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Combined venom gland cDNA sequencing and

venomics of the New Guinea small-eyed snake,Micropechis ikaheka

Owen Paivaa,1, Davinia Plab,1, Christine E. Wrightc,d, Markus Beutlerc,d, Libia Sanzb,José María Gutiérreze, David J. Williamsa,d,⁎, Juan J. Calveteb,⁎⁎aSchool of Medicine & Health Sciences, University of Papua New Guinea, Boroko, NCD, Papua New GuineabInstituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, SpaincCardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, AustraliadAustralian Venom Research Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, AustraliaeInstituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica

A R T I C L E I N F O

⁎ Correspondence to: D.J. Williams, Charles CGuinea, P.O. Box 168 Konedobu, NCD 125, Pa⁎⁎ Correspondence to: J.J. Calvete, Instituto de Bfax: +34 96369 0800.

E-mail addresses: david.williams@unime1 These authors have contributed equally t

http://dx.doi.org/10.1016/j.jprot.2014.07.0191874-3919/© 2014 Elsevier B.V. All rights rese

A B S T R A C T

Article history:Received 1 June 2014Accepted 14 July 2014Available online 8 August 2014

The venomarsenal of theNewGuinea small-eyed snake,Micropechis ikaheka, was investigated bya joint cDNA sequencing and venomics approach. Twenty-seven full-length DNA sequencesencoding novel venom proteins were recovered in this study. Using this cDNA dataset weachieved locus-specific resolution for 19 out of the approximately 50 reverse-phase- and SDS-PAGE-separated venom proteins. The venom proteome ofM. ikaheka is dominated by at least 29D49-phospholipase A2 (PLA2) and 14 short and long neurotoxins of the three-finger toxin (3FTx)family. These protein classes represent, respectively, 80% and 9.2% of the total venom proteins.Two PIII-metalloproteinase (SVMP) molecules (7.6%), three CRISP isoforms (1.8%), and a singleKunitz-type inhibitor, vespryn, 5′-nucleotidase, serine proteinase and LAO molecules, none ofwhich represents more than 0.7% of the total venom proteome, complete the protein arsenal ofM. ikaheka. In concordancewith clinical observations, this venomcomposition points to a centralrole for post-synaptically-acting neurotoxic toxins in the envenomation strategy developed bythis species. PLA2 molecules represent the main myotoxic components of M. ikahekavenom. In addition, the estimated LD50 for mice of the reverse-phase-isolated 3FTx(0.22 mg/kg) and PLA2 (1.62 mg/kg) enriched fractions, strongly suggests that these twotoxin classes contribute synergistically to venom lethality, with the 3FTxs playing adominant role. The high structural and functional conservation exhibited by M. ikahekaand Australian elapid venomsmay underlay the positive clinical outcomes of envenomingresulting from bites by M. ikaheka that have been documented through the use of bioCSLpolyvalent antivenom.

Keywords:Micropechis ikahekaSnake venomicsVenom gland cDNAPharmacological effects ofsnake venomThree-finger toxinPhospholipase A2

ampbell Toxinology Centre, School of Medicine & Health Sciences, University of Papua Newpua New Guinea.iomedicina de Valencia, C.S.I.C., Jaume Roig 11, 46010 Valencia, Spain. Tel.: +34 96 339 1778;

lb.edu.au (D.J. Williams), jcalvete@ibv.csic.es (J.J. Calvete).o this work and both should be considered first author.

rved.

Fig. 1 – The New Guinea small‐eyed snakNew Guinea and Irian Jaya (West Papua).photo: David Williams).

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Biological significanceThe poorly understood venom proteome of the New Guinea small-eyed snake, Micropechisikaheka, a large and powerfully built elapid endemic to Papua New Guinea and IndonesianWest Papua province, was investigated through a combined venomics and venom glandtranscriptomics approach. Although M. ikaheka accounts for only a small proportion ofsnakebites on the mainland, 40% of snakebites on Karkar Island are attributed to bites bythis snake. Major effects of envenomings include life-threatening post-synaptic neuromus-cular blockade resulting in respiratory paralysis, myotoxicity, severe bleeding, hypotensionand cardiovascular abnormalities. We have investigated the contribution of 3FTxs and PLA

2molecules in venom lethality, myotoxicity, and cardiovascular function. Our work providesimportant correlations between venom composition and its pharmacological activity. Inconjunction with the antivenomics work reported in the companion paper, our study maycontribute to improve treatment outcomes for snakebite victims ofM. ikaheka.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Molecular evidence indicates a rapid late-Miocene radiation ofAustralasian venomous snakes [1] that began 12–15 millionyears before present (MYBP). Over this time the Australo-Melanesian Hydrophiinae radiation has generated over 160species recognized in ~50 genera of terrestrial and marineelapids [2]. However, research into Australasian elapid venomshas mainly focused on the genera of greatest clinical signifi-cance, including Acanthophis, Hoplocephalus, Notechis, Oxyuranus,Pseudechis, Pseudonaja and Tropidechis.

The New Guinea small-eyed snake or Ikaheka snake,Micropechis ikaheka [3] (Fig. 1), is a basal endemic species thathas variously been considered a sister taxon to the AustralianPseudechis, Pseudonaja, Oxyuranus and Demansia and a sister

e. Distribution of the NPicture: M. ikaheka from

taxon to the Laticauda [4]. The latter study estimated the ap-pearance of Micropechis at 11.5 MYBP and considered it basalto all other hydrophiines. This species is large (average length,1.2–1.7 m; maximum length, 2.1–2.3 m), heavyset and extreme-ly excitable when disturbed. It is restricted entirely to PapuaNew Guinea and Indonesian Papua province on the islands ofNew Guinea where it is widely distributed throughout themainland and several of the offshore islands along the Madangand Sepik coasts, including Karkar Island and Manam Island(Madangprovince),Mushu, Kairiru,Walis, Tarawai, Tumleo andother small islets (Sanduan& East Sepik provinces), and theAruIslands to the west [5] (Fig. 1). Wild-caught snakes sometimesregurgitate partly digested snakes, and in captivity the speciesshows a preference for snakes and lizards, but can occasionallybe conditioned to accept rodents. It has been said to prey on a

ew Guinea small-eyed snake (Micropechis ikaheka) in PapuaKaviak Plantation, Karkar Island, Madang province (Map &

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wide variety of small ground dwelling animals, particularlylizards, frogs, small rodents and other snakes, including its ownspecies [6,7]. Specimens have been collected in a variety ofhabitats, at elevations from sea level to over 1500 m, rangingfrom the hot, humid lowland floodplains with grasslands,rainforests, swamps and marshes through wet monsoonalforests into cool, moist highland montane forests. Favouredmanmade microhabitats include copra, cocoa and oil palmplantations, as well as village gardens.

This fossorial species is shy and inoffensive unless dis-turbed, andwhile seldom seen by day, it is often encountered atnight when it emerges from shelter to forage for food, and canbe very defensive if touched, stepped on, or able to sensemove-ment nearby. It is a medically important species that has beenimplicated in human fatalities in both PNG and IndonesianPapua [8]. One of its favoured habitats is old coconut husk pilesin and around copra plantations. M. ikaheka shelter in thesepiles of decaying husks during the day [9] and bites typicallyoccur when plantation workers disturb snakes entering orleaving these refugia, as people arrive for work, or are walkinghome in the mornings or late afternoons. The New Guineasmall-eyed snake accounts for only a small proportion ofsnakebites on the mainland, whereas approximately 40% ofsnakebites on Karkar Island could be attributed to bites by M.ikaheka [8]. The subcutaneous lethal doseofM. ikahekavenomper25 g mouse has been reported to be 3.4 μg [10]. This venom hasstrong neurotoxic and myotoxic activities, as well as anticoag-ulant, platelet-aggregation-inhibiting, and insulin-secretion-stimulating effects [11–13]. In vivo studies have focused on thecardiovascular and haematological effects of this venom. It hasbeen reported to cause pulmonary hypertension and depressionof cardiac output accompanied by increased free-haemoglobinplasma levels (more than 50-fold) [11]. Envenoming byM. ikahekais known to provoke life-threatening post-synaptic neuromus-cular blockade resulting in respiratory paralysis, frequently ac-companied by myotoxicity resulting in rhabdomyolysis andmyoglobinuria, and sometimes coagulopathy and spontaneoussevere and life-threatening bleeding, hypotension and cardio-vascular abnormalities [8,11].

