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Comparative proteomic analysis of the venom of the taipansnake, Oxyuranus scutellatus, from Papua New Guinea andAustralia: Role of neurotoxic and procoagulant effects invenom toxicity

María Herreraa, Julián Fernándeza, Mariángela Vargasa, Mauren Villaltaa, Álvaro Seguraa,Guillermo Leóna, Yamileth Anguloa, Owen Paivab, Teatulohi Matainahob,Simon D. Jensenb, c, Kenneth D. Winkelc, Juan J. Calveted,David J. Williamsb, c, e, José María Gutiérreza,⁎aInstituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa RicabSchool of Medicine & Health Sciences, University of Papua New Guinea, Port Moresby, Papua New GuineacAustralian Venom Research Unit, Department of Pharmacology, University of Melbourne, Parkville, VIC, AustraliadInstituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaume Roig 11, 46010 Valencia, SpaineNossal Institute for Global Health, University of Melbourne, Parkville, VIC, Australia

A R T I C L E I N F O

⁎ Corresponding author. Tel.: +506 2229 3135;E-mail address: jose.gutierrez@ucr.ac.cr (J

1874-3919/$ – see front matter © 2012 Elseviedoi:10.1016/j.jprot.2012.01.006

A B S T R A C T

Article history:Received 14 December 2011Accepted 8 January 2012Available online 14 January 2012

The venom proteomes of populations of the highly venomous taipan snake, Oxyuranusscutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dis-sociation tandemmass spectrometry of tryptic peptides. Proteins belonging to the followingseven protein families were identified in the two venoms: phospholipase A2 (PLA2), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase,cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition,C-type lectin/lectin-like protein and venom natriuretic peptide were identified in thevenom of specimens from PNG. PLA2s comprised more than 65% of the venoms of thesetwo populations. Antivenoms generated against the venoms of these populations showeda pattern of cross-neutralization, corroborating the immunological kinship of thesevenoms. Toxicity experiments performed in mice suggest that, at low venom doses, neuro-toxicity leading to respiratory paralysis represents the predominant mechanism of prey im-mobilization and death. However, at high doses, such as those injected in natural bites,intravascular thrombosis due to the action of the prothrombin activator may constitute apotent and very rapid mechanism for killing prey.

© 2012 Elsevier B.V. All rights reserved.

Keywords:TaipanOxyuranus scutellatusVenomTaipoxinProteomeAntivenom

fax: +506 2292 0485..M. Gutiérrez).

r B.V. All rights reserved.

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1. Introduction

Australia and New Guinea harbor a rich biodiversity of venom-ous snakes of the family Elapidae, which include species posses-sing some of the most toxic venoms in the world [1]. Amongthem, the taipans (Oxyuranus spp.), are distributed in Australia,Papua New Guinea and Indonesian Papua, and produce someof the world's most toxic snake venoms [2]. Combined withtheir agility, speed, large adult body size (up to 3m forOxyuranusscutellatus), high venom output (>100mg) and nervous disposi-tions, taipans are extremely dangerous snakes to encounter.Snakebites by Oxyuranus spp. often result in severe neurotoxicenvenoming which may be fatal in the absence of prompt anti-venom treatment [3,4]. There are three species of taipans: (a) O.scutellatus, with populations in northern Australia, southernPapua New Guinea and southern Indonesian Papua [1,5,6]; (b)Oxyuranus microlepidotus, the inland taipan, which occurs insouth-western Queensland and north-eastern to central-northern South Australia [7]; and (c) Oxyuranus temporalis,known from a handful of specimens collected in the south-eastern deserts of Western Australia [7]. O. scutellatus occurs ina wide variety of habitats including sugarcane fields and wood-lands in the eastern and north coast of Australia and in savan-nah regions of southern New Guinea [5]. Phenotypic differencesbetween theNewGuinean andAustralian populations of the tai-pan had prompted their classification as separate subspecies,Oxyuranus scutellatus canni and Oxyuranus s. scutellatus, respec-tively [8]. However, recent evidence from mitochondrial DNAanalysis revealed high similarities between these populations,demonstrating very recent genetic exchange among them, thusquestioning their subspecific status [6,7]. Hence, further studiesare required to assess the intraspecies variation between thesepopulations.

Human envenomings by O. scutellatus are not common innorthern Australia [1]. However, this species inflicts many se-vere bites in the southern regions of Papua New Guinea (PNG)[4]. These envenomings are predominantly characterized by aneurotoxic effect of rapid onset, which often leads to respira-tory paralysis in the absence of timely administration of anti-venom [3,9]. In addition, coagulopathy associated withspontaneous bleeding has been described in these patients[10], together with myotoxicity and cardiac disturbances insome cases [11]. The toxins responsible for these effectshave been isolated and characterized, such as the potent neu-rotoxic and myotoxic heterotrimeric phospholipase A2 (PLA2)complex taipoxin (named cannitoxin for the West Papuanpopulation) [12,13], two monomeric PLA2s, named OS1 andOS2 [14], Oscutarin-C, a prothrombin activator responsiblefor the characteristic coagulopathy [15], post-synapticallyacting α-neurotoxins [16], and taicatoxin, a blocker ofvoltage-dependent calcium channels [17]. In addition, cDNAanalysis derived from venom gland transcripts detectedvarious putative toxin genes, including PLA2s, neurotoxins,cysteine-rich secretory proteins (CRISPs), a venom natriureticpeptide and a nerve growth factor [18]. However, besides theproteins previously characterized from this venom, thepresence in the venom of these additional componentsencoded by these transcripts, and their possible biologicalactivity, remain unknown.

The present work presents a comparative proteomic analysisof the venomsofO. scutellatus fromAustralia and PNG, in order toascertain whether there are phenotypic differences in thevenom composition of these populations. In parallel, the mostrelevant biological activities of these venoms were determined,and correlated with the venom proteome. Owing to the acceler-ated processes that characterize the evolution of snake venomtoxins [19–21], it was of interest to assess whether the venomproteome of these very similar but allopatric populations differs,in order to predict possible variations in their biological/toxico-logical profiles, and in the immunologically cross-reactivity ofantivenoms raised against these venoms. In addition, the poten-tial role of neurotoxic and procoagulant components of thesevenoms in prey immobilizationwas explored in amousemodel.

2. Materials and methods

2.1. Venom fractionation

The venom of O. scutellatus from PNG was a pool obtainedfrom twelve healthy, adult specimens collected in PNG'sMilne Bay Province and Central Province. These snakes weremaintained in a purpose-built serpentarium at the Universityof PNG, and venom was collected at 21 day intervals. Venomwas obtained using Parafilm-covered Eppendorf tubes. Sam-ples contaminated by blood were discarded, and all sampleswere handled using plastic pipettes and tubes. Venom wassnap-frozen to −80 °C, before being freeze-dried and storedaway from light at −20 °C. The venom of Australian O. scutella-tus was obtained from Venom Supplies Pty Limited (Tanunda,South Australia). It was stored at −20 °C until used. Forreverse-phase HPLC separations, 5 mg venom was dissolvedin 200 μL of 0.1% trifluoroacetic acid (TFA) and 5% acetonitrile(buffer A). Insoluble material was removed by centrifugationat 13,000 g for 10 min at room temperature and then the su-pernatant was loaded on a C18 column (250×4.6 mm, 5 μmparticle size; Agilent) using an Agilent 1100 chromatograph.For the elution of proteins, a flow of 1 mL of solvent/min wasused, with the following conditions: 5% B (B: 95% acetonitrile,0.1% TFA) for 10 min, followed by 5–15% B over 20 min, 15–45%B over 120 min, and 45–70% B over 20 min. Proteins weredetected at 215 nm. Protein peaks were collected manuallyand dried in a Speed Vac (Savant). The relative abundances(% of the total venom proteins) of the different protein fami-lies in the venoms were estimated from the relation of thesum of the areas of the chromatographic peaks containingproteins from the same family to the total area of venom pro-tein peaks. ChemStation v.B.04.01 (Agilent) software was usedfor the integration of the chromatogram. If a chromatographicpeak contained two or more proteins, their relative distribu-tion was estimated by densitometry of the SDS-PAGE-separated bands using ImageJ v.1.4 (http://rsb.info.nih.gov/ij/).

