families with Zn2þ-dependent metalloproteinases, phospholipase A2 (PLA2), C-type lectin-like proteins and disintegrins present in highest concentration. Correlation between the number of proteins identified in the proteome with those predicted by the transcriptome was only fair suggesting that the final composition of the venom may be determined by complex transcriptional and posttranslational regulatory mechanisms. This study was complemented by analysis of venom gland transcriptomes of E. pyramidum leakeyi, E. coloratus, and E. carinatus sochureki that revealed substantial intrageneric venom variation (Casewell et al., 2009). However, transcripts encoding metalloproteinases, C-type lectins, PLA2, serine proteases, L-amino acid oxidases, and growth factors were obtained from all four transcriptomes. There is a constant need for new types of anti-cancer agents particularly in cases where the tumor is not responsive to conventional pharmaceutical therapy (Chen and Tiwari, 2011). The presence of proteins with antitumor activity in venoms has been described for a wide range of snake species belonging to the Viperidae family (Marcinkiewicz et al., 2003; Roberto et al., 2004; Swenson et al., 2005; Galán et al., 2008; Grozio et al., 2008; Bazaa et al., 2009; El-Refaei and Sarkar, 2009; Park et al., 2009; Rodrigues et al., 2009; Khunsap et al., 2011; Klein et al., 2011; Hayashi et al., 2012). The initial impetus for this study was to examine venom samples from an Echis species from each of the four species groups (E. ocellatus, E. pyramidum leakeyi, E. carinatus sochureki and E. coloratus) for the presence of components with anti-cancer activity using human non-small cell lung adenocarcinoma A549 cells (Foster et al., 1998). Selectivity for tumor cells was assessed by measuring cytotoxicity against non-neoplastic human umbilical vein endothelial cells (HUVECs) (Park et al., 2006) and human erythrocytes. Several snake venoms are associated with broad-spectrum antibacterial activity (Perumal Samy et al., 2007, 2008). The presence of proteins with antibacterial activity in the Echis venoms was assessed

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using reference strains of Gram-negative Escherichia coli and the Gram-positive Staphylococcus aureus bacteria. 2. Materials and methods 2.1. Venom collection All lyophilized venom samples were supplied by the Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, U.K. E. ocellatus venom (58.2 mg) was collected in Nigeria, E. coloratus venom (28.5 mg) was collected in Egypt, E. pyramidum leakeyi venom (33. 9 mg) was collected in Kenya, and E. carinatus sochureki venom (27.1 mg) was collected in the United Arab Emirates. In each case, the samples represented a pool of venoms from 10 snakes collected in the same locality. 2.2. Cytotoxicity assays Human non-small cell lung adenocarcinoma A549 cells were maintained at 37  C in RPMI 1640 medium containing 2 mM L-glutamine and supplemented with 10% fetal calf serum (FCS, Biowest, Nouaille, France), and antibiotics (penicillin 50U/mL; streptomycin 50 mg/mL). EndoGRO human umbilical vein endothelial cells (HUVECs) were maintained in EndoGRO MV-VEGF Complete Media Kit (Millipore, Temecula, CA, USA). In all experiments, cell viability was determined by trypan blue dye exclusion to be higher than 99%. Cells were seeded in 96-well plates at a density of 5  103 cells/well. After 24 h, the cells were treated for 24 h with freeze-dried aliquots (100 mL) of chromatographic effluent. Cell viability was determined by measurement of ATP concentrations using a CellTiter-Glo Luminescent Cell Viability assay (Promega Corporation, Madison, WI, USA). Luminescent signals were measured using a GLOMAX Luminometer system. LC50 values were determined by incubating either A549 or HUVEC cells (5  103 cells/well) with increasing concentrations of the purified [Ser49]PLA2 proteins (0.3–10 mM) in triplicate. LC50 was taken as the mean concentration of peptide producing 50% cell death in three independent experiments. 2.3. Hemolysis assay Freeze-dried aliquots (100 mL) of chromatographic effluent were incubated with washed human erythrocytes (2  107 cells) from a healthy donor in Dulbecco’s phosphate-buffered saline, pH 7.4 (100 mL) for 1 h at 37  C. After centrifugation (1500  g for 30 s), the absorbance at 450 nm of the supernatant was measured. A parallel incubation in the presence of 1% v/v Tween-20 was carried out to determine the absorbance associated with 100% hemolysis. The LC50 value of the purified [Ser49]PLA2 from E. ocellatus was measured in the concentration range of 6.25–100 mM as previously described (Conlon et al., 2012). 2.4. Antimicrobial assays Reference strains of microorganisms were purchased from the American Type Culture Collection (Rockville, MD, USA). The ability of freeze-dried aliquots (100 mL) of