The venom composition ofM. ikaheka is poorly understood,and has been studied by only a handful of researchers. Fourgroups of IB PLA2s (MiPLA-1, MiPLA-2 [PDB code 1PWO], MiPLA-3[1OZY], and MiPLA-4 [1P7O]), which exhibited different levels ofmyotoxic and anticoagulant activities, have been isolated fromM. ikaheka venom [14–17], and the effects of venom on skeletalmuscle and venom-induced contraction of smoothmuscle havebeen shown to be dependent on PLA2 activity [18]. Some toxinmolecules potentially involved in the clinical effects ofM. ikahekavenom have been isolated and characterized structurally andfunctionally. Thus, blood clotting alterations have been attribut-ed to the action of mikarin, a single-chain Ca2+-independentmetalloproteinase, and group I prothrombin activator of 47 kDaisolated from the venomofM. ikaheka [19] (UniProtKB/Swiss-Protaccession code P0DJ43) and to the anticoagulant activity of somePLA2 [15,16]. Several short- and long-chain post-synaptic neuro-toxins of 6–8 kDahave also been isolated fromM. ikaheka venom.One of these, mikatoxin, has been found to produce neuromus-cular paralysis through irreversible nicotinic AChR antagonism[20,21]. However, the overall composition and relative abun-dance of the toxins of M. ikaheka venom, necessary figures to

establish structure–function correlations, have not been report-ed yet. To fill this gap, we have conducted a combined study ofvenom gland cDNA sequencing and venom proteomics of M.ikaheka, together with the analysis of the toxin profile of venomfractions. In conjunction with the functional venomics andantivenomics work reported in the companion paper [22], ourwork provides important correlations between the compositionof the venom and its pharmacological activity that mightimprove treatment outcomes for snakebite victims.

2. Materials and methods

2.1. Ethical approval

Experiments involving mice were approved by the Institu-tional Committee for the Care and Use of Laboratory Animals(CICUA) of the University of Costa Rica, and adhere to theInternational Guiding Principles for Biomedical ResearchInvolving Animals of the Council of International Organiza-tions of Medical Sciences (CIOMS). For studies undertakenwith rat tissues, the University of Melbourne Animal EthicsCommittee approved experiments in accordance with theAustralian Code for the Care and Use of Animals for ScientificPurposes (8th edition, 2013, National Health and MedicalResearch Council, Canberra). Venomous snakes maintainedat the University of Papua New Guinea in Port Moresby, PNG,are collected, kept and used in experiments in accordancewith approvals from the UPNG SMHS Ethics Committee, and thePNGGovernment's Department of Environment&Conservation.

2.2. Venom and venom glands

A pool of venom fromM. ikahekawas obtained from adult spec-imens maintained alive at the Charles Campbell ToxinologyCentre's Serpentarium, at the University of Papua New Guinea(UPNG) School of Medicine & Health Sciences. The venom wasfrozen at −80 °C, lyophilized later, placed in 5 mL, capped glassvials and stored at −80 °C prior to use. A M. ikaheka specimenwas euthanized and the two venom glands were carefullydissected out, immediately placed in RNAse later® (Bio-Rad) andstored at −80 °C.

2.3. RNA isolation and complementary DNA (cDNA) templatesynthesis

The snap frozen venom glands were later partially thawed,weighed, placed in a 5 mL test tube and homogenized with apolytron prior to RNA isolation. RNA isolation was carried outby the Tri Reagent method, using 1 mL of Tri reagent solution(Sigma-Aldrich, Castle Hill, Australia) per 50–100 mg venomgland tissue and following the manufacturer's protocols. Toconfirm successful isolation of RNA from M. ikaheka venomglands, electrophoresis of a small quantity of the ‘RNA’ pelletsample solution was carried out on a 1% Tris-Acetate EDTA(TAE) agarose gel run at 70 V for about 45 min or until thetracking dye reached halfway through the gel. Followingconfirmation of RNA isolation on TAE agarose gel, RNA wasprecipitated in isopropanol, washed with 70% ethanol andfinally resuspended in DEPC-treated water. First strand cDNA

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was then synthesized from 1 μg of total RNA with an oligo(dT)12–18 primer via reverse transcription with 200 U of Super-script II RNAse H Reverse Transcriptase (Invitrogen, Mt.Waverly, Australia). Ethanol was then precipitated from thesample and the template cDNA was resuspended in sterilewater and stored at −20 °C until used.

2.4. Amplification of toxin sequences from M. ikaheka venomgland cDNA

Specific forward (F) and reverse (R) primers designed fromtranscripts encoding sequences previously identified fromscreens of cDNA libraries prepared from venom glands ofvarious Australian elapid snakes [23–27] were: CRISP (F:5′-GGA GTT ACA CTG GGG CTC-3′; R: 5′-ACT GAA TGG GAGATC AGC-3′); 5′-nucleotidase (5′-NUC) (F: 5′-ATG ACA ACTTCT TGG AGT G-3′; R: 5′-TTA TTC TTC TTC TTC CTC-3′); short-chain neurotoxins (SNTXs) (the single-gene-specific primer5′-GGT CGT CGA TGG ATG AGA GCA AAA CTC-3′); long-chainneurotoxins (LNTXs) (F: 5′-ATG AAA ACT CTG CTG CTGACC-3′; R: 5′-GTC GAG ATG TCA AAG ACG CA-3′); serineprotease (SP) (F: 5′-ATG GCT CCT CAA CTA CTC CTC TG-3′; R:5′-TTA GAG CCG ACC AGT GCT TGA CTC-3′); PLA2 (F: 5′-TGCTTG CAG CTT CAC CAC TGA C-3′; R: 5′-TCC TCG CGC TGA AGCCTC TCA AA-3′); snake venom metalloproteinase (SVMP) (F:5′-ATG GCT CCT CAA CTA CTC CTC TG-3′; R: 5′-TTA GAG CCGACC AGT GCT TGA CTC-3′); and vespryn (VES) (F: 5′-ATG CTCCTG TTC ACA CTA TGC T-3′; R: 5′-TTA AAG ATT TGT GAGTGA AAC ACC T-3′). PCR amplifications were carried out fromthe M. ikaheka cDNA template using AmpliTaq™ Gold Taqpolymerase (Applied Biosystems, Foster City, USA), to identifyfull-length coding sequences. PCR reactionmixtures weremadeup to a final volumeof 25 μL containing approximately 200 ng ofcDNA template, 25 pmol of the forward and reverse primers,1 unit of AmpliTaq Gold (Applied Biosystems, Foster City, CA) in1× buffer, 2.25 mM MgCl2, and 200 μM deoxyribonucleosidetriphosphates (dNTPs). The PCR amplification (cycling) condi-tions for eachproteinof interestwere carriedout separately on a2720 Thermocycler (Applied Biosystems) according to methodsby St. Pierre and colleagues (2005a, 2005b, 2007a, and 2007b): forCRISP, the reaction was thermocycled at 95 °C for 8 minfollowed by 20 cycles of denaturation (95 °C for 20 s), annealing(51 °C for 20 s) and extension (72 °C for 30 s), and 15 cycles of95 °C for 20 s, 54 °C for 20 s, and 72 °C for 60 s, and a finalextension step at 72 °C for 3 min; for 5′-NUC, 95 °C for 60 sfollowed by 30 cycles of 95 °C for 20 s, 52 °C for 20 s, 72 °C for150 s, and a final extension at 72 °C for 3 min; for SNTX andLNTX, 95 °C for 8 min followed by 30 cycles of 95 °C for 20 s,59 °C for 20 s, 72 °C for 40 s, and a final extension at 72 °C for3 min; for SP, denaturation at 95 °C for 8.5 min followed by35 cycles of 95 °C for 30 s, 52 °C for 60 s, 72 °C for 3 min, and afinal extension step at 72 °C for 12 min; for PLA2, 95 °C for 8 minfollowed by 30 cycles of 95 °C for 20 s, 61 °C for 20 s, 72 °C for40 s, and a final extension step at 72 °C for 12 min; for SVMP,95 °C for 8.5 min followed by 35 cycles of 95 °C for 30 s, 52 °C for60 s, 72 °C for 3 min, a final extension step at 72 °C for 12 min;and for VES, 95 °C for 1 min followed by 30 cycles of 95 °C for20 s, 52 °C for 20 s, 72 °C for 2.5 min, and a final extension stepat 72 °C for 3 min. All PCR reactions were run with appropriateno template controls. Following PCR, the PCR products were run

out on 1% TAE agarose gels and single gel bands positive forcDNAs of interest were excised. Each excised gel band was thenpurified using the QIAEX II gel extraction kit (QIAGEN, Hilden,Germany) and the product cloned using the pGEM®-T vectorsystem (Promega, Madison, Wisconsin) in either Top10 or D5HαEscherichia coli cloning systems. Multiple clones were then se-quenced with an ABI Big Dye Terminal cycle sequence readyreaction kit (Perkin-Elmer, Norwalk, USA).

2.5. Reverse-phase and SDS-PAGE analyses

For venomics characterisation, 2–2.5 mg of crude, lyophilizedvenom proteins was dissolved in 200 μL of water containing0.1% trifluoroacetic acid (TFA), centrifuged to remove debris,and separated by reverse-phase HPLC using an ETTANTM LCHPLC system (Amersham Biosciences) and a LichrosphereRP100 C18 column (250 × 4 mm, 5 μm particle size) eluted at1 mL/min with a linear gradient of 0.1% TFA in water (solutionA) and acetonitrile (solution B) (5% B for 10 min, followed by 5–15% B over 20 min, 15–45% B over 120 min, and 45–70% B over20 min). Protein detection was carried out at 214 nm with areference wavelength of 400 nm. Fractions were collected man-ually, dried in a vacuum centrifuge (Savant), redissolved inwater, and submitted to SDS-PAGE analysis in 12% polyacryl-amide gels, under reducing conditions. Gels were stained withCoomassie blue R-250.