2.2. Characterization of protein fractions

Protein fractions were separated by 15% SDS-PAGE (reducingconditions) and stained with Coomassie Blue R-250. Some iso-lated protein fractions were subjected to N-terminal sequenceanalysis (using a Procise instrument, Applied Biosystems,

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Foster City, CA) according to the manufacturer's instructions.Amino acid sequence similarity searches were performedagainst the available databanks using the BLAST program[22]. In addition, Coomassie-stained bands were excisedfrom gels and subjected to reduction with dithiothreitol andalkylation with iodoacetamide, followed by in-gel digestionwith sequencing grade bovine trypsin on an automated di-gester (ProGest, Digilab), according to manufacturer's instruc-tions. Each tryptic digest was then analyzed in a MALDI-TOF–TOF Applied Biosystems 4800-Plus mass spectrometer, afterspotting 0.5 μL of α-cyano-4-hydroxycinnamic acid and 0.5 μLof each sample onto an Opti-TOF 384-well plate. For spectraacquisition, 1625 shots were accumulated in reflector positivemode, using a laser intensity of 3000. CalMix-5 (ABSciex) pep-tides were used as external standards. The twelve most in-tense ions of each MS spectrum were selected as precursorsfor automated collision-induced dissociation MS/MS (2KV,positive mode) using 500 shots per sample and a laser intensi-ty of 3900. The resulting spectra were analyzed using Protein-Pilot v.2.0.1 (ABSciex) to identify proteins, at a confidence levelof 99%. Tryptic digests not identified by MALDI-TOF–TOF weresubjected to nano-electrospray ionization (nESI)-MS/MS anal-ysis on a Q-Trap 3200 instrument (Applied Biosystems).Doubly- or triply-charged ions of peptides selected from theMALDI-TOF mass fingerprint spectra were analyzed in En-hanced Resolution mode (250 amu/s), and the monoisotopicions were fragmented using the Enhanced Product Ion toolwith Q0 trapping. Settings for MS/MS analyses were: Q1, unitresolution; collision energy, 25–40 eV; linear ion trap Q3 filltime, 250 ms; and Q3 scan rate, 1000 amu/s. ResultingCollision-Induced Dissociation (CID) spectra were interpretedwith the aid of the BioAnalyst v.1.5 (http://www.absciex.com/Products/Software/BioAnalyst-Software) manual se-quencing tool, and the obtained sequences were first com-pared to all of the known Oxyuranus spp. sequences with theMEGA 5 alignment and analysis tool (http://www.megasoftware.net) using a genus level toxin sequence data-base prepared from BLAST (http://blast.ncbi.nlm.nih.gov)search data before being submitted to BLAST for a generalizedsearch against all snake venom sequences if no genus levelidentity matches were found.

2.3. Biological and enzymatic activities of venoms

2.3.1. Lethal activityTwo types of experiments were performed concerning lethal-ity: In the first set, the Median Lethal Dose (LD50) of venomswas assessed in CD-1 mice (18–20 g) by the intravenous (i.v.)route, in order to determine whether the differences invenom proteomes translate into variations in the systemictoxicity. Various doses of venoms were injected in the caudalvein and deaths occurring within 24 h were recorded. LD50

was estimated by the Spearman–Karber method [23]. In an-other set of experiments, an experimental design was usedto determine the probable cause of death in mice whenusing low and high venom doses, in order to discern themode of death in rodents when a large dose of venom isinjected, as occurs in natural bites. In these experiments,mice were injected by the i.v. route, with specific low andhigh doses of either venom or purified taipoxin; the signs of

toxicity shown by mice were observed and the time of deathwas recorded. In some experiments, tissue samples were col-lected from the lung immediately after death, and placed informalin fixative solution. After routine processing, sampleswere embedded in paraffin, and 5–8 μm sections were pre-pared and stained with hematoxylin–eosin for histologicalanalysis. In the case of mice injected with PBS, they weresacrificed by an overdose of anesthetic (a mixture of xylazineand ketamine), and tissue samples were processed as de-scribed. The experimental protocols involving the use of ani-mals in this study were approved by the InstitutionalCommittee for the Care and Use of Laboratory Animals(CICUA) of the University of Costa Rica.

2.3.2. Coagulant activity in vitroThemethod described by TheakstonandReid [24]was followed,with the modifications described by Vargas et al. [25]. Variousamounts of venom, dissolved in 100 μL of 0.15 M NaCl, wereadded to aliquots of 200 μL of human citratedplasmapreviouslyincubated at 37 °C for 5 min. CaCl2 was added (15 μL of 0.2 MCaCl2 to 200 μL plasma) immediately before addition of thevenom. Experiments were run in quadruplicate. Clotting timeswere recorded, and the Minimum Coagulant Concentration(MCC) was determined. TheMCC corresponds to the concentra-tion of venom that induced plasma clotting in 60 s.

2.3.3. PLA2 activityPLA2 activity was determined titrimetrically, using egg yolkphospholipids as substrates [26]. Activity was expressed asμEq fatty acid released per mg protein per min.

2.4. Neutralization by antivenoms

In order to assess the cross-neutralization of venom lethaland coagulant activities by antivenoms raised against thevenoms of O. scutellatus from PNG and Australia, monospecifictaipan antivenoms produced by Instituto Clodomiro Picado(batch 4511209 ICP; expiry date November 2012) and by CSLLimited (CSL), Melbourne, Victoria, Australia (batch B0548-06301; expiry date March 2012) were used. The former wasraised against the venom of O. scutellatus from PNG and thelatter against venom from specimens collected in Australia.The production methods and physicochemical characteristicsof these antivenoms were previously described [25]. For neu-tralization assays, a fixed dose of venom (4 LD50s for lethalityand 2 MCCs for coagulant effect) was incubated with varyingvolumes of antivenom, at 37 °C, for either 30 min (for lethalactivity) or 3 min (for coagulant activity). Controls includedsolutions of venom incubated with saline solution instead ofantivenom. After incubation, lethal and coagulant activitieswere assessed as described above. Neutralization wasexpressed as either Effective Dose 50% (ED50), for lethality, orEffective Dose (ED), for coagulant effect, corresponding to theratio mg venom neutralized per mL antivenom at which neu-tralization was achieved, as previously described [25].

2.5. Statistical analysis

In some cases, the significance of the differences betweenmean values of two experimental groups was assessed by

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the Student's t test, using Instat statistics software (GraphPadsoftware, La Jolla, CA).

3. Results and discussion

3.1. Venomics

The RP-HPLC elution profiles of O. scutellatus venoms fromPNG and Australia, and the SDS-PAGE migration of the pro-teins present in each fraction are shown in Figs. 1 and 2. Al-though the two chromatograms had a similar number ofpeaks, 23 and 25, the elution patterns revealed differences.Proteins related to seven and nine protein families were iden-tified by mass spectrometry in the venoms of Australian andPNG O. scutellatus, respectively. These families of proteinsidentified in both venoms correspond to: phospholipase A2

(PLA2), BPTI/Kunitz-type inhibitor, metalloproteinase (SVMP),three-finger toxin (3FTx), cysteine-rich secretory proteins(CRISP), and subunits of the factor Va-Xa-like prothrombin ac-tivator Oscutarin C (Tables 1 and 2; Fig. 3). Three sequenceswith identity to thrombin-like, serine proteinase enzymes(TLE) were observed in Australian O. scutellatus venom. In

Fig. 1 – Elution profiles of venom proteins ofO. scutellatus from Papof proteins in each peak (B). Five milligram venom was fractionateFractions were analyzed by SDS-PAGE under reducing conditionsor nESI-MS/MS tryptic peptide MS/MS de novo sequencing, as sumleft lanes of each gel.

addition, C-type lectin/lectin-like proteins, and venom natri-uretic peptides were identified in the venom of PNG O.scutellatus.