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chromatographic effluent to inhibit the growth of E. coli (ATCC 25726) and S. aureus (ATCC 25923) was determined in duplicate using 96-well microtiter cell-culture plates. After reconstitution in Mueller–Hinton broth (50 mL), the fractions were incubated with an inoculum (50 mL of 106 colony forming units/mL) from a log-phase culture of reference strains of the bacteria for 18 h at 37  C in a humidified atmosphere of air. After incubation, the absorbance at 630 nm of each well was determined using a microtiter plate reader. Minimum inhibitory concentrations (MICs) of the purified [Ser49]PLA2 from E. ocellatus against S. aureus (ATCC 25923) and E. coli (ATCC 25726) were measured in the concentration range of 6.25–100 mM by a standard double dilution method (Clinical Laboratory and Standards Institute, 2008) and were taken as the lowest concentration of peptide where no visible growth was observed. The values were confirmed by measurement of absorbance at 630 nm. In order to monitor the validity and reproducibility of the assays, incubations were carried out in parallel with increasing concentrations of ampicillin as previously described (Conlon et al., 2012).

with an equal volume of a 1:10 (v/v) dilution of a saturated solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile containing 0.1% TFA, dried, and analyzed with a Voyager-DE Pro MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA) operated in delayed extraction and reflectron modes. Tryptic peptide mass fingerprints (PMF) were matched against a combined Echis (E. ocellatus, E. pyramidum leakeyi, E. carinatus sochureki and E. coloratus) transcriptome database (Casewell et al., 2009; Wagstaff et al., 2009). PMF-based protein identifications were validated by sequencing selected peptide ions in a Waters nanoAquity uPLC-SYNAPTÔ G2 mass spectrometry system. Collision-induced dissociation (CID) spectra were interpreted manually or using MassLynxÔ searches against the Echis transcriptome database. For both, PMF- and CIDbased searches, mass tolerance was set to 0.6 Da, and carbamidomethyl cysteine and oxidation of methionine were fixed and variable modifications, respectively. 3. Results 3.1. Purification of the PLA2 proteins

2.5. Protein purification The lyophilized venom samples were redissolved in 0.1% (v/v) trifluoroacetic acid (TFA)/water (2 mL) and injected onto a (2.2 cm  25 cm) Vydac 218TP1022 (C-18) reversedphase HPLC column (Grace, Deerfield, IL, USA) equilibrated with 0.1% (v/v) TFA/water at a flow rate of 6.0 mL/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients. Absorbance was monitored at 214 nm and 280 nm, and fractions (1 min) were collected. Freeze-dried aliquots (100 mL) of the fractions were reconstituted in RPMI 1640 medium (100 mL) and their abilities to produce cytolysis of lung adenocarcinoma A549 cells were determined as described in the previous section. Fractions containing peptides with cytotoxic activity were successively chromatographed on a (1 cm  25 cm) Vydac 214TP510 (C-4) column and a (1 cm  25 cm) Vydac 219TP510 (phenyl) column. The concentration of acetonitrile in the eluting solvent was raised from 21% to 56% over 50 min and the flow rate was 2.0 mL/min. 2.6. Protein identification Electrospray-ionization mass spectrometry of the purified PLA2 proteins was carried out using an Agilent 6310 Series ion trap instrument (Agilent Technologies, Santa Clara, CA, USA) in positive ion mode as previously described (Zahid et al., 2011). For protein identification, the purified PLA2 proteins were initially analyzed by SDS-PAGE (on 12 or 15% polyacrylamide gels under reducing and non-reducing conditions) and the protein bands were excised from Coomassie Brilliant Blue-stained gels and subjected to automated reduction, alkylation, and in-gel digestion with sequencing grade porcine pancreatic trypsin using a ProgestÔ digestor (Genomic Solutions, Ann Arbor, MI, USA). The tryptic peptide mixtures were dried in a SpeedVac and dissolved in 5 mL of 50% acetonitrile and 0.1% TFA. 0.85 mL of digest were spotted onto a MALDI-TOF sample holder, mixed