2.6. Characterization of the venom proteome

Protein bands of interest were excised from a CoomassieBrilliant Blue-stained SDS-PAGE gel and subjected to in-gelreduction (10 mM dithiothreitol) and alkylation (50 mMiodoacetamide), followed by overnight sequencing-gradetrypsin digestion (66 ng/μL in 25 mM ammonium bicarbonate,10% acetonitrile; 0.25 μg/sample) in an automated processor(using a Genomics Solution ProGest Protein Digestion Worksta-tion) following the manufacturer's instructions. Tryptic digestswere dried in a SpeedVac, redissolved in 15 μL of 0.1% formicacid in water, and submitted to LC-MS/MS. To this end, trypticpeptides were separated by nano-Acquity UltraPerformanceLC® (UPLC®) using BEH130 C18 (100 μm × 100 mm, 1.7 μmparticle size) column in-line with a Waters SYNAPT G2 HighDefinition Mass Spectrometry System. The flow rate was set to0.6 μL/minand the columnwas developedwith a linear gradientof 0.1% formic acid in water (solution A) and 0.1% formic acid inacetonitrile (solution B), isocratically 1% B for 1 min, followed by1–12% B for 1 min, 12–40% B for 15 min, and 40–85% B for 2 min.Doubly and triply charged ions were selected for collision-induced dissociation (CID) MS/MS. Fragmentation spectra wereinterpreted (a) manually (de novo sequencing), (b) using theon-line form of the MASCOT program at http://www.matrixscience.com against the NCBI non-redundant data-base, and (c) processed in Waters Corporation's ProteinLynxGlobal SERVER 2013 version 2.5.2. (with Expression version2.0) against the species-specific venom gland cDNA-derivedtoxin sequences. MS/MS mass tolerance was set to ±0.6 Da.Carbamidomethyl cysteine and oxidation of methionine wereselected as fixed and variable modifications, respectively.

The relative abundances (expressed as percentage of thetotal venom proteins) of the different protein families were

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calculated from the relationship of the sum of the areas of thereverse-phase chromatographic peaks containing proteinsfrom the same family to the total area of venom proteinpeaks in the reverse-phase chromatogram [28,29].

2.7. Fractionation of M. ikaheka venom into 3FTx- and PLA2-rich fractions

Two mg-aliquots of M. ikaheka whole venom were fractionat-ed by reverse-phase HPLC using a Teknokroma Europa C18(0.4 cm × 25 cm, 5 μm particle size, 300 Å pore size) columnand an Agilent LC 1100 High Pressure Gradient Systemequipped with DAD detector and micro-Auto-sampler. Theflow rate was set to 1 mL/min and the column was developedwith a linear gradient of 0.1% TFA in water (solution A) andacetonitrile (solution B), isocratically (5% B) for 5 min, follow-ed by 5–25% B for 10 min, 25–45% B for 60 min, and 45–70% for10 min. Protein detection was carried out at 214 nm with areference wavelength of 400 nm. Fractions eluting between 8and 12 min and between 13 and 25 min, enriched in 3FTxsand D49-PLA2 molecules, respectively, and were separatelypooled and used to correlate venom lethality and myotoxicactivity to particular toxin classes. The toxin profile of the 3FTx-and D49-PLA2-enriched fractions was checked by analyticalreverse-phase HPLC using a Discovery® BIO Wide Pore C18(15 cm × 2.1 mm, 3 μm particle size, 300 Å pore size) columnandanAgilent LC1100HighPressureGradient Systemequippedwith DAD detector. The flow ratewas set to 0.4 mL/min and thecolumn was developed with a linear gradient of 0.1% TFA inwater (solution A) and 0.1% TFA in acetonitrile (solution B):isocratically (5% B) for 1 min, followed by 5–25% B for 5 min, 25–45% B for 35 min, and 45–70% for 5 min. Protein detection wascarried out at 214 nmwith a reference wavelength of 400 nm.

2.8. Estimation of the median lethal dose (LD50) of venomfractions

Various doses of the 3FTx or the PLA2 fractions, dissolved in atotal volume of 100 μL of 0.14 MNaCl, 0.04 M phosphate, pH 7.2(PBS) were injected into groups of 4 CD-1 mice (16–18 g) by theintravenous route. Deaths occurring during 24 hwere recorded,and the Median Lethal Dose (LD50) was estimated by probitanalysis.

2.9. Myotoxic activity of the PLA2 fraction

Groups of four CD-1 mice (18–20 g) were injected, intramus-cularly in the right gastrocnemius, with two doses of thePLA2 fraction (30 or 60 μg), dissolved in 50 μL of PBS. Controlmice were injected with 50 μL of PBS alone. Three hours afterinjection, a blood sample was collected into heparinisedmicrocapillary tubes. The creatine kinase (CK) activity of plasmawas determined using a commercial kit (CK LIQUI-UV, StanbioLab., Texas, USA). Activity was expressed in units/L [30].

2.10. Inhibition of PLA2, lethal and myotoxic activities by p-bromophenacyl bromide (pBPB)

To assess the role of PLA2 activity in the lethal and myotoxiceffects of this venom, 1 mL of a solution of venom (3 mg/mL

in 0.1 M Tris, 0.7 M EDTA, pH 8.0, buffer) was incubated with150 μL of a solution of pBPB (3 mg/mL ethanol). Incubationwas carried out at room temperature for 24 h. Controlsincluded venom solution incubated with ethanol withoutpBPB, and pBPB solution incubated with buffer Tris. Abroga-tion of PLA2 activity was assessed by incubating variousamounts of control or inactivated venom, for 20 min at 37 °C,with chicken egg yolk suspension in a 0.1 M Tris, 10 mMCaCl2, 1% Triton X-100, pH 8.5, buffer. Then, fatty acids wereextracted and titrated with 0.018 M NaOH [31]. The sampleswere also tested for lethal and myotoxic activities, as describedabove. In the case of myotoxicity, in addition to plasma CKactivity quantification,micewere euthanized by CO2 inhalationand samples of injected gastrocnemius muscles were dissectedout and immersed in 10% formalin solution for fixation. Afterroutine processing for embedding in paraffin, 5–8 μm sectionswere obtained and stained with haematoxylin–eosin for micro-scopic observation [30].

2.11. Pharmacological effects in rat isolated cardiac andvascular tissues

Male Sprague–Dawley rats (339 ± 5 g) were exposed to 5%isoflurane (Baxter Healthcare Pty. Ltd.; NSW, Australia) and95% O2. When deeply anaesthetised, they were killed by rapidheart excision. Tissues were immediately placed in Krebs'physiological salt solution (PSS) and bubbled with carbogen(O2 95%; CO2 5%) at pH 7.4. PSS is composed as follows (inmmol/L): NaCl 119; KCl 4.69; MgSO4·7H2O 1.17; KH2PO4 1.18;glucose 11 (5.5 for mesenteric artery); NaHCO3 25; CaCl2·6H2O2.5; and EDTA 0.026.

2.12. Mesenteric arteries

Third order mesenteric arterial branch ring segments (2 mmlength; 327 ± 5 μm i.d.) were dissected from the mesentericfan. Ring segments were set up in Mulvany–Halpern isomet-ric myograph baths (620 M; DanishMyo Technology; Aarhus,Denmark). Force of contraction wasmeasured via a PowerLab 4/30 amplifier and computer (Chart v5.5.6 forMac; ADInstrumentsPty. Ltd.; Bella Vista, NSW, Australia). Vessel segments werenormalized to 100 mm Hg and adjusted to a passive tensionequivalent to a transmural pressure of 90 mm Hg [32]. Arteryviability was determined by potassium depolarisation withdepolarising isotonic high K+-containing physiological salinesolution (KPSS), in which Na+ in Krebs' solution was replaced byK+ ([K+]KPSS = 124 mM), and noradrenaline (10 μM). Tissueswerethen washed with physiological saline solution (PSS) andallowed to return to their resting contractile tone. Responseswere expressed as % of KPSS maximum contraction.

For assessment of venom-induced relaxation, arteries werepre-contracted with endothelin-1 (ET-1 tone, 1–3 nM) to 70–80%of KPSSmaximumcontraction. Venomwas added in cumulativehalf-log10 increments (starting at 0.9 μg/mL) allowing time foreach response to reach plateau between additions. Tissues werepre-treated for 30 min with vehicle (PSS), indomethacin (3 μM),atropine (3 μM), Nω-nitro-L-arginine methyl ester (L-NAME;100 μM) or glibenclamide (3 μM) prior to addition of venom.