Phospholipase A2 species, including taipoxin, were themost abundant proteins in both venoms (Fig. 3). Proteomicanalysis highlights the abundance of the neurotoxic heterotri-mer taipoxin in both venoms, albeit with quantitative differ-ences, since a significantly higher concentration (56% vs20%) was observed in the venom of PNG O. scutellatus(Tables 1 and 2). The higher proportion of this very potenttoxin may explain the observed lower LD50 of PNG O. scutella-tus venom inmurine testing (Table 3). This PLA2 oligomer con-stitutes one of the most toxic components yet isolated fromsnake venoms [12,27]. It induces presynaptic neurotoxicity as-sociated with the depletion of transmitter from the motornerve terminals and other ultrastructural manifestations ofterminal nerve damage [12,28,29], caused by the ability ofthis toxin to hydrolyze phospholipids in the plasma mem-brane of nerve terminals [30]. In addition, taipoxin inducesmyotoxicity owing to its ability to disrupt the integrity of skel-etal muscle plasma membrane [31].

Taipoxinwasoriginally isolated fromthevenomofAustralianO. scutellatus [12]. More recently, an analogous toxin, named

ua NewGuinea by RP-HPLC (A), and electrophoretic separationd on a C18 column, as described in Materials and methods.and characterized by N-terminal sequencing, MALDI-TOF/TOFmarized in Table 1. Molecular massmarkers are shown in the

Fig. 2 – Elutionprofiles of venomproteins ofO. scutellatus fromAustralia by RP-HPLC (A), and electrophoretic separation of proteinsin each peak (B). Five milligram venomwas fractionated on a C18 column, as described inMaterials andmethods. Fractions wereanalyzed by SDS-PAGE under reducing conditions and characterized by N-terminal sequencing, MALDI-TOF/TOF or nESI-MS/MStryptic peptideMS/MSdenovo sequencing, as summarized inTable 2.Molecularmassmarkers are shown in the left lanes of eachgel.

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cannitoxin, was characterized from the venom of PapuanO. scutellatus [13], although this can be regarded as taipoxingiven that the PNG and Australian populations belong to thesame species, and indeed, we identified the N-terminal se-quence for taipoxin (NLLQFGFMIR), rather than cannitoxin(NLLQFGYMIR), in venom of our PNG O. scutellatus suggestingthat both isoforms are likely to exist in this venom. In bothpopulations, the trimers are comprised of subunit α, which isan active neurotoxic PLA2, and by subunits β and γ, which arePLA2 homologues without enzymatic activity [12,13,32–34]. De-spite the lack of catalytic and toxic activities of subunits β andγ, they significantly potentiate the toxicity of subunit α [27,35].As in the case of other oligomeric neurotoxic PLA2s, the non-toxic subunits play the role of chaperonemolecules, preventingthe binding of the toxic subunit to non-specific binding sites incell membranes, thus allowing the complex to reach high-affinity binding sites on the membranes of nerve terminalsand skeletalmuscle cells, where the α subunit exerts its toxicityby hydrolyzing phospholipids in these membranes [27,30]. Thisrepresents a highly successful strategy to enhance the toxicityof neurotoxic and myotoxic PLA2s.

In addition to this powerful presynaptic neurotoxic mech-anism, the venoms of the two populations of O. scutellatuscontain small quantities of post-synaptic long- and short-

chain ‘three-finger’ α-neurotoxins which block the nicotiniccholinergic receptor at the motor end-plate, inducing a flaccidparalysis [16,36]. Interestingly, pharmacological studies onnerve-muscle preparations have demonstrated differencesbetween some of the post-synaptic neurotoxins of these twopopulations of O. scutellatus [16]. The adaptive implicationsof this difference are unclear, but it is likely they are minor,owing to the high content and potency of taipoxin, which rep-resents a powerful immobilizing mechanism, regardless ofthe effects of post-synaptic neurotoxins. The presence of var-ious neurotoxic mechanisms in the same venom is likely toconstitute a redundant strategy for prey immobilization, al-though, in the case of taipan venom, presynaptic neurotoxic-ity largely dominates as the mechanism of paralysis.

Besides taipoxin, the venoms of Australian and PNG popu-lations of O. scutellatus contain a number of additional PLA2

components in different proportions, with Australian O. scu-tellatus having a higher overall PLA2 composition (79.7% vs.68.3%) than the PNG population, despite having much less tai-poxin. Some of these other PLA2s included sequences withidentity to the monomeric enzymes OS1, OS2, OS3 and OS4previously isolated from this venom [14,37]. OS1 and OS2have been used in the characterization of themost thoroughlystudied PLA2 receptors, named N- and M-type receptors

Table 1 – Assignment of the reverse-phase chromatographic fractions from the venom of Oxyuranus scutellatus, from Papua New Guinea, to protein families by N-terminalsequencing, MALDI-TOF–TOF mass spectrometry and nESI-MS/MS of selected peptide ions from in-gel digested protein bands.Peak ID % Mass

kDaPeptide ion MS/MS-derived or N-terminal (Nt)