The elution profiles on a preparative Vydac C-18 column of venoms from E. ocellatus, E. pyramidum leakeyi, E. carinatus sochureki and E. coloratus are shown in Fig. 1(A–D). In each case, only the prominent peak designated by the asterisk contained material with appreciable cytotoxic activity (>50% cell death under the conditions of assay) against human lung adenocarcinoma A549 cells. Studied at the same concentration, no fraction contained material that inhibited growth of E. coli and S. aureus or produced appreciable hemolysis of human erythrocytes (>10% cell lysis). Rechromatography of the peaks containing the cytotoxic components from E. ocellatus, E. pyramidum leakeyi, and E. carinatus sochureki on semi-preparative Vydac C-4 column revealed that the material was already >90% pure and purification to near homogeneity was accomplished by a final chromatography on a Vydac phenyl column (data not shown). The yields of the purified proteins, subsequently shown to be [Ser49]PLA2, were E. ocellatus 1172 mg (2.01%), E. pyramidum leakeyi 378 mg (1.11%), and E. carinatus sochureki 344 mg (1.27%). The figures in parentheses show the % of the total mass of lyophilized venom. In contrast, chromatography of the peak with cytotoxic activity from E. coloratus on a Vydac C4 column indicated the presence of four proteins in relatively high abundance (Fig. 2). The major component (peak 1), subsequently shown to be [Ser49]PLA2 was purified to near homogeneity on a Vydac phenyl column. The final yield of pure protein was 450 mg representing 1.59% of the total mass of lyophilized venom. Peaks 2 and 3 were subsequently shown to contain isoforms of PLA2 and peak 4 contained a protein of mass 24,682 Da that was identified as a cysteine-rich secretory protein (CRISP) (transcript ECO_00025) that has a calculated molecular mass [MþH]þ of 24,683.8 Da (Casewell et al., 2009). 3.2. Identification of the PLA2 proteins The identities of the cytotoxic proteins were determined by PMF and CID-MS/MS analysis of peptides generated by in-

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Fig. 1. Reversed-phase HPLC on a preparative Vydac C-18 column of venom from (A) E. ocellatus, (B) E. pyramidum leakeyi, (C) E. carinatus sochureki, and (D) E. coloratus. The peaks designated by the asterisk displayed strong cytotoxic activity against lung adenocarcinoma A549 cells and were purified further. The dashed line shows the concentration of acetonitrile in the eluting solvent.

Fig. 2. Purification of PLA2 proteins from E. coloratus on a semi-preparative Vydac C-4 column. Peak 1 contained [Ser49]PLA2, peak 2 contained an isoform of the component in peak 1, peak 3 contained an [Asp49] PLA2 protein related to transcript ECO_00086, and peak 4 contained a cysteine-rich secretory protein (CRISP) (transcript ECO_00025). The arrowheads show where peak collection began and ended. The dashed line shows the concentration of acetonitrile in the eluting solvent.