For assessment of intramural sympathetic nerve-mediatedresponses, arteries were subjected to a selective α2-adrenoceptor

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inhibition protocol to enhance the contractile responses toelectrical field stimulation, i.e. to prevent auto-inhibition ofneurotransmitter release by presynaptic α2-adrenoceptors[33]. The vessels were electrically stimulated by square waveelectrical field stimulation via platinum electrodes connectedto a low-output-resistance stimulator (Grass SD9/S88, Quin-cy, MA, USA). A control stimulation episode, without venom,consisting of 3 trains (3 s burst, 0.25 ms pulsewidth; 30 V) at 3different frequencies – 6.25, 12.5 and 25 Hz – was performed.M. ikaheka venom was then added (90, 270, 360 and 450 μg/mL).Each venom concentration was incubated for 10 min followedby a stimulation episode. Finally, the venomwas washed out (3times within 10 min) and a final stimulation episode wasapplied. Contractions caused by electrical stimulation wereexpressed as % of KPSS maximum contraction.

2.13. Rat right and left atria

The beating heart was placed in an acrylic bottom Petri dishfilled with cold PSS (full glucose) and bubbled with carbogen.The left and right atria were then separated and pierced bytwo stainless steel hooks (made from 30 G needles) at theopposing ends of each atrium and connected to an acrylicorgan bath leg and an isometric force transducer (GrassInstruments FT03C) in 20 mL organ baths filled with 15 mLPSS and continuously bubbled with carbogen at 37 °C. Forcetransducers were connected to a 6-channel amplifier (OctalBridge Amp, ADInstruments). The input was recorded withChart (v5.5.6) for Mac.

Both atriawere passively stretched to a force of 1 g (9.81 mN).Electrical field stimulation at 1 Hz, for 0.3 ms at 150% thresholdvoltage (minimum voltage that causes contraction) via twoplatinum field electrodes connected to an electrical stimulator(S88, Grass Instruments) was applied to stimulate the leftatrium, whereas the right atrium beats spontaneously. After a60 min resting period with washouts every 30 min a stablebaseline was established. Isoprenaline (0.1 μM) was applied tocheck atrial viability. Venom was then added in cumulativehalf-log10 increments (starting at 0.009 μg/mL) allowing time forthe response to plateau between additions without or with30 min pre-treatment with the β-adrenoceptor antagonistpropranolol (1 μM) or calcitonin gene-related peptide (CGRP)antagonist CGRP8–37 (1 μM). To assess the role of phospholipaseA2 activity, venom was treated with p-bromophenacyl bromide(pBPB) using the method of Díaz-Oreiro and Gutiérrez [34].Separate aliquots of venom were kept overnight at room tem-perature to serve as controls. The right atrial rate was expressedin beats/min, whereas the left atrial force was shown as changefrom the resting force (g).

2.14. Statistical analysis

Data are expressed as the mean ± 1 standard error of themean (SEM) of n experiments. For mesenteric arteries, theinitial tone was compared between venom treatment groupsby one-way ANOVA. Maximum relaxation to venom in eachpre-treatment group was compared to the venom alonegroup using one-way ANOVA, with Dunnett's post hoc test(Prism 6; GraphPad Software, San Diego, CA, USA). Relaxationwithin each group was analysed with repeated measures

(RM) ANOVA; a Dunnett post hoc test was used to compareeach concentration with baseline. The pre- and post-treatmentright atrial rates were compared within groups by Student'spaired t-test; post-treatment baselines were compared betweengroups using one-way ANOVA,with Dunnett's post hoc test. Thesigmoidal concentration–response curves were fitted usingPrism 6 for each individual experiment (where applicable). TheEC50 (the concentration required to cause 50% of the maximumresponse) and maximum responses of each treatment groupwere compared to the control group (venom alone) by one-wayANOVA, with Dunnett's post hoc test. Average SEM withintissues was calculated by RMANOVA using the pooled estimateof error from the residual mean square as [error mean square/number of tissues]0.5 after subtracting the sums of squaresbetween tissues and between concentrations from the totalsum of squares for each treatment [35]. A p value < 0.05 wasconsidered significant.

3. Results and discussion

3.1. Novel full-length sequences of M. ikaheka venom glandcDNA clones

Twenty-seven full-length DNA sequences encoding novelvenom proteins were identified from the M. ikaheka venomgland cDNA library. The translated full-length amino acidsequences include three cysteine-rich secretory proteins(CRISPs; AHZ08820–22: 238 residues), one 5′-nucleotidase (5′NUC; AHZ08799: 559 amino acids), two serine proteinases(SPs; AHZ08800–01: 242 amino acids each), three shortneurotoxins of 62 residues (SNTXs; AHZ08816–18), threelong neurotoxins (LNTXs; AHZ08823–25: 83–84 residues),twelve D49-phospholipase A2 (PLA2) molecules (AHZ08804–12, AHZ08814–15: 124 amino acid residues; AHZ08813: 119residues), a PIII snake venom metalloprotease [PIII-SVMP;AHZ08819: 613 residues] and two vespryn isoforms (AHZ08802–03: 218 amino acids) differing in just an amino acid positionwithin the propeptide sequence.

The primers used to generate the M. ikaheka venom glandcDNA library were designed by other workers [23–27] and arebased on sequences in the venoms of Australian elapid snakes.These primers were chosen because they have been previouslyused successfully against non-target Australo-Papuan elapidtemplates. For example, the serine protease primers weredesigned by St Pierre et al. [25] from a Pseudonaja textilis FactorX-like protease (AY631238), where the forward primer matchespositions 131–153 of the target template and any unintendedtemplates, and the reverse primermatches positions 1522–1499of the target template and positions 1534–1511 on potentialunintended templates. The products produced by the primersspan a conserved calcium-binding EGF-like domain containingtheCa2+ binding sites, another EGF-like Factor Xa inhibitory site,as well as cleavage sites and part of the active sites in thetrypsin-like serine protease domain. The deduced protein se-quence of the M. ikaheka serine proteases AHZ08800 andAHZ08801 has greatest similarity (90%) to A8QL56, α- andβ-fibrinogenase (OhS1) from the Asian king cobra (Ophiophagushannah) venom and to Q5MCS0, a fibrinogenolytic serineprotease known as harobin from spine-bellied sea snake

215J O U R N A L O F P R O T E O M I C S 1 1 0 ( 2 0 1 4 ) 2 0 9 – 2 2 9

(Hydrophis hardwickii) venom. Similarly while PLA2 primerswere designed by St Pierre et al. 2005 [23] from an Oxyuranusscutellatus PLA2 sequence, the PLA2 sequenced in this currentstudy from an M. ikaheka cDNA library has greatest identitynot to PLA2 from O. scutellatus, but to 1OZY and 1PWO — bothknownPLA2s fromM. ikaheka. Anovel PLA2 (AHZ08813) generatedfrom the same primers had 80% identity and 86% similarity toP00609 (toxin II-5) fromNotechis scutatus venom, rather than anO.scutellatus PLA2. We feel these examples underscore the fact thatthe primer source species has not influenced the results reportedin the current study.

A 238 amino residue M. ikaheka CRISP [AHZ08822] com-prised of a 19 residue propeptide and a 219 mature protein isidentical to kaouthin-2 [P84808], a CRISP fromAsianmonocellatecobra (Naja kaouthia) venom [36]. The other two CRISP sequences[AHZ08820 and AHZ08821] differed from each other only in avaline for alanine substitution at position 2 in themature peptide.The closest (87–89% identity) reported homologs of these CRISPmolecules are ETE62137 (O. hannah), ACE73577 and ACE73578(Bungarus candidus), and kaouthin-2 [P84808] (N. kaouthia).

The full-length protein sequence of a 5′-nucleotidase fromM. ikaheka [AHZ08799] exhibits 99% identity to the venomgland 5′-nucleotidase from the black whip snake, Demansiavestigiata [ABK63558], and 95% identity to a cytosolic purine5′-nucleotidase [XP_003218558] from the American anole lizard(Anolis carolinensis), but only 26–29% identity with three partial5′-nucleotidase sequences [ETE69544, ETE58819, ETE67404] fromthe venom of the king cobra (O. hannah).

The two highly homologous serine proteinases [AHZ08800–01] show highest similarity (90%) to the α- and β-fibrinogenaseOhS1 from the venom of king cobra (O. hannah) [A8QL56], andthe fibrinogenolytic serine protease harobin from the spine-bellied sea snake (H. hardwickii) [Q5MCS0].