sequenceType Related

proteinProtein description

m/z z

OsPG1 2.8 6787.6 a N-ter MTCYNQQSSEAKTTT 3FTXb GI|123910874 SNTX-1 (O. scutellatus)OsPG2 0.9 ~7 1338.4 1 CPPGBEVCYTK 3FTX GI|254772668 LNTX-1 (O. scutellatus)OsPG3 0.5 7910.3 a N-ter RRCFTTPSVRSERCP 3FTX GI|254772668 LNTX-1 (O. scutellatus)OsPG4a 2.9 ~7 1055.3 1 FYYNPHSK BPTI/Kunitz GI:239977265 Venom protease inhibitor 1 (O. scutellatus)OsPG4b 1465.5 1 FCHXPPBPGPCR BPTI/Kunitz GI|263546 Taicatoxin serine protease inhibitor (O. scutellatus)OsPG5 6 7222.6 a N-ter KDRPKFCHLPPKPGP BPTI/Kunitz GI|263546 Taicatoxin serine protease inhibitor (O. scutellatus)OsPG6 0.7 6677.8 a N-ter KDRPKFCHLPPKPGP BPTI/Kunitz GI|263546 Taicatoxin serine protease inhibitor (O. scutellatus)OsPG7a 2.6 ~7 1055.3 1 FYYNPHSK BPTI/Kunitz GI:239977265 Venom protease inhibitor 1 (O. scutellatus)OsPG7b 1465.5 1 FCHXPPBPGPCR BPTI/Kunitz GI|263546 Taicatoxin serine protease inhibitor (O. scutellatus)OsPG8a 0.2 ~7 1055.3 1 FYYNPHSK BPTI/Kunitz GI:239977265 Venom protease inhibitor 1 (O. scutellatus)OsPG8b 1465.5 1 FCHXPPBPGPCR BPTI/Kunitz GI|263546 Taicatoxin serine protease inhibitor (O. scutellatus)OsPG9a 0.6 ~7 1055.3 1 FYYNPHSK BPTI/Kunitz GI:239977265 Venom protease inhibitor 1 (O. scutellatus)OsPG9b 1465.5 1 FCHXPPBPGPCR BPTI/Kunitz GI|263546 Taicatoxin serine protease inhibitor (O. scutellatus)OsPG13 0.2 ~10 996 2 YYXAAEEVXWDYSP (291.1) Oscutarin C GI|82073021 Oscutarin C non-catalytic subunit (O. scutellatus)OsPG14a 2.7 ~15 1201.6 1 GGSGTPVDBXDR PLA2 GI|66475092 OS7 precursor (O. scutellatus)OsPG14b 1.9 ~4 1352.7 1 XGNGCFGFPXDR Natriuretic GI|32363246 Oxs SNP c (O. scutellatus)OsPG15a 22.7 ~13 1930.7 1 HGCYPSXTTYTWECR PLA2 GI|129435 Taipoxin beta chain (O. scutellatus)OsPG15b 1.1 ~16 1876.9 1 TBCEVFVCACDFAAAK PLA2 GI|129435 Taipoxin beta chain (O. scutellatus)OsPG16a 4.9 ~16 1490.5 1 CAPoxYWTXYSWK PLA2 GI|71066728 PLA-6 precursor (O. scutellatus)OsPG16b 4.9 ~14 1238.5 1 NXXBFGFMXR PLA2 GI|129413 Taipoxin alpha chain (O. scutellatus)OsPG16c 2 ~10 1238.5 1 NXXBFGFMXR PLA2 GI|129413 Taipoxin alpha chain (O. scutellatus)OsPG17a 11.8 ~14 1238.5 1 NXXBFGFMXR PLA2 GI|129413 Taipoxin alpha chain (O. scutellatus)OsPG17b.1 5.1 ~17 965.6 2 (201.2)XNFANXXECANHGTR PLA2 GI|913013 OS1 PLA2 (O. scutellatus)OsPG17b.2 857.5 2 (228.2)VBFGFFXECAXR PLA2 GI|71066724 PLA-4 precursor (O. scutellatus)OsPG17c 3.1 ~15 1839.8 1 THDECYAEAGBXSACK PLA2 GI|129446 Gamma-taipoxin (O. scutellatus)OsPG18a 0.6 ~30 1839.8 1 THDECYAEAGBXSACK PLA2 GI|129446 Gamma-taipoxin (O. scutellatus)OsPG18b 2.9 ~15 1406.4 1 THDECYAEAGK PLA2 GI|129446 Gamma-taipoxin (O. scutellatus)OsPG19a 0.9 ~30 1238.7 1 NXXBFGFMXR PLA2 GI|129413 Taipoxin alpha chain (O. scutellatus)OsPG19b.1 0.3 ~15 1486.5 1 TNCPASCFCBNK CRISP ACE73574 CRISP (Viridovipera stejnegeri)OsPG19b.2 2859.8 1 TAVTCFAGAPYNDDXYNXGMXECHK PLA2 GI|129446 Gamma-taipoxin (O. scutellatus)OsPG19c 3.8 ~15 1851.2 1 RPAXDFMNYGCYCGK PLA2 GI|129435 Taipoxin beta chain (O. scutellatus)OsPG20a 0.3 ~100 964.5 1 CBTEWXK CRISP GI|123916495 Pseudechetoxin-like protein (O. scutellatus)OsPG20b.1 1.4 ~30 1839.8 1 THDECYAEAGBXSACK PLA2 GI|129446 Gamma-taipoxin (O. scutellatus)OsPG20b.2 536.2 2 CPATCFCR CRISP GI|123916495 Pseudechetoxin-like protein (O. scutellatus)OsPG20c 0.2 ~26 1238.6 1 EXVDKHNDXR CRISP GI|123916495 Pseudechetoxin-like protein(O. scutellatus)OsPG20d 0.4 ~23 2859.8 1 TAVTCFAGAPYNDDXYNXGMXECHK PLA2 GI|129446 Gamma-taipoxin (O. scutellatus)OsPG20e 0.2 ~20 1419.7 1 AEVDDVXEVRFK Oscutarin C GI|82073021 Oscutarin C non-catalytic subunit (O. scutellatus)OsPG21a 1.3 ~30 1182.6 1 QDFGXVSGFGR Oscutarin C GI|82073037 Oscutarin C FVa-like subunit (O. scutellatus)OsPG21b.1 0.8 ~30 1182.6 1 QDFGXVSGFGR Oscutarin C GI|82073037 Oscutarin C FVa-like subunit (O. scutellatus)OsPG21b.2 ~15 1839.8 1 THDECYAEAGBXSACK PLA2 GI|129446 Gamma-taipoxin (O. scutellatus)OsPG21c 0.6 ~20 1986.7 1 DCPSDWSSYDXYCYK C-type Lectin GI|332278153 CTL (Trimeresurus albolabris)OsPG23a 1.9 ~50 2225.8 1 XHSWVECESGECCEBCR SVMP GI|145982762 Scutellatease-1 (O. scutellatus)OsPG23b.1 7 ~30 1396.7 1 XGXFVDHGMoxYTK SVMP GI|118643 SVMP (Trimeresurus gramineus)OsPG23b.2 2265.1 1 TSHDHABXXTATXFNGNVXGR SVMP GI|469190 SVMP (Gloydius halys)

a In these cases the molecular mass was determined in Da by ESI-MS.b Abbreviations: 3FTx: three-finger toxin; PLA2: phospholipase A2; SVMP: metalloproteinase; SP: serine proteinase; CRISP: cysteine-rich secretory proteins; CTL: c-type lectin. Cysteine residues deter-mined in MS/MS analyses are carbamidomethylated, unless otherwise stated. X: Leu/Ile; B: Lys/Gln; Mox: oxidized M; Pox: oxidized P.

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[14,38]. OS2 is also a highly toxic component which inducesvarious effects, such as myotoxicity and neurotoxicity[14,39], whereas OS1 displays lower toxicity [37]. Other se-quences with broad identity to previously characterized O.scutellatus PLA2 were also found, and it is clear that there aremany PLA2s in both of these venoms that are yet to be fully se-quenced and described. The presence of additional toxicPLA2s is compatible with the description of abundant PLA2

transcripts described for the venom gland of O. scutellatus[18] and very likely reflects an undergoing evolutionary pro-cess of gene duplication and accelerated evolution describedfor this family of venom toxins [19]. The existence of abun-dant PLA2 isoforms fits into the redundant toxin paradigm ofvenom composition described above. In addition, monomericPLA2s of low toxicity but high enzymatic activity may play apredominantly digestive role.

The adaptive role of myotoxicity in taipoxin [31] and othermyotoxic PLA2s, such as OS2 [39], deserves consideration. Al-though it may be a by-product of the acquisition of strongneurotoxicity (i.e. a consequence of the ability of these toxinsto interact with binding sites in the plasma membranes ofnerve terminals and other excitable cells, such as myocytes),myotoxicity is likely to have a relevant adaptive role associat-ed with the digestion of musclemass. Since O. scutellatus feedslargely onmammals [40], characterized by a largemass to sur-face ratio, i.e. type III prey [41], this poses a problem for the di-gestion of prey characterized by a large mass of muscle tissue.In the absence of significant proteinase content in thesevenoms, the acquisition of myotoxicity is likely to contributeto muscle protein digestion. The action of myotoxic PLA2s onthe plasma membrane of muscle fibers facilitates the releaseof cytosolic proteins and the access to myofilament proteins[42]. In addition, myonecrosis, with the associated incrementsof cytosolic Ca2+ levels following the disruption of the perme-ability barrier of the plasma membrane, results in activationof intracellular proteinases, such as m- and μ-calpains, withthe consequent hydrolysis of cytoskeletal proteins present inmyofibrils [43,44]. This, in turn, may facilitate the further di-gestion of the predominant myofilament proteins, i.e. actinand myosin, by proteinases present in the gastric and pancre-atic secretions. It is likely that one of the adaptive roles ofmyotoxic PLA2s in snake venoms may be associated with di-gestion of muscle mass [42,45].

O. scutellatus venom contains potent Group C prothrombinactivators [46]. These activators require a phospholipid sub-strate and free Ca2+ ions as cofactors. Our proteomic datarevealed four peaks in PNG taipan venom with constituentsthat had identity with the Factor Va-Xa-like prothrombin acti-vator Oscutarin C, comprising 2.5% of the venom, compared tojust two peaks making up 1.3% of Australian taipan venom[15]. The percentage of the prothrombin activator in taipanvenom has previously been estimated to be at most 5% [15].However, despite its relatively low concentration, the coagu-lant activity of taipan venoms is very high (see below) and dis-turbances of hemostasis are a common feature of taipanenvenoming. The higher concentration of Oscutarin C inPNG O. scutellatus venom helps to explain why antivenomraised against Australian O. scutellatus venom is less effectiveagainst this toxin than antivenom raised using venom fromPNG O. scutellatus (Table 3). It may also explain why clinical

bleeding after taipan bites is more commonly reported in thevictims of snakebite in Papua New Guinea [10] than it isamong taipan bite patients in Australia. Oscutarin C is a mul-timeric protein comprised of a factor Xa-like proteinase, a fac-tor Va-like protein, and additional subunits [15]. From theclinical standpoint, these procoagulants activate prothrom-bin, thus leading to defibrinogenation, blood factor depletionand blood incoagulability, which induce bleeding in a numberof patients [10]. From the biological standpoint, however, therole of these potent procoagulants in the natural prey of tai-pans may be associated with intravascular thrombosis, an ef-fect that provokes rapid death (see below).