gel tryptic digestion. As shown in Fig. 3, identification of multiple peptides from the proteins from E. ocellatus, E. pyramidum leakeyi, E. carinatus sochureki, the peak 1 and peak 2 proteins from E. coloratus demonstrated identity with [Ser49]PLA2. The average molecular masses ([MþH]þ) of the purified [Ser49]PLA2s determined by electrospray mass spectrometry were consistent with their proposed structures: E. ocellatus Mr obs 13,825, Mr calc 13,826; E. pyramidum leakeyi Mr obs 13,697, Mr calc 13,698; E. carinatus sochureki Mr obs 13,850, Mr calc 13,852; E. coloratus peak 1 Mr obs 13,692, Mr calc 13693; E. coloratus peak 2 Mr obs 13,720, Mr calc 13,720. Mr obs refers to the observed molecular mass in Daltons and Mr calc refers to the molecular mass calculated from the proposed sequences shown in Fig. 3. The average molecular mass of the peak 3 component from E. coloratus was 13,798 Da. This mass does not correspond exactly with that of any PLA2 proteins present in the E. coloratus venom transcriptome database (Casewell et al., 2009). However, analysis of its tryptic PMF indicated that the peak 3 protein is structurally similar to the [Asp49] PLA2 transcript sequence ECO_00086 (Fig. 3). The calculated mass of the ECO_00086 protein is 13,818 suggesting

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E. ocellatus (EO_00015) SVVELGKMIIQETGKSPFPSYTSYGCFCGGGEKGTPKDATDRCCFVHSCCYDKLPDC SPKTDRYKYQRENGEIICENSTSCKKRICECDKAVAVCLRENLQTYNKKYTYYPNFL CKGEPEKC E. pyramidum (EPL_00195) SVIELGKMIIQLTNKTPASYVSYGCFCGGGDKGKPKDATDRCCFVHSCCYDTLPDCS PKTDQYKYKWENGEIICENSTSCKKRICECDKAVAICLRDNLNTYNKKYRIYPNFLC RGDPDKC E. carinatus sochureki (ECS_00014) SIVELGKMIIQETGKSPFPSYTSYGCFCGGGERGPPLDATDRCCLAH SCCYDTLPDCS PKTDRYKYKRENGEIICENSTSCKKRICECDKAMAVCLRKNLNTYNKKYTYYPNFW CKGDIEKC E. coloratus 1 (Ec_01C02_ECO00035) SVIELGKMIVQLTNKTPASYVSYGCFCGGGDRGKPKDATDRCCFVHSCCYDTLPDCS PKTDQYKYKWENGEIICENSTSCKKRICECDKAVAICLRENLKTYNKKYKIYPNILCR GEPDKC E. coloratus 2 (Ec_04H07_ECO00035) SVIELGKMIVQLTNKTPVSYVSYGCFCGGGDRGKPKDATDRCCFVHSCCYDTLPDCS PKTDQYKYKWENGEIICENSTSCKKRICECDKAVAICLRENLKTYNKKYKIYPNILCR GEPDKC E. coloratus 3 ( ECO_00086), HLLQFENMIYQKTGKFAIIAYSNYGCYCGWGGKgkpqdatdrCCFVHDCCYGRVNgcdpk ADSYSYSFENGDIVCGDDDPCRRavcecdrVAANCFAENLKtynkkYWLSSIIDCKeesekc E. coloratus peak 4 (ECO_00025) NVDFDSESPRKPEIQNEIIDLHNSLRRSVNPTASNMLRMEWYPEAAANAERWAFRCT LNHSPRDSRVIDGIKCGENIYMSPYPIKWTAIIHKWHDEKKNFVYGIGASPANAVIGH YTQIVWYKSYRGGCAAAYCPSSAYKYFYVCQYCPAGNIIGKTATPYKSGPPCGDCPS ACDNGLCTNPCTREDEFINCNDLVKQGCQTDYLKSNCAASCFCHSEIK Fig. 3. Primary structures of the PLA2 and CRISP proteins from E. ocellatus (EO), E. pyramidum leakeyi (EPL), E. carinatus sochureki (ECS), and E. coloratus (ECO) venoms. Tryptic peptides sequenced by mass spectrometry are underlined. The proteins are identified by their venom gland transcriptome database codes. The symbol “w” denotes “similar to” and the amino acid residues shown in lower case have not been confirmed by sequence analysis. Residue at position 49, which defines the PLA2 subfamily, is displayed in boldface. E. coloratus CRISP was identified by its molecular mass determined by electrospray mass spectrometry.