The cDNA-encoded full-length SNTXs and LNTXs share anidentical 21-residue propeptide sequence (1MKTLLLTLVVVTIVCLDLGYT21). The three short neurotoxins [AHZ08816–18] show70–71% amino acid sequence identity (78–79% similarity) tothree Pseudechis post-synaptic 3FTxs (A8HDJ4 from the red-bellied black snake, Pseudechis porphyriacus; BAN63794 andA8HDJ3 from the mulga snake, Pseudechis australis), and to thepost-synaptic short neurotoxin II [P10457] from the yellow-ipped sea krait, Laticauda colubrina. On the other hand, twoof theM. ikaheka long neurotoxins [AHZ08823 and AHZ08824] exhibithighest protein sequence identity (82–85%) to the post-synapticα-elapitoxin-Nss2a [P01384] from the venom of the Australiantiger snake N. scutatus, while the third long neurotoxin [AHZ08825] amino acid sequence exhibits 79% identity to the post-synaptic long neurotoxin ABX58167 from the Australian pygmycopperhead, Austrelaps labialis.

PLA2 toxins are an important component of the venom ofmanyAustralian elapid snakes. Twelve PLA2 clones [AHZ08804–15] were amplified and sequenced. The translated maturesequences of AHZ08804–12 and AHZ08814–15 comprise 124amino acid residues and display high identity to previouslyreported M. ikaheka venom PLA2s [14,15,17]. Thus, AHZ08808,AHZ08804, AHZ08809, AHZ08807, AHZ08806, and AHZ08810 are,respectively, 100%, 99%, 99%, 90%, 89%, and 88% identicalto MiPLA3 [1OZY], while AHZ08812, AHZ08815, AHZ08805,AHZ08811 and AHZ08814 share 94%, 93%, 90%, 88% and 88%amino acid sequence identity with MiPLA2 [1PWO]. MiPLA-J

[AHZ08813] represents a new acidic PLA2 composed of 119amino acid residues that exhibit greatest similarity (80%identity; 86% similarity) to a basic 119 amino acid residueneurotoxic PLA2 (toxin II-5) from N. scutatus venom [P00609][37,38].

The vespryn (Venom PRY-SPRY domain-containing proteins)precursor isoform sequences, AHZ08802 and AHZ08803, displayhighhomology (>90% identity) to a number of vesprynmoleculesfrom Australian elapid snakes, including Cryptophis nigrescens[ABW74872andABW74873],P. porphyriacus [ABW74875],Drysdaliacoronoides [AEH95532-34], P. australis [ABW74876], N. scutatus[ABW74866], D. vestigiata [ABK63561], and others.

We obtained the complete amino acid sequence of a PIII-SVMP [AHZ08819]whichhas 84% similarity tomicrolepidotease-1 from Oxyuranus microlepidotus [ABQ01137], textilease-1 from P.textilis [ABQ01140], and scutellatease-1 from O. scutellatus[ABQ01136]. Other PIII-SVMPs with high similarity includedcarinatease-1 (83%) from Tropidechis carinatus [ABQ01132],scutatease-1 (82%) from N. scutatus [ABQ01138], australease-1[81%] from P. australis [ABQ01134] and porphyriacase-1 [80%]from Pseudechis porphyriacus [ABQ01133]. The PIII-SVMP [AHZ08819] sequence also exhibits sequence similarity with peptidesequences of the prothrombin activator, Mikarin, isolated fromM. ikaheka venom [14] (Fig. 2).

3.2. The venom proteome of M. ikaheka

The venom of M. ikaheka was separated by RP-HPLC into 30fractions (Fig. 3A). Each chromatographic fraction was analysedby SDS-polyacrylamide gel electrophoresis (Fig. 3A, insert) andelectrospray ionization (ESI) mass spectrometry (Table 1).Protein bands were excised from SDS-polyacrylamide gel andsubmitted to venomics analysis [28,29]. Production spectrawereinterpreted manually or processed in MASCOT or ProteoLinxagainst the NCBI non-redundant database and the species-specific venom gland cDNA-derived toxin sequences. The data,listed inTable 1, identified ~50 protein species belonging toninedifferent snake venom protein families, whose relative abun-dances are displayed in Fig. 3B.

The venom proteome is dominated by a diversity of D49-PLA2 and 3FTx molecules. These protein classes represent,respectively, 80% and 9.2% of the total venom proteins. Wedetected 29 PLA2s and 14 3FTx bands, including uniquepeptide evidence for eight PLA2 [AHZ08804, 05, 07, 09–13] andfive 3FTx [AHZ08816–18, 24–25] venom gland amplified cDNAs(Table 1). Although peptide ions matching AHZ08806 andAHZ08808were sequenced, we were unable to unambiguouslyidentify these proteins because the MS/MS-derived peptidesequences did not bear the resolution capable of distinguishingamongst individual isoforms.

PIII-SVMP fragments with identity to a novel SVMP [AHZ08819] and the partial sequence of a PIII-SVMP prothrombinactivator ‘Mikarin’ [P0DJ43] accounted for 7.6% of the totalvenom proteome. Most tryptic peptides sequences gatheredfrom the PIII-SVMP molecules eluted in RP-HPLC peaks 24, 25,26, 27 and 30 are included in both Mikarin and AHZ08819(Fig. 2). However, two of them, m/z 736.7(2+) EAQCDSGECCEKand 577.7(2+) CGDGMVCSNR are only present in AHZ08819(Fig. 2), indicating that this novel PIII-SVMP, rather thanMikarin, may represent the major PIII-SVMP of the M. ikaheka

Fig. 2 – Alignment of the partial amino acid sequences of Mikarin [P0DJ43] and the full-length sequence of AHZ08819 (thiswork). Non-identical positions are highlighted in red, and internal tryptic peptide sequenced by MS/MS that match thecDNA-deduced amino acid sequence of AHZ08819 are shown in boldface and underlined. MS/MS-derived peptide sequencesthat correspond neither to P0DJ43 nor to AHZ08819 are displayed in lower case.

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venom sampled. In addition, minor tryptic peptide ions se-quenced from the 52 kDa band of fractions 25–30 exhibitedsimilarity to other elapid SVMPs (Table 1), suggesting that, inaddition to Mikarin and AHZ08819,M. ikaheka venom containsuncharacterized PIII-SVMP(s).

Three CRISP isoforms, and single Kunitz-type inhibitor,vespryns, 5′-nucleotidase, serine proteinases, and L-aminoacid oxidase (LAAO) molecules, complete the protein arsenalof M. ikaheka venom (Table 1, Fig. 3B). None of these toxinfamilies is more than 1.8% of the total venom proteome of M.ikaheka. Except for the LAAO molecule, the fragments ofwhich can be assembled into a partial sequence with 40%identity to LAAO from O. scutellatus [Q4JHE3] and N. scutatus[Q4JHE2] and 39% to LAAO from Bungarus fasciatus [A8QL52]and B. multicinctus [A8QL51], and a novel BPTI/Kunitz-typeinhibitor with a sequence PADTGPCK–FPAFYNPVQR that had76% identity to several Kunitz-type inhibitors from Austrelapssuperbus [B5KL38–39] Drysdalia coronoides [ACR78497–98 ACR78500, F8J2F3] P. porphyriacus [B5KL31] and P. rossignolii [BAJ76674] whichwere not represented in the pool of cDNA clonesamplified from M. ikaheka venom gland, all the other minorproteins correspond to a full-length putative toxin translatedfrom the venom gland cDNA library.

Australia and Papua New Guinea are home to a richbiodiversity of venomous terrestrial and marine snakes ofthe family Elapidae, which include species possessing some

of the most toxic venoms in the world [5]. Neurotoxicityleading to respiratory paralysis represents the predominantmechanism of prey immobilization and death caused bymost Australian and Papuan snakes [12,39]. Clinical obser-vations indicate that human envenoming by M. ikaheka ispredominantly characterized by neurotoxicity, i.e. muscleparalysis, systemic myotoxicity and haematological alter-ations [8]. The venom arsenal ofM. ikaheka strongly suggestsa central role for neurotoxins in the envenomation strategydeveloped by the New Guinea small-eyed snake. Catalyti-cally active (D49) PLA2 proteins are present in the venom ofmany terrestrial and marine Australo-Papuan elapid snakesand typically exhibit presynaptic neurotoxic activity, myotoxicactivity, or both, although some forms exhibit antiplateletactivity [40–43]. Members of the three-finger toxin (3FTx) familyare also abundant proteins in the venoms of many elapids(cobras, kraits and mambas), hydrophiids (sea snakes), andcolubrids [41,44–48]. Despite their pronounced structural simi-larity, the proteins of this family are amongst the mostfunctionally diverse groups of snake venom toxins, exhibitinga wide variety of pharmacological effects including neurotoxic,cytotoxic, cardiotoxic, anticoagulant, and antiplatelet activities[47,49]. The α-neurotoxins found in Australian elapids are post-synaptically acting short or long-chain neurotoxins. Short andlong-chain neurotoxins have similar effects and bind with highaffinity to nicotinic acetylcholine receptors of skeletal muscle

Fig. 3 – Characterisation of the venom proteome ofM. ikaheka. Panel A, elution profiles of 2 mg ofM. ikaheka venom proteins byRP-HPLC, and electrophoretic analysis of -mercaptoethanol-reduced proteins eluted in each peak (insert). Protein bands weresubmitted to in-gel tryptic digestion followed by LC-nESI-MS/MS tryptic ion sequencing, as described inMaterials andmethodssummarized in Table 1. Panel B, relative occurrence of proteins from different toxin families in the venom ofM. ikaheka. CRISP,cysteine-rich secretory protein; LAO, L-amino acid oxidase; PIII-SVMP, snake venom Zn2+-metalloproteinase (SVMP) of classesIII; DC, disintegrin-like/cysteine-rich fragment of PIII-SVMP; 5′NT, 5′-nucleotidase; SP, serine proteinase; vespryn, venom PRY–SPRY domain-containing protein; 3FTX, three-finger toxin; Kunitz, Kunitz-type serine proteinase inhibitor; D49-PLA2,D49-phospholipase A2. For details of the individual proteins consult Table 1.