The venoms of the two populations of taipan also con-tained additional venom components, whose biological rolesremain largely unknown (Tables 1 and 2; Fig. 3). After PLA2s,the two most abundant components in both venoms areKunitz-type inhibitors and SVMPs. A number of peaks inboth venoms had sequence identity to BPTI/Kunitz-type in-hibitors previously identified in O. scutellatus venom, includ-ing a subunit of taicatoxin, which is an oligomer of threedifferent proteins: an α-neurotoxin-like peptide, a neurotoxicPLA2 and a Kunitz-type inhibitor of 7 kDa, linked by non-covalent bonds at an approximate stoichiometry of 1:1:4 [17].Originally, taicatoxin was shown to block the high thresholdCa2+ channel current of excitable membranes from the heart[17], inducing a series of alterations in cardiac myocyte cellfunctions [47], although it was later shown also to interactwith apamin-sensitive, small conductance, Ca2+-activated po-tassium channels [48]. The potential toxic role of taicatoxin intaipan envenomings is not known, although it has been sug-gested that it may be associated with the electrocardiographicabnormalities described in human patients envenomed bytaipans in PNG [11]. SVMPs comprise between 5 and 9% ofthe proteomes of these two venoms. Elapid venoms typicallycontain a relatively low abundance of SVMPs [49–52], whosetoxicity has not been well characterized. SVMPs isolatedfrom elapid venoms belong to the class P-III, i.e. the matureproteins are comprised by metalloproteinase, disintegrin-likeand cysteine-rich domains [53,54]. Some SVMPs isolatedfrom elapid venoms of Naja species induce hydrolysis of thecomplement system, von Willebrand factor and pro-TNF-α[55–58]. However, these activities have only been demonstrat-ed in vitro and the actual biological role of these effects re-mains unknown. A hemorrhagic SVMP of high molecularmass was characterized from the venom of Ophiophagus han-nah which induces hemorrhage in some species only [59]. AP-III SVMP, Mikarin, in the venom of Micropechis ikaheka, theNew Guinea small-eyed snake, was found to be a Group I(Ca2+-independent) prothrombin activator [60]. In the case ofO. scutellatus venoms, the role of SVMPs has not been elucidat-ed, and they do not seem to be associated with the predomi-nant toxic activities of the venom, i.e. neurotoxicity,myotoxicity and coagulopathy and, consequently, may playa predominantly digestive role.

Several bands in taipan venoms corresponded to CRISPshaving identity to a pseudechetoxin-like protein (GI|123916495)from O. scutellatus venom and the related protein pseudecin,previously characterized from the venom of the Australianred-bellied blacksnake Pseudechis porphyriacus [18,61]. TheseCRISPs bind with high affinity to the cyclic nucleotide-gated

Table 2 – Assignment of the reverse-phase chromatographic fractions from the venom of Oxyuranus scutellatus, from Australia, to protein families by N-terminal sequencing,MALDI-TOF–TOF mass spectrometry and nESI-MS/MS of selected peptide ions from in-gel digested protein bands.

2135JO

UR

NA

LO

FPR

OT

EO

MIC

S75

(2012)

2128–2140

Fig. 3 – Overall composition of the venoms of O. scutellatus from Papua New Guinea and Australia according to protein families.Phospholipases A2 (PLA2), three-finger toxins (3FTx), C-type lectin/lectin-like protein (CTL), proteinase inhibitor (BPTI/Kunitz),serine proteinases (SP), venom natriuretic peptide (VNP), snake venom metalloproteinase (SVMP), cysteine-rich secretoryproteins (CRISP), factor V-like protein (FV-like).

2136 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 2 1 2 8 – 2 1 4 0

ion channels, occluding the entrance of the pore and inhibitingthe flow of current [62]. These proteins have proved to be valu-able tools in the study of these channels, although their physio-logical/toxicological role in the venom remains unknown. Inaddition, our analysis revealed the presence of other minorcomponents, such as serine proteinases, C-type lectin/lectin-

Table 3 – Lethality and coagulant activity of venoms of O.neutralization by monospecific antivenoms.

Lethality

Ne

Venom LD50 (μg per mouse) b Antivenom against O. s

O. scutellatus (PNG) 0.08±0.01 5.6±1.7O. scutellatus (AUS) 0.50±0.10 11.0±2.8

Coagulant activity

Venom MCC (μg/mL) c Antivenom against O. scut

O. scutellatus (PNG) 0.33±0.13 2.37±0.08O. scutellatus (AUS) 1.30±0.60 10.0±2.5

a Neutralization is expressed as the amount of mg of venom neutralizeddoses used corresponded to 4 LD50s (for lethality) and 2 MCC (for coagulab The Median Lethal Dose (LD50) was determined in CD-1 mice (16–18 g) bthality in 50% of the injected mice within 24 h of injection.c The Minimum Coagulant Concentration (MCC) was estimated in citratemethods). It corresponds to the venom concentration that induces clottin

like components and venom natriuretic peptides (Tables 1 and2, Fig. 3). A toxicologically-relevant serine proteinase in thisvenom corresponds to the factor Xa-like subunit of the pro-thrombin activator [15]. The possible biological role in thisvenom of the C-type lectin/lectin-like proteins and venom na-triuretic peptides, previously characterized in the venoms of

scutellatus from Papua New Guinea and Australia, and

utralization (mg venom/mL antivenom) a

cutellatus (PNG) Antivenom against O. scutellatus (Australia)

5.9±0.1316.4±2.1

Neutralization (mg venom/mL antivenom)a

ellatus (PNG) Antivenom against O. scutellatus (Australia)

0.45±0.174.1±1.4

by 1 mL of antivenom. For neutralization experiments, the challengent activity).y the i.v. route. It corresponds to the dose of venom that induces le-

d human plasma with the addition of calcium (see Materials andg of plasma in 60 s.

2137J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 2 1 2 8 – 2 1 4 0

these populations [63], remains unknown, although it is likelyto be minor in the overall pattern of envenoming owing to thelow concentration of these components in the venom.

3.2. Correlation between the proteomes and the toxicologi-cal and immunological profiles of the venoms

The populations of taipan of Australia and PNG were split intosubspecies on the basis of morphological differences [8]. How-ever, more recent molecular analysis has not supported suchdifferentiation and suggested the occurrence of gene flow be-tween these populations as recently as the late Pleistocene [6].Venom phenotyping constitutes an interesting area of analysisof geographical differentiation between populations. Our prote-omic data showed that the proteomes of the venoms of thesetwo populations present the same set of protein components,with similar concentration of the main families of proteins.However, they show differences in their HPLC separation pro-files and in the concentration of taipoxin, since higher amountswere found in the venom of PNG taipans, in agreement with itshigher lethal activity (Table 3). The percentage of taipoxin in thevenome of AustralianO. scutellatus (20%) roughly corresponds tothe previously described yield of this toxin [12]. In contrast, thepercentage of taipoxin (56%) in PNG O. scutellatus deduced byproteomic analysis ismuch higher than the 16% yield of “canni-toxin” obtained fromPapuanO. scutellatus [13]. This discrepancymight suggest additional variation between the venoms of theeastern PNG and western New Guinea (Western Province ofPNG and IndonesianWest Papua) populations which are physi-cally disjoint from one another, an issue that deserves furtherinvestigation.