that the peak 3 component contains one or more amino acid substitutions in the regions denoted by the lower case letters in Fig. 3 compared with the [Asp49]PLA2 in the database. 3.3. Cytotoxic activities of the [Ser49]PLA2 proteins The effect of increasing concentration of the purified [Ser49]PLA2 proteins on the viability of human lung adenocarcinoma A549 cells and human umbilical vein endothelial HUVEC cells is shown in Fig. 4. The LC50 values are shown in Table 1. The LC50 value for [Ser49]PLA2 from E. ocellatus against human erythrocytes was >100 mM and the MIC values against E. coli and S. aureus were >100 mM. 4. Discussion The multi-functional PLA2 superfamily of enzymes (EC 3.1.1.4) is widely distributed in nature and the proteins have been divided into 16 distinct groups on the basis of

their primary structures (Schaloske and Dennis, 2006). The secretory PLA2 proteins present in venoms from the Viperidae are classified as group II (Six and Dennis, 2000). The PLA2 enzymes were originally identified by their ability to hydrolyze the ester bond at position 2 of 1,2-diacyl-sn-3phosphoglycerides but snake venom PLA2 proteins exhibit a wide range of pharmacological properties. These include myonecrosis, neurotoxicity, cardiotoxicity, anti-coagulant, hemorrhagic, hemolytic, and anti-angiogenic activities, and the ability to inhibit platelet aggregation [reviewed in Kini, 2005]. Although the amino acid sequences of the group II PLA2 proteins from Viperidae are moderately well conserved, their biological properties differ appreciably. Enzymes with an aspartic acid residue at position 49 of the catalytically active site show strong esterolytic activity and Ca2þ-dependent myotoxic activity whereas lysine-49 homologs have lost their ability to bind Ca2þ and so are catalytic inactive but retain myotoxic activity by a Ca2þ-independent mechanism (Ownby et al., 1999; Gutiérrez and Ownby, 2003; Gallacci and Cavalcante, 2010;

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Fig. 4. Effects on the viability of A549 human lung adenocarcinoma cells and HUVEC human umbilical vein cells of a 24 h exposure to [Ser49]PLA2 from E. ocellatus (panels A & B), E. coloratus (panels C & D), E. carinatus sochureki (panels E & F), and E. pyramidum leakeyi (panel G & H). All experiments were repeated three times. Columns: mean; bars: SEM.

Lomonte and Rangel, 2012; Gutiérrez and Lomonte, 2013). PLA2 variants with a serine residue at position 49 have been isolated from the venoms of Vipera ammodytes (Kri zaj et al., 1991) and E. carinatus sochureki (Polgar et al., 1996; Zhou et al., 2008). These proteins show potent Ca2þ-

Table 1 Cytotoxicities of [Ser49]PLA2 isolated from four species of saw-scaled viper against human non-small cell lung adenocarcinoma A549 cells and human umbilical vein endothelial HUVEC cells. A549 E. E. E. E.

ocellatus coloratus carinatus sochureki pyramidum leakeyi

Data show LC50 values (mM)  S.E.M.