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cells. In addition, L-type Ca2+ channel antagonists of the 3FTxfamily may act synergistically with muscarinic three-fingertoxins to promote hypotension [50].

Vespryns (Venom PRY–SPRY domain-containing proteins)are a recently identified family of proteins [51–53]. Ohaninisolated from king cobra venom is a well-characterized vesprynmolecule, which is known to induce hypolocomotion andhyperalgesia in mice [54]. A primary function of L-amino acidoxidases (LAAO) is probably to promote prey hypotension byactivating soluble guanylate cyclase in the presence of super-oxide dismutase [50]; CRISPs arewidelydistributedmolecules in

snake venoms (reviewed by Yamazaki and Morita [55]; butconsult also [56]). Neurotoxic cysteine-rich secreted proteinshave been identified and characterized from the Australianelapids P. australis and P. porphyriacus. Thesemolecules targetcyclic nucleotide-gated ion channels and inhibit smoothmuscle contraction [57,58].

First reported in 1938 [59], 5′ nucleotidases have since beenfound in a number of snake venoms [60]. These hydrolyticenzymes play a central role in liberating adenosine whichmay help in prey immobilization [50]. The identification offree purines as endogenous constituents of venoms has

Table 1 – Assignment of the reverse-phase fractions from the venom of the NewGuinean small-eyed snake,Micropechis ikaheka (Mi-), isolated as in Fig. 2a, to protein familiesby nESI-MS/MS collision-induced dissociation of peptide ions obtained from in-gel digested protein bands separated by SDS-PAGE (insert in Fig. 2a). X = Ile or Leu; Mox,methionine sulphoxide. Cysteine residues are carbamidomethylated. Nter, N-terminal sequence determined by automated Edman degradation. Apparent molecular masses(Mapp in kDa) were estimated from SDS-PAGE analyses of β-mercaptoethanol-reduced samples. Quasimolecular isotope-averaged masses (M + H+) were determined byESI-MS (QTrap2000), and those matching masses calculated for full-length cDNA sequences are underlined.

Spot ID % Mapp/M + H+ m/z z Peptide sequence Best match Protein family

Mi- cDNA sequence NCBI/UniProtKB

1 0.4 6682.6 457.6 3 TWNGIHSITER AHZ08816–18 3FTx (short α-neurotoxin)517.7 2 GCGCPTVKR AHZ08817–18 ~P80548 3FTx (short α-neurotoxin)769.3 2 LCCNQQSSQPK AHZ08817 ~P01434, ~ACY68694 3FTx (short α-neurotoxin)840.3 2 GISLMCCHADECNN AHZ08817–18 3FTx (short α-neurotoxin)

6609.2 749.9 2 KTWNGIHGSITER AHZ08816 [22–83] 3FTx (short α-neurotoxin)6668.6 749.8 2 KTWNGIHGSITER AHZ08818 [22–83] 3FTx (short α-neurotoxin)

840.4 2 GISLMCCHADECNN7826.6 1427.6 1 KTWCDAWCGSR AHZ08824–25 [22–92] 1NTN_A, P01382 3FTx (long α-neurotoxin)7552.3 749.8 2 KTWNGIHGSITER AHZ08825 [18–88] 3FTx (long α-neurotoxin)

840.4 2 GISLMCCHADECNN618.3 2 SCSGGETSCYK

2 0.7 7.5 kDa 503.2 2 (171.1)PADTGPCK B5KL39, ACR78500 BPTI/Kunitz-type inhibitor701.4 2 FPAFYYNPVQR ~B5KL31, BAJ76674 BPTI/Kunitz-type inhibitor

3 2.1 6910.8 Nter: LICYLSPKDTQIAPP ~P0C8R6 3FTx (long α-neurotoxin)941.3 2 FCXTESWCDGFCGSR ~AHZ08823 ~A8HDK7 3FTx (long α-neurotoxin)569.8 2 NGENXCFKR ~AHZ08816–18 ~P29179 3FTx (short α-neurotoxin)659.3 2 GCAATCPEAKPR AHZ08825 ADN67572 3FTx (long α-neurotoxin)570.3 2 NGENICKFKR ~P29179 3FTx (weak neurotoxin)642.8 2 SWCDAWCGSR ~AHZ08824–25 ~P82662 3FTx (long α-neurotoxin)566.7 2 (217,2)QTCPPADK ~AHZ08825 ~Q8UW28 3FTx (α-neurotoxin)688.3 2 VDXGCAATCPXAK AHZ08823–24 0512217A, P01384 3FTx (long α-neurotoxin)

4 0.05 15 kDa 601.8 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

549.3 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

671.3 2 AFTDYGCYCGK AHZ08804–12, 14–15 1PWO_A D49-PLA2

0.05 7 kDa 695.3 2 VDXGCAATCPTPK ~AHZ08823–24 ~Q9W7J5, A8HDK7–K9 3FTx (long α-neurotoxin)688.3 2 VDXGCAATCPXAK AHZ08823–24 0512217A, P01384 3FTx (long α-neurotoxin)497.3 2 LICYLSPK ~P01384 3FTx (long α-neurotoxin)941.3 2 FCXTESWCDGFCGSR ~AHZ08823 ~A8HDK7 3FTx (long α-neurotoxin)

5 0.1 16 kDa 601.8 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

548.7 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

734.7 2 YIEANNHIDPKR D49-PLA2

1010.5 2 GILSRPYVNTYAYDCTK AHZ08805 1PWO_A D49-PLA2

507.7 2 FVCNCDAK ~AHZ08804–15 AAB33760, AAZ22635, 37, 40–41, 43 D49-PLA2

0.1 7 kDa 695.3 2 VDXGCAATCPTPK ~AHZ08823–24 ~Q9W7J5, A8HDK7–K9 3FTx (long α-neurotoxin)757.9 2 (199.2)EXGCAATCPXPK AHZ08823–24 0512217A, P01384 3FTx (long α-neurotoxin)

6 0.4 7 kDa 649.8 2 TWCDAWCGSR AHZ08824–25 1NTN_A, P01382, Q53B57 3FTx (long α-neurotoxin)720.3 2 TCPPGENLCYTK AHZ08823–25 A1IVR7–R9, F8J2E2 3FTx (long α-neurotoxin)757.9 2 VVELGCAATCPIPK AHZ08823–24 0512217A, P01384 3FTx (long α-neurotoxin)516.8 2 ICLSTPDVK AHZ08823–25 ~BAN66265 3FTx (long α-neurotoxin)

2638.2 1 HYEDVICCSTDNCNPFPTRPR AHZ08824 ~P01384 3FTx (long α-neurotoxin)

218JO

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209–229

7 4.1 17 kDa 876.9 2 TP(146.2)TCPPGEVXCFTK AHZ08823–25 ~B2BRQ6 3FTx (long α-neurotoxin)517.3 2 APYIDANNR AHZ08812, AHZ08815 ~1PWO_A D49-PLA2

1.2 17 + 7 kDa 720.3 2 TCPPGENLCYTK AHZ08823–25 A1IVR7–R9, F8J2E2 3FTx (long α-neurotoxin)757.9 2 VVELGCAATCPIPK AHZ08823–24 0512217A, P01384 3FTx (long α-neurotoxin)497.3 2 LICYLSPK ~P0C8R6 3FTx (long α-neurotoxin)695.3 2 VDXGCAATCPTPK ~AHZ08823–24 ~Q9W7J5, A8HDK7–K9 3FTx (long α-neurotoxin)

8, 9 2.2 14–15.5 kDa 886.8 2 AVCDCDVEAAECFAR AHZ08813 ~ACY68711 D49-PLA2

1994.7 1 GTMYDYYCSSDGPYCR AHZ08813 ~ABK63573, ~ACY68711 D49-PLA2

802.8 2 TPYNNDFYNIDTK AHZ08813 ~Q8UUH8–H9 D49-PLA2

668.8 2 IHDDCYADAEK AHZ08813 ~P00609, ~F8J2D2 D49-PLA2

601.8 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14-15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

1.1 7 kDa 649.8 2 TWCDAWCGSR AHZ08824–25 1NTN_A, P01382, Q53B57 3FTx (long α-neurotoxin)720.3 2 TCPPGENLCYTK AHZ08823–25 A1IVR7-R9, F8J2E2 3FTx (long α-neurotoxin)