The functional implications of the variations in the venomproteomes were assessed by determining the main toxic andenzymatic activities of these venoms. In terms of lethality,the venom of PNG taipans is 6.25 times more potent thanthe venom of Australian taipans (Table 3), in agreement withthe higher concentration of taipoxin in the former. Thus, thedifferential expression of taipoxin in these venoms representsa mechanism to regulate the overall toxicity, owing to the po-tency of this toxin. On the other hand, coagulant activity invitro is also slightly higher in the venom from PNG specimens(Table 3), and this again may be due to the higher percentageof this toxin in PNG venom. In addition, PLA2 activity was 240and 660 μEq/mg/min fatty acid for the venoms of specimensfrom PNG and Australia, respectively. The higher activity ofAustralian venom correlates with the higher proportion of‘non-taipoxin’ PLA2s in this venom.

In agreement with the proteomic similarities observed be-tween these venoms, antivenoms raised in horses againstvenoms from these populations showed a noteworthy patternof cross-neutralization (Table 3). Both antivenoms were effec-tive in the neutralization of lethal and coagulant activities ofthe venoms from both populations of taipans, thus revealinga similar antigenic makeup of these venoms regarding neuro-toxic and coagulant components. Interestingly, both antive-noms had a higher neutralizing potency against lethality ofthe venom of Australian specimens than against the venomfrom PNG, probably reflecting the higher content of the potentneurotoxin taipoxin in the latter, thus making neutralizationmore difficult to achieve.

3.3. Assessing the toxicity of taipan venom from a biologicalperspective: which is the predominant mechanism of immobili-zation and death of mice?

The high toxicity ofO. scutellatus venoms, together with the char-acteristic biting pattern of ‘snap and release’ [40], represents ad-aptations for feeding on relatively large mammalian prey,which are capable of retaliation. The high concentration of tai-poxin in these venomssuggests that taipoxin-mediatedparalysismight be the main mechanism involved in rapid prey immobili-zation and death. Nevertheless, the possible role of the potentprocoagulant activity of this venom also has to be considered.To approach this issue, we analyzed the time and the circum-stances of death in mice injected with O. scutellatus venom andpurified taipoxin, at different doses, using mice as abiologically-relevant model, since taipans mostly feed on mam-mals, including mice [40]. Mice injected i.v. with a lethal, but rel-atively low dose of venom from PNG specimens, i.e. 5 LD50s, diedat 62±11min (n=5) after injection, with evident manifestationsof limb paralysis and respiratory difficulties, i.e. with neurotoxicmanifestations. However, when a higher dose of venom (50 μg,corresponding to 625 LD50s) was administered i.v., mice had animmediate collapse, and death occurred in 228±163 s (n=5). Re-markably, thesemicedidnot showparalyticmanifestations, sug-gesting that other mechanisms of death may be at work at thishigh dose. In order to comparatively assess the effect of a highdose of taipoxin, 50 μg of this neurotoxin (corresponding to 1250LD50s, [12]) were injected i.v. The time of death was 709±68 s(n=5), which was significantly more prolonged (p<0.05) whencompared with mice injected with the same dose of venom;mice injected with this high dose of taipoxin died with evidentsigns of paralysis and respiratory distress.

Since taipan venom has a potent procoagulant activity, thepossibility that death at high doses is due to intravascularthrombosis was considered. A histological analysis of lung tis-sue was performed immediately after death of mice injectedi.v.with 50 μg venom,which died rapidly uponvenom injection.Abundant thrombi were observed in pulmonary vessels (Fig. 4),suggesting a rapid thrombotic effect as themost plausible causeof death in these circumstances, as has been observed after thei.v. injection of a prothrombin activator from the venom ofBothrops asper [64]. Clearly, therefore, the rapid cause of deathin mice injected with a high dose of venom does not seem tobe due to the action of taipoxin, but very likely to the thromboticeffect of the prothrombin activator. Since this snake is able toinject a large dose of venom in their prey, estimated to bearound 20mg in mice [65], it is likely that high doses of venomreach the bloodstream in a natural bite in mammalian prey,thus raising the possibility that the prothrombin activatorplays a relevant role in rapid prey immobilization and death.

From a biological standpoint, it appears that prey immobili-zation and death induced by taipan venom may occur by twodifferent and highly effective mechanisms: intravascularthrombosis provoked by the prothrombin activator, OscutarinC, and paralysis leading to respiratory failure induced by thePLA2 trimer, taipoxin. It is somehow puzzling that a venomthat has a high concentration of one of the most potent neuro-toxic components yet described in any snake species may usean alternative mechanism (intravascular coagulation), basedon the action of procoagulants present in low relative amounts

Fig. 4 – Light micrographs of sections of lung tissue injectedwith either PBS (A) or O. scutellatus venom from PNG (B and C)by the intravenous route. Mice were injected with either100 μL of PBS or with 50μg venom, dissolved in 100μL PBS.Animals injected with venom had an immediate collapse anddied within the first min of injection. A sample of lung tissuewas collected immediately after death and processed forhistological evaluation. Control mice were sacrificed with anoverdose of anesthetic and lung tissue was collected andprocessed. Thrombi are observed in large and small bloodvessels in mice injected with venom (arrows in B and C),whereas control mice injected with PBS did not show anyintravascular thrombi, and their vessels were filled witherythrocytes (arrow, A). Magnification: 200× in A and B, and400× in C.

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in the venom, for immobilizing and killing prey in natural con-ditions. Thismay constitute an example of a highly effective re-dundancy mechanism for prey immobilization in a snake

venom. From a clinical perspective, on the other hand, themain cause of death in humans is neurotoxicity leading to re-spiratory paralysis [3], whereas the action of prothrombin acti-vators largely results in defibrinogenation, factor depletionand incoagulable blood, in some cases with active bleeding, in-stead of thrombosis [10]. At the same time, however, up to23%of patients bittenby PNG taipans inone study reported sud-den collapse and transient loss of consciousness in the firstmi-nutes after the bite [3]. In Australia similar events have beenreported in rapidly fatal cases of brown snake (genus Pseudo-naja) envenoming [66], another species with Group C prothrom-bin activators. It could be suggested, based on our rodentexperiments, that at veryhighvenomdoses the rapid formationof intravascular clots may have a role in these collapses, a hy-pothesis that deserves further consideration.

In conclusion, the venom proteomes of O. scutellatus fromAustralia and PNG are highly similar, although differencesin the amounts of taipoxin occur. The venom of PNG taipanshas a higher concentration of this neurotoxin, in agreementwith its higher lethal activity in mice. These venoms alsopresent immunological similarities, revealed by the abilityof monospecific antivenoms to neutralize venoms of thetwo populations. It is suggested that the cause of death inmice injected with O. scutellatus venom depends on thedose of venom injected; at low doses, neurotoxic paralysispredominates, whereas at high doses, such as those injectedin natural bites, intravascular thrombosis occurs, thusrepresenting a rapid and effective mechanism for killingrodents.

Acknowledgments

The authors thank Dr Andrés Hernández and B.Sc. Julissa Fon-seca (Instituto Clodomiro Picado) for their collaboration, aswell as Dr Bruno Lomonte for performing some of the massspectrometry analyses. This study was supported by CONARE,Vicerrectoría de Investigación (Universidad de Costa Rica) (pro-jects 741-A7-611 and 741-A9-506), CRUSA-CSIC (Project2009CR0021), Ministerio de Ciencia e Innovación, Madrid,Spain (grant BFU2010-17373), Generalitat Valenciana, Valencia,Spain (grant PROMETEO/2010/005), the PNG Office of HigherEducation, CTP Limited (Milne Bay Estates), and the AustralianVenom Research Unit (University of Melbourne), which isfunded by the Australian Government Department of Healthand Ageing, the Australia Pacific Science Foundation andSnowy Nominees. Analyses performed at the Proteomics Labo-ratory of Instituto ClodomiroPicadowere supportedbyCONAREand Vicerrectoría de Investigación, Universidad de Costa Rica.