5.2 3.5 8.5 2.9

   

HUVEC 0.3 0.4 0.3 0.2

5.0 4.9 12.2 2.5

   

0.3 0.5 0.3 0.2

independent myotoxicity but, unlike the Lys49 homologs, also display weak esterolytic activity (Petan et al., 2007). The present study has shown that, under the conditions of assay, [Ser49]PLA2 is the component with the greatest cytotoxicity against human non-small cell lung adenocarcinoma A549 cells in the venom of four species of sawscaled viper. It should be pointed out, however, that the proteins were isolated under the relatively harsh elution conditions of reversed-phase HPLC so that it is possible that the cytotoxicity of other venom proteins was reduced, or even abolished, by denaturation. These [Ser49]PLA2 components from E. ocellatus, E. carinatus sochureki, and E. pyramidum leakeyi differ from the PLA2 proteins from the same species whose primary structures were predicted from the nucleotide sequences of cDNAs and contain an aspartic acid at position 49 (Bharati et al., 2003). Similarly, the [Ser49]PLA2 with a mass of 13,850 Da isolated from

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venom of E. carinatus sochureki from the U.A.E. in this study is clearly different from the ortholog with mass 13,804 Da (termed ecarpholin S) identified by Polgar et al. (1996) in commercially available venom from the same species whose geographical origin was not stated. The major [Ser49]PLA2 component in E. coloratus venom is the same as a protein previously identified in the E. coloratus venom gland transcriptome database (Casewell et al., 2009) The minor [Ser49]PLA2 component with mass 13,720 Da differs from the major component by a single Ala / Val substitution suggestive of a very recent gene duplication event. However, in view of the fact that the sample studied was prepared from a pool of venoms from 10 snakes, the possibility that the isoform represented a difference in allelic expression or a mutation in the gene of an individual cannot be excluded. Appreciable intra- as well as interspecies heterogeneity is often found in the composition of snake venoms (including Echis) and can result in impaired efficacy of anti-venoms (Calvete et al., 2009; Casewell et al., 2010). The partially characterized 13,798 Da [Asp49]PLA2 has not been described previously. The primary structures of the [Ser49]PLA2 proteins isolated in this study are compared in Fig. 5. Previous studies have identified snake venom PLA2 proteins as potential anti-cancer agents (Rodrigues et al., 2009). Examples include [Asp49]PLA2 and [Lys49]PLA2s from Bothrops brazili active against human T-cell leukemia JURKAT cells (Costa et al., 2008), a [Lys49]PLA2 from Protobothrops flavoviridis also active against human leukemia cells (Murakami et al., 2011), and a purified but incompletely characterized PLA2 from Daboia russelii siamensis venom that was cytotoxic to human skin melanoma SKMEL-28 cells in vitro and inhibited lung tumor colonization by murine skin melanoma B16F10 cells in BALB/c mice (Khunsap et al., 2011). The Echis [Ser49]PLA2s are cytotoxic

to A549 lung adenocarcinoma with LC50 values 8.5 mM (Table 1) but show only very weak hemolytic activity against human erythrocytes (LC50 > 100 mM for the E. ocellatus protein). However, their therapeutic potential as anti-tumor agents is severely restricted by their strong cytotoxic activities against non-neoplastic HUVEC (LC50  12.5 mM). The [Ser49]PLA2 proteins are present in high concentration in the venom of each species are so it is probable that they make an important contribution to the local tissue necrosis observed at the site of the snakebite. The cytotoxicity against cells derived from human vein tissue indicate that these proteins may also be responsible, along with the metalloproteinases in the venom (Sajevic et al., 2011), for the observed vascular damage and disruption of capillary vessels that leads to local hemorrhage following envenomation (Baldo et al., 2010; Escalante et al., 2011). Our present findings identifying [Ser49]PLA2 molecules as the major cytotoxic factors of Echis venoms broaden the understanding of the action of non-[Asp49] PLA2 enzymes, and should be helpful in the structureguided development of new drugs for the topical treatment of necrosis caused by saw-scaled viper bites (Warrell, 1995; Mahadeswaraswamy et al., 2008). Group II secreted PLA2 proteins are a component of the system of innate immunity in mammals and show potent growth-inhibitory activity against Gram-positive bacteria but are generally incapable of penetrating the lipopolysaccharide envelope of Gram-negative bacteria [reviewed in Nevalainen et al., 2008]. In contrast, both [Asp49] and [Lys49]PLA2 from Bothrops asper venom showed broadspectrum anti-bactericidal activity and a synthetic 13residue peptide representing a region near the C-terminal loop of the [Lys49]PLA2 reproduced the bactericidal effect of the intact protein (Páramo et al., 1998; Santamaría et al., 2005). Similarly, an incompletely characterized [Asp49]