2638.2 1 HYEDVICCSTDNCNPFPTRPR AHZ08824 ~P01384 3FTx (long α-neurotoxin)569.8 2 NGENXCFKR ~P29179 3FTx (weak toxin)659.3 2 GCAATCPEAKPR ~AHZ08825 ADN67572, P85520 3FTx (long α-neurotoxin)497.3 2 LICYLSPK ~P01384 3FTx (long α-neurotoxin)

9 0.3 25 kDa 1100.9 2 YFVEVGEECDCGPPQVCR ~AHZ08819 ~A8QL49 PIII-SVMP725.8 2 DDCDLPEICTGR ~AHZ08819 P0DJ43, ~ABQ01137 PIII-SVMP506.2 2 CPTDSFQR AHZ08819 ABQ01132–35, 39 PIII-SVMP728.8 2 EDLDYG(Mox)VEPGTK ~AHZ08819 ~P0DJ43, ~ABQ01132 PIII-SVMP542.3 2 C(IΔ-16)ALWGPGVK AHZ08819 ~3K7L_A, ~Q9PVK7 PIII-SVMP

10a 4.8 13,898.5 1237.5 2 CTIPGREPLLAFSNYGCYCGK AHZ08806, 12, 15 1P7O_A D49-PLA2

517.3 2 APYIDANNR AHZ08812, AHZ08815 ~1PWO_A D49-PLA2

601.8 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

549.3 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

889.9 2 EPLLAFSNYGCYCGK AHZ08804–12, 14–15 1OZY_A, 1P7O_A D49-PLA2

14,014.1 517.3 2 APYIDANNR AHZ08812, AHZ08815 ~1PWO_A D49-PLA2

1011.3 2 GILSRPYVNTYAYDCTK AHZ08805 ~1PWO_A D49-PLA2

Nter: NLIQFSYLIQCANHG AHZ08813 ACY68711, ~2114420A D49-PLA2

10b 4.7 13,367.1 886.8 2 AVCDCDVEAAECFAR AHZ08813 ~ACY68711 D49-PLA2

802.9 2 TPYNNDFYNIDTK AHZ08813 ~Q8UUH8–H9 D49-PLA2

866.4 2 TPYNNDFYNIDTKK AHZ08813 ~Q8UUH8–H9 D49-PLA2

668.8 2 IHDDCYADAEK AHZ08813 ~P00609, ~F8J2D2 D49-PLA2

998.4 2 GTMYDYYCSSDGPYCR AHZ08813 ~ABK63573, ~ACY68711 D49-PLA2

11 5.2 [14,039.1, 13908.7] 747.8 2 PYVNTYAYDCTK AHZ08805 ~1PWO_A D49-PLA2

1011.3 2 GILSRPYVNTYAYDCTK AHZ08805 ~1PWO_A D49-PLA2

601.8 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

524.3 2 APYIEANNR AHZ08806–07, 10 1PWO_A D49-PLA2

1055.5 2 GILSPGYVNTYSYDCTDGK ~AHZ08812 ~1PWO_A D49-PLA2

12 7.2 14039.2 Nter: NLYQFRNMIICTIPDR AHZ08805, 11, 14 ~1PWO_A D49-PLA2

840.4 1 NLYQFR AHZ08805, 11, 14 ~1PWO_A D49-PLA2

532.8 2 GKITCNDQK AHZ08805 ~1OZY_A, ~1PWO_A D49-PLA2

1011.3 2 GILSRPYVNTYAYDCTK AHZ08805 ~1PWO_A D49-PLA2

656.8 2 YIEANNHIDPK AHZ08805 1PWO_A D49-PLA2

549.3 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

450.2 2 TAAMCFAK AHZ08804–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

601.8 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14–15 1OZY_A, 1PWO_A, 1P7O_A D49-PLA2

(continued on next page)

219JO

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NA

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FPR

OT

EO

MIC

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Table 1 (continued)

Spot ID % Mapp/M + H+ m/z z Peptide sequence Best match Protein family

Mi- cDNA sequence N BI/UniProtKB

13 2.7 15 kDa 594.8 2 GGSGTPVDDXDR AHZ08810 AET85561 AAZ29512 D49-PLA2

741.4 2 (244.1)APYXEANN(219)K AHZ08806–07, 10 1PWO_A D49-PLA2

1068.9 2 GILSPGYXNTYSYDCDNGK AHZ08811 1PWO_A, P7O_A D49-PLA2

628.8 2 APYIEANN(384.2) AHZ08806–07, 10 1PWO_A D49-PLA2

14 2.5 13773.4 524.3 2 APYIEANNR AHZ08806 1PWO_A D49-PLA2

601.8 2 GGSGTPVDELDELR AHZ08806, 11 1PWO_A, P7O_A D49-PLA2

807.4 3 GILSPGYXNTYSYDCDNGK AHZ08811 1PWO_A, P7O_A D49-PLA2

684.8 2 (CΔ-16)KLFICNCDR AHZ08804–07, 09–12, 14-15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

13848.8 580.8 2 GGSGTPVDDLDK AHZ08810 AET85561 AAZ29512 D49-PLA2

1209.1 2 CTIPGIEPLLAFSNYGCYCGK AHZ08804, 07–10 1OZY_A D49-PLA2

873.8 2 CCQTHDYCYDEAK AHZ08806–07, 10 ~1OZY_A ~ABK63564 D49-PLA2

15 4.9 14026.1 601.7 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14-15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

549.2 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

615.7 2 GGDGTPVDELDR ~AHZ08804–09, 11–12, 14–15 ~P59069, ADF50037 D49-PLA2

16 kDa Nter: NLYQFRNMIICTIP AHZ08805, 11, 14 ~1PWO_A D49-PLA2

2992.4 1 NMIICTIPDREPLLAFSNYGCYCGK AHZ08805, 11, 14 ~1PWO_A D49-PLA2

3006.6 1 LFICNCDRTAAMCFAKAPYIEANNR AHZ08806–07, 10 ~1PWO_A D49-PLA2

1209.1 2 CTIPGIEPLLAFSNYGCYCGK AHZ08804, 07–10 1OZY_A D49-PLA2

13877.9 689.1 3 GILSGPSFNTYAYDCTDGK AHZ08804, 08–09 1OZY_A D49-PLA2

17 3.1 14016.6 524.3 2 APYIEANNR AHZ08806–07, 10 1PWO_A D49-PLA2

549.3 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, PWO_A, 1P7O_A D49-PLA21210.6 2 CTIPGIEPLLAFSNYGCYCGK AHZ08804, 07–10 1OZY_A D49-PLA2

1749.6 1 CCQTHDYCYDEAK AHZ08806–07, 10 ~1OZY_A ~ABK63564 D49-PLA2

1778.8 1 EPLLAFSNYGCYCGK AHZ08804–12, 14–15 1OZY_A, P7O_A D49-PLA2

18 13.2 23 + 16 kDa Nter: NLIQFRKMIKCTIPG AHZ08804, 08–09 1OZY_A D49-PLA2

13773.7 524.3 2 APYIEANNR AHZ08806–07, 10 1PWO_A D49-PLA2

549.3 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

1210.6 2 CTIPGIEPLLAFSNYGCYCGK AHZ08804, 07–10 1OZY_A D49-PLA2

13848.8 875.3 2 CCQTHDYCYDEAK AHZ08806–07, 10 ~1OZY_A ~ABK63564 D49-PLA2

580.8 2 GGSGTPVDDLDK AHZ08810 AET85561 AAZ29512 D49-PLA2

19 0.4 16 kDa 524.3 2 APYIEANNR AHZ08806–07, 10 1PWO_A D49-PLA2

601.7 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14–15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

549.3 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

1210.6 2 CTIPGIEPLLAFSNYGCYCGK AHZ08804, 07–10 1OZY_A D49-PLA2

20 1.5 13775.7 524.3 2 APYIEANNR AHZ08806–07, 10 1PWO_A D49-PLA2

601.7 2 GGSGTPVDELDR AHZ08804–09, 11–12, 14–15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

549.3 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

1210.6 2 CTIPGIEPLLAFSNYGCYCGK AHZ08804, 07–10 1OZY_A D49-PLA2

21 11.4 13903.3 Nter: NLLQFRKMIKCTIPG AHZ08804, 08–09 1OZY_A D49-PLA2

13875.4 1011.3 2 GILSRPYVNTYAYDCTK AHZ08805 1PWO_A D49-PLA2

13932.9 549.3 2 LFICNCDR AHZ08804–07, 09–12, 14–15 1OZY_A, PWO_A, 1P7O_A D49-PLA2

2962.3 1 EPLLAFSNYGCYCGKGGSGTPVDELDR AHZ08804–09, 11–12, 14–15 1OZY_A, P7O_A D49-PLA2

22 1.8 23431.6 889.8 2 YLYVCQYCPAGNIR AHZ08820–21 [28–235] ~2DDB_A ACN93671 CRISP597.3 2 QIVDKHNALR AHZ08820–21 [28–235], AHZ08822 [26–237] P81993, A N93671 CRISP