R E F E R E N C E S

[1] White J. Clinical toxicology of snakebite in Australia and NewGuinea. In: Meier J, White J, editors. Handbook of clinicaltoxicology of animal venoms and poisons. Boca Raton: CRCPress; 1995. p. 595–617.

[2] Broad AJ, Sutherland SK, Coulter AR. The lethality in mice ofdangerous Australian and other snake venoms. Toxicon1979;17:661–4.

2139J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 2 1 2 8 – 2 1 4 0

[3] Lalloo DG, Trevett AJ, Korinhona A, Nwokolo N, Laurenson IF,Paul M, et al. Snake bites by the Papuan taipan (Oxyuranusscutellatus canni): paralysis, hemostatic andelectrocardiographic abnormalities, and effects ofantivenom. Am J Trop Med Hyg 1995;52:525–31.

[4] Williams D. Snakebite in Papua New Guinea. In: Williams DJ,Jensen SD, Nimorakiotakis B, Winkel KD, editors. Venomousbites and stings in Papua New Guinea: a treatment guide forhealth workers and doctors. Melbourne: University ofMelbourne; 2005. p. 5–32.

[5] O'Shea M. A guide to the snakes of Papua New Guinea. PortMoresby: Independent Publishing; 1996.

[6] Wüster W, Dumbrell AJ, Hay C, Pook CE, Williams DJ, Fry BG.Snakes across the Strait: Trans-Torresian phylogeographicrelationships in three genera of Australasian snakes(Serpentes: Elapidae: Acantophis, Oxyuranus, andPseudechis). Mol Phylogenet Evol 2005;34:1–14.

[7] Doughty P, Maryan B, Donnellan SC, Hutchinson MN. A newspecies of taipan (Elapidae: Oxyuranus) from central Australia.Zootaxa 2007;1422:45–58.

[8] Slater KR. On the New Guinea taipan. Mem Natl Mus Vic1956;20:201–5.

[9] Connolly S, Trevett AJ, Nwokolo NC, Lalloo DG, Naraqi S,Mantle D, et al. Neuromuscular effects of Papuan Taipansnake venom. Ann Neurol 1995;38:916–20.

[10] Lalloo DG, Trevett AJ, Owens D, Minei J, Naraqi S, Saweri A,et al. Coagulopathy following bites by the Papuan taipan(Oxyuranus scutellatus canni). Blood Coagul Fibrinolysis 1995;6:65–72.

[11] Lalloo DG, Trevett AJ, Nwokolo N, Laurenson IF, Naraqi S,Kevau I, et al. Electrocardiographic abnormalities in patientsbitten by taipans (Oxyuranus scutellatus canni) and other elapidsnakes in Papua New Guinea. Trans R Soc Trop Med Hyg1997;91:53–6.

[12] Fohlman J, Eaker D, Karlsson E, Thesleff S. Taipoxin, anextremely potent presynaptic neurotoxin from the venom ofthe Australian snake taipan (Oxyuranus s. scutellatus).Isolation, characterization, quaternary structure andpharmacological properties. Eur J Biochem 1976;68:457–69.

[13] Kuruppu S, Reeve S, Banerjee Y, Kini RM, Smith AI, HodgsonWC. Isolation and pharmacological characterization ofcannitoxin, a presynaptic neurotoxin from the venom of thePapuan Taipan (Oxyuranus scutellatus canni). J Pharmacol ExpTher 2005;315:1196–202.

[14] Lambeau G, Barhanin J, Schweitz H, Qar J, Lazdunski M.Identification and properties of very high affinity brainmembrane-binding sites for a neurotoxic phospholipasefrom the taipan venom. J Biol Chem 1989;264:11503–10.

[15] Speijer H, Govers-Riemslag JW, Zwaal RF, Rosing J.Prothrombin activation by an activator from the venom ofOxyuranus scutellatus (Taipan snake). J Biol Chem 1986;261:13258–67.

[16] Kornhauser R, Hart AJ, Reeve S, Smith AI, Fry BG, HodgsonWC. Variations in the pharmacological profile ofpost-synaptic neurotoxins isolated from the venoms of thePapuan (Oxyuranus scutellatus canni) and coastal (Oxyuranusscutellatus scutellatus) taipans. Neurotoxicology 2010;31:239–43.

[17] Possani LD, Martin BM, Yatani A, Mochca-Morales J, ZamudioFZ, Gurrola GB, et al. Isolation and physiologicalcharacterization of taicatoxin, a complex toxin with specificeffects on calcium channels. Toxicon 1992;30:1343–64.

[18] St Pierre L, Woods R, Earl S, Masci PP, Lavin MF. Identificationand analysis of venom gland-specific genes from the coastaltaipan (Oxyuranus scutellatus) and related species. Cell MolLife Sci 2005;62:2679–93.

[19] Ohno M, Ménez R, Ogawa T, Danse JM, Shimohigashi Y,Fromen C, et al. Molecular evolution of snake toxins: is thefunctional diversity of snake toxins associated with a

mechanism of accelerated evolution? In: Moldave K, editor.Progress in nucleic acid research and molecular biology. NewYork: Academic Press; 1998. p. 307–64.

[20] Ohno M, Chijiwa T, Oda-Ueda N, Ogawa T, Hattori S.Molecular evolution of myotoxic phospholipases A2 fromsnake venom. Toxicon 2003;42:841–54.

[21] Fry B, Scheib H, van der Weerd L, Young B, McNaughtan J,Ramjan SF, et al. Evolution of an arsenal: structural andfunctional diversification of the venom system in theadvanced snakes (Caenophidia). Mol Cell Proteomics 2008;7:215–46.

[22] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, MillerW, et al. Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs. Nucleic Acid Res 1997;25:3389–402.

[23] World Health Organization. Progress in the characterizationof venoms and standardization of antivenoms. WHO OffsetPublication, No. 58. Geneva: World Health Organization; 1981.

[24] Theakston RDG, Reid HA. Development of simple standardassay procedures for the characterization of snake venoms.Bull World Health Org 1983;61:949–56.

[25] Vargas M, Segura A, Herrera M, Villalta M, Estrada R, CerdasM, et al. Preclinical evaluation of caprylic acid-fractionatedIgG antivenom for the treatment of Taipan (Oxyuranusscutellatus) envenoming in Papua New Guinea. PLoS Negl TropDis 2011;5:e1144.

[26] Gutiérrez JM, Lomonte B, Chaves F, Moreno E, Cerdas L.Pharmacological activities of a toxic phospholipase A isolatedfrom the venom of the snake Bothrops asper. Comp BiochemPhysiol C 1986;84:159–64.

[27] Montecucco C, Rossetto O. On the quaternary structure oftaipoxin and textilotoxin: the advantage of being multiple.Toxicon 2008;51:1560–2.

[28] Cull-Candy SG, Fohlman J, Gustavsson D, Lüllmann-Rauch R,Thesleff S. The effects of taipoxin and notexin on thefunction and fine structure of the murine neuromuscularjunction. Neuroscience 1976;1:175–80.

[29] Harris JB, Grubb BD, Maltin CA, Dixon R. The neurotoxicity ofthe venom phospholipases A2, notexin and taipoxin. ExpNeurol 2000;161:517–26.

[30] Paoli M, Rigoni M, Koster G, Montecucco C, Postle AD. Massspectrometry analysis of the phospholipaseA2 activity of snakepre-synaptic neurotoxins in cultured neurons. J Neurochem2009;111:737–44.

[31] Harris JB, Maltin CA. Myotoxic activity of the crude venomand the principal neurotoxin, taipoxin, of the Australiantaipan, Oxyuranus scutellatus. Br J Pharmacol 1982:61–75.

[32] Fohlman J, Lind P, Eaker D. Taipoxin, an extremely potentpresynaptic snake venom neurotoxin. Elucidation of theprimary structure of the acidic carbohydrate-containingtaipoxin subunit, a phospholipase homolog. FEBS Lett1977;84:367–71.