Fig. 5. A comparison of the primary structures of the [Ser49]PLA2 proteins isolated from E. ocellatus, E. pyramidum leakeyi, E. carinatus sochureki, and E. coloratus venoms. Amino acids that are fully conserved in all four species are shown in bold type. A gap, denoted by *, has been introduced in some sequences to maximize structural similarity. The Ala / Val substitution in the E. coloratus paralogs is underlined.

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PLA2 isolated from commercially available E. carinatus venom showed strong antibacterial activity against the Gram-negative bacteria Burkholderia pseudomallei and Enterobacter aerogenes but activity against a range of other clinically relevant microorganisms was weak (Perumal Samy et al., 2010). Snake venom PLA2 molecules have potential as therapeutic agents to treat infections produced by antibiotic-resistant bacteria such as methicillin-resistant S. aureus (MRSA) and extended-spectrum b-lactamase (ESBL) producing E. coli. However, the [Ser49]PLA2 from E. ocellatus showed no growth inhibitory activity against reference strains of either E. coli or S. aureus at concentrations up to 100 mM. There was not sufficiently pure material to determine MIC values for the [Ser49]PLA2 proteins from E. coloratus, E. pyramidum leakeyi, and E. carinatus sochureki against E. coli and S. aureus but analysis of the chromatographic fractions of venom from these species (Fig. 1) did not indicate the presence of a component with potent activity against these microorganisms. Ethical statement The study did not involve any work with live animals or human subjects. Acknowledgments This work was supported by a Faculty Support Grant and a University/National Research Foundation Grant from U.A.E. University and an award from the Terry Fox Fund for Cancer Research (to JMC and SA), by grant NE/J018678/1 from the Natural Environment Research Council (NERC), UK (to NRC) and grant BFU2010-17373 from the Ministries of Science and Innovation (MICINN) and Economy and Competitivity (MINECO), Madrid, Spain (to JJC). The authors thank Dr M. Meetani, Department of Chemistry, U.A.E. University for mass spectrometry measurements, Mr Paul Rowley, Liverpool School of Tropical Medicine for his herpetological expertise, and Dr. Katarina Hostanska, Department of Internal Medicine, Institute for Complementary Medicine, University Hospital Zurich, Switzerland for providing the A549 cell line. Conflict of interest The authors declare no conflict of interest. References Baldo, C., Jamora, C., Yamanouye, N., Zorn, T.M., Moura-da-Silva, A.M., 2010. Mechanisms of vascular damage by hemorrhagic snake venom metalloproteinases: tissue distribution and in situ hydrolysis. PLoS Negl. Trop. Dis. 4, e727. Barlow, A., Pook, C.E., Harrison, R.A., Wüster, W., 2009. Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Proc. Roy. Soc. B 276, 2443–2449. Bazaa, A., Luis, J., Srairi-Abid, N., Kallech-Ziri, O., Kessentini-Zouari, R., Defilles, C., Lissitzky, J.C., El Ayeb, M., Marrakchi, N., 2009. MVL-PLA2, a phospholipase A2 from Macrovipera lebetina transmediterranea venom, inhibits tumor cells adhesion and migration. Matrix Biol. 28, 188–193. Bharati, K., Hasson, S.S., Oliver, J., Laing, G.D., Theakston, R.D., Harrison, R.A., 2003. Molecular cloning of phospholipases A2 from venom glands of Echis carpet vipers. Toxicon 41, 941–947.

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