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1

,1

1

,,

11

11

11,C

23523.7 654.3 2 CSFAHSPPHLR AHZ08820–21 [28–235] ACN93671, P84808 CRISP23 0.3 35 kDa 465.8 2 EWAVGLAGK AHZ08802–03 P82885 Vespryn

23 kDa 888.9 2 YLYVCQYCPAGNIR AHZ08820–21 [28–235] ~2DDB_A, ACN93671 CRISP447.2 2 DSIATPY AHZ08820–21 [28–235] ~2DDB_A CRISP697.3 2 VIQAWYDENKK AHZ08820–21 [28–235] ~ACN93671 CRISP

16 kDa 403.7 2 HFFEVK AHZ08802–03 P82885 Vespryn465.8 2 EWAVGLAGK AHZ08802–03 P82885 Vespryn924.9 2 TVKNVGVPQVVPDNPER AHZ08802–03 ~ABW74874 Vespryn

24 <0.1 52 kDa 725.8 2 DDCDLPEICTGR AHZ08819 P0DJ43 PIII-SVMP796.4 2 TNTPEQDRYLQVK AHZ08819 P0DJ43 PIII-SVMP

<0.1 37 kDa 619.8 2 EVVKF(Mox)NSXR ~ F8S0Z7 5′-nucleotidase476.3 2 VGXXGYTTK ~AHJ80886 5′-nucleotidase523.3 2 VPTYVPLEK ~BAN89427 5′-nucleotidase

24, 25 <0.1 29 kDa 549.3 3 GVIAGNSNVICPSDSR AHZ08800–01 ETE66745 Serine protease615.9 2 NILCAGVLEGGK AHZ08800–01 A8QL53 Serine protease

25, 26 3.0 52 kDa 796.4 2 TNTPEQDRYLQVK AHZ08819 P0DJ43 PIII-SVMP571.7 2 SACCNAATCK AHZ08819 P0DJ43 PIII-SVMP725.8 2 DDCDLPEICTGR AHZ08819 P0DJ43 PIII-SVMP728.3 2 VAPDICFTYNQK AHZ08819 ~ABQ01133 PIII-SVMP736.8 2 EAQCDSGECCEK AHZ08819 PIII-SVMP860.9 2 AAKDDCDLPEICTGR AHZ08819 PIII-SVMP505.7 2 CPTDSFQR AHZ08819 PIII-SVMP492.2 2 VQGCGFCR AHZ08819 PIII-SVMP577.7 2 CGDGMVCSNR AHZ08819 PIII-SVMP

27 3.2 48 kDa 796.4 2 TNTPEQDRYLQVK AHZ08819 P0DJ43 PIII-SVMP571.7 2 SACCNAATCK AHZ08819 P0DJ43 PIII-SVMP725.8 2 DDCDLPEICTGR AHZ08819 P0DJ43728.3 2 VAPDICFTYNQK AHZ08819 ~ABQ01133 PIII-SVMP492.2 2 VQGCGFCR AHZ08819 PIII-SVMP577.7 2 CGDGMVCSNR AHZ08819 PIII-SVMP955.9 2 NGHPCQNNKGYCYNGK AHZ08819 P82942 PIII-SVMP

1945.1 1 CPIMTNQCIALWGPGVK AHZ08819 3K7L_A PIII-SVMP28, 29 0.3 56 kDa 509.2 2 VTXXEASER Q4JHE3, Q4JHE2 LAAO

567.3 2 YPVKPSEEGK412.7 2 SASQXYR678.9 2 RXYFEPPXPPK608.8 2 FWEADGXHGGK659.7 2 TSADXVXNDXSXXHQLPK726.9 2 EXQAXCYPSMXK595.8 2 XYFAGEYTAR578.3 2 VHGWXDSTXK408.7 2 RVWEVK439.2 2 YDTYSTK960.9 2 TLSYVTADYVXVCSSSR989.1 3 TSADXVXNDXSXXHQXPK451.3 2 XXEEXKR565.8 2 NDDXFSYEK438.7 2 STTDLPSR

(continued on next page)

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Table 1 (continued)

Spot ID % Mapp/M + H+ m/z z Peptide sequence Best match Protein family

Mi- cDNA sequence N BI/UniProtKB

30 1.1 52 + 48 kDa 637.6 3 NGHPCQNNKGYCYNGK AHZ08819 PIII-SVMP505.7 2 CPTDSFQR ABQ0113 PIII-SVMP796.4 2 TNTPEQDRYLQVK AHZ08819 P0DJ43 PIII-SVMP736.8 2 EAQCDSGECCEK AHZ08819 PIII-SVMP725.8 2 DDCDLPEICTGR AHZ08819 PIII-SVMP505.7 2 CPTDSFQR AHZ08819 PIII-SVMP577.7 2 CGDGMVCSNR AHZ08819 PIII-SVMP

25–30 52 kDa 664.4 2 XNXEPDVSVTXK ~F8RKV9 PIII-SVMP671.3 2 XNXEPEVSVTXK ~F8RKV9 PIII-SVMP414.2 2 ETVXXPR ~ADD140 PIII-SVMP

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further supported the role of purinergic signalling in enven-omation. Purines are known to potentiate venom-inducedhypotension and paralysis via purinergic receptors. In addition,ATP released from skeletal muscle by the myotoxic action ofPLA2s acts as a “danger signal” stimulating purinergic receptorsto enhance and spread both the muscle damage caused by themyotoxins and pain [61]. High concentrations of adenosinegenerated by the combined action of venom myotoxins andnucleotidases may play an important role in envenomationvia its hypotensive, paralyzing and anticoagulant activities[62].

3.3. Lethal activity of M. ikaheka venom fractions

The finding that the venomics profile of M. ikaheka is stronglybased on PLA2s and 3FTxs prompted us to investigate the roleof these toxin classes in venom lethality in mice. To this end,the venom was fractionated by reverse-phase HPLC andfractions eluting between 8 and 12 min (3FTxs) and between13 and 25 min (PLA2s) were separately pooled (Fig. 4) and usedto assess lethality and myotoxic activity. The 3FTx fractionhad an estimated LD50 of 0.22 mg/kg (95% confidence limits:0.03–0.41 mg/kg) by the i.v. route. Mice injected with doses of0.59 mg/kg or higher died within one hour of injection. Animalsreceiving this fraction showed signs of respiratory and limbparalysis. On the other hand, the PLA2 fractions showed a lowertoxicity, with an estimated LD50 of 1.62 mg/kg (95% confidencelimits: 0.90–2.51 mg/kg). These mice also showed signs of res-piratory difficulties, although the time of death was more

Fig. 4 – RP-HPLC fractionation of M. ikaheka venom into 3FTx- anreverse-phase HPLC separation of 75 μg of whole venom protein(3FTxs) and between 13 and 25 (D49-PLA2s) were separately poolereverse-phase HPLC separation of 75 μg of pooled 3FTxs fromM. iC, reverse-phase HPLC separation of 50 μg of pooled PLA2s from

prolonged (more than 12 h) when compared to mice injectedwith the 3FTx fraction.

The LD50 of the M. ikaheka PLA2 venom fraction was muchhigher than those of typical presynaptically active PLA2s fromAustralian elapid venoms, such as notexin [63], textilotoxin[64], and taipoxin [65]. Hence, although mice injected with thepooled PLA2s ofM. ikaheka venom developed signs of neurotox-icity, i.e. limb paralysis and respiratory distress, these PLA2mol-ecules do not seem to play a predominant role in the overalllethal effect of the venom. Furthermore, inhibition of PLA2 bychemical modification with pBPB affected the venom's lethalityonly to apartial extent. LD50s of native andpBPB-inhibitedvenomwere 0.62 mg/kg (95% confidence limits: 0.43–0.88 mg/kg), and1.24 mg/kg (95% confidence limits: 0.87–1.60 mg/kg). Hence, ourresults support the hypothesis that post-synaptically-acting3FTxs play the predominant role in the neurotoxic effect inducedby M. ikaheka venom. However, despite their lower toxicityas compared to 3FTxs, PLA2s might contribute to the overalllethality of the venom. The estimated LD50 of M. ikahekavenom was 0.62 mg/kg. As judged from proteomic analysis,this amount of venom contains 0.057 mg of 3FTxs and0.5 mg of PLA2s. These figures represent 26% and 30% of theestimated LD50 of the 3FTx and the PLA2 fractions, respec-tively, thus suggesting that these two toxin classes maycontribute synergistically to venom lethality. This hypothesis issupported by the antivenomics outcome reported in theaccompanying paper [22], showing that Australian antivenomsexhibited strong immunorecognition of α-neurotoxins of the3FTx family and neutralized the lethal, i.e. neurotoxic, and

d PLA2-rich fractions. Panel A, reference analyticals