[33] Lind P. Amino-acid sequence of the β1 isosubunit of taipoxin,an extremely potent presynaptic neurotoxin from theAustralian snake taipan (Oxyuranus s. scutellatus). Eur JBiochem 1982;128:71–5.

[34] Lind P, Eaker D. Amino-acid sequence of the α-subunit oftaipoxin, an extremely potent presynaptic neurotoxin fromthe Australian snake taipan (Oxyuranus s. scutellatus). Eur JBiochem 1982;124:441–7.

[35] Bon C. Multicomponent neurotoxic phospholipases A2. In:Kini RM, editor. Venom phospholipase A2 enzymes.Structure, function and mechanism. Chichester: Wiley; 1997.p. 269–85.

[36] Zamudio F, Wolf KM, Martin BM, Possani LD, Chiapinelli VA.Two novel α-neurotoxins isolated from the taipan snake,Oxyuranus scutellatus, exhibit reduced affinity for nicotinicacetylcholine receptors in brain and skeletal muscle.Biochemistry 1996;35:7910–6.

2140 J O U R N A L O F P R O T E O M I C S 7 5 ( 2 0 1 2 ) 2 1 2 8 – 2 1 4 0

[37] Lambeau G, Cupillard L, Lazdunski M. Membrane receptorsfor venom phospholipases A2. In: Kini RM, editor. Venomphospholipase A2 enzymes. Structure, function andmechanism. Chichester: Wiley; 1997. p. 389–412.

[38] Lambeau G, Lazdunski M. Receptors for a growing family ofsecreted phospholipases A2. Trends Pharmacol Sci 1999;20:162–70.

[39] Rouault M, Rash LD, Escoubas P, Boilard E, Bollinger J,Lomonte B, et al. Neurotoxicity and other pharmacologicalactivities of the snake venom phospholipase A2 OS2: theN-terminal region is more important than enzymatic activity.Biochemistry 2006;45:5800–16.

[40] Shine R, Covacevich J. Ecology of highly venomous snakes:the Australian genus Oxyuranus (Elapidae). J Herpetol 1983;17:60–9.

[41] Cundall D, Green HW. Feeding in snakes. In: Schwenk K,editor. Feeding. Form, function and evolution in tetrapodvertebratesSan Diego: Academic Press; 2000. p. 293–333.

[42] Nicholson J, Mirtschin P, Madaras F, Venning M, Kokkinn M.Digestive properties of the venom of the Australian CoastalTaipan, Oxyuranus scutellatus (Peters, 1867). Toxicon 2006;48:422–8.

[43] Sorimachi H, Ishiura S, Suzuki K. Structure and physiologicalfunction of calpains. Biochem J 1997;328:721–32.

[44] Gutiérrez JM, Ownby CL. Skeletal muscle degenerationinduced by venom phospholipases A2: insights into themechanisms of local and systemic myotoxicity. Toxicon2003;42:915–31.

[45] Lomonte B, Angulo Y, Sasa M, Gutiérrez JM. Thephospholipase A2 homologues of snake venoms: biologicalactivities and their possible adaptive roles. Protein Pept Lett2009;16:860–76.

[46] Kini RM. The intriguing world of prothrombin activators fromsnake venom. Toxicon 2005;45:1133–45.

[47] Fantini E, Athias P, Tirosh R, Pinson A. Effect of taicatoxin(TCX) on the electrophysiological, mechanical andbiochemical characteristics of spontaneously beatingventricular cardiomyocytes. Mol Cell Biochem 1996;160–161:61–6.

[48] Doorty KB, Bevan S, Wadsworth JD, Strong PN. A novel smallconductance Ca2+-activated K+ channel blocker fromOxyuranus scutellatus taipan venom. Re-evaluation oftaicatoxin as a selective Ca2+ channel probe. J Biol Chem1997;272:19925–30.

[49] Nawarak J, Sinchaukul S, Wu MY, Phutrakul S, Chen ST.Proteomics of snake venoms from Elapidae and Viperidaefamilies by multidimensional chromatographic methods.Electrophoresis 2003;24:2838–54.

[50] Li S, Wang X, Zhang Y, Ren N, Wang K, Zhao X, et al.Proteomic characterization of two snake venoms: Naja najaatra and Agkistrodon halys. Biochem J 2004;384:119–27.

[51] Kulkeaw K, Chaicumpa W, Sakolvaree Y, Tongtawe P,Tapchaisri P. Proteome and immunome of the venom of theThai cobra, Naja kaouthia. Toxicon 2007;49:1026–41.

[52] Fernández J, Alape-Girón A, Angulo Y, Sanz L, Gutiérrez JM,Calvete JJ, et al. Venomic and antivenomic analyses of theCentral American coral snake,Micrurus nigrocinctus (Elapidae).J Proteome Res 2011;10:1816–27.

[53] Fox JW, Serrano SMT. Structural considerations of the snakevenom metalloproteinases, key members of the M12reprolysin family of metalloproteinases. Toxicon 2005;45:969–85.

[54] Gutiérrez JM, Rucavado A, Escalante T. Snake venommetalloproteinases. Biological roles and participation in thepathophysiology of envenomation. In: Mackessy SP, editor.Handbook of venoms and toxins of reptiles. Boca Raton: CRCPress; 2010. p. 115–38.

[55] Ward CM, Vinogradov DV, Andrews RK, Berndt MC.Characterization of mocarhagin, a cobra venommetalloproteinase from Naja mocambique mocambique, andrelated proteins from other Elapidae venoms. Toxicon1996;34:1203–6.

[56] Hamako J, Matsui T, Nishida S, Nomura S, Fujimura Y, Ito M,et al. Purification and characterization of kaouthiagin, a vonWillebrand factor-binding and -cleaving metalloproteinasefrom Naja kaouthia cobra venom. Thromb Haemost 1998;80:499–505.

[57] Sun QY, Bao J. Purification, cloning and characterization of ametalloproteinase from Naja atra venom. Toxicon 2010;56:1459–69.

[58] Guan HH, Goh KS, Davamani F, Wu PL, Huang YW,Jeyakanthan J, et al. Structures of two elapid snake venommetalloproteinases with distinct activities highlight thedisulfide patterns in the D domain of ADAMalysin familyproteins. J Struct Biol 2010;169:294–303.

[59] Weissenberg S, Ovadia M, Kochva E. Species specificsensitivity towards the hemorrhagin of Ophiophagus hannah(Elapidae). Toxicon 1987;25:475–81.

[60] Gao R, Kini RM, Gopalakrishnakone P. A novel prothrombinactivator from the venom of Micropechis ikaheka: isolation andcharacterization. Arch Biochem Biophys 2002;408:87–92.

[61] Yamazaki Y, Brown RL, Morita T. Purification and cloning oftoxins from elapid venoms that target cyclic nucleotide-gatedion channels. Biochemistry 2002;41:11331–7.

[62] Brown RL, Lynch LL, Haley TL, Arsanjani R. Pseudechetoxinbinds to the pore turret of cyclic nucleotide-gated ionchannels. J Gen Physiol 2003;122:749–60.

[63] Fry BG, Wickramaratana JC, Lemme S, Beuve A, Garbers D,Hodgson WC, et al. Novel natriuretic peptides from thevenom of the inland taipan (Oxyuranus microlepidotus):isolation, chemical and biological characterization. BiochemBiophys Res Commun 2005;327:1011–5.

[64] Loría GD, Rucavado A, Kamiguti AS, Theakston RDG, Fox JW,Alape A, et al. Characterization of ‘basparin A’, aprothrombin-activating metalloproteinase, from the venomof the snake Bothrops asper that inhibits platelet aggregationand induces defibrination and thrombosis. Arch BiochemBiophys 2003;418:13–24.

[65] Morrison JJ, Peam JH, Coulter AR. The mass of venom injectedby two Elapidae: the taipan (Oxyuranus scutellatus) and theAustralian tiger snake (Notechis scutatus). Toxicon 1982;20:739–45.

[66] White J. Why do people still die from brown snake bites?Emerg Med 2000;12:204–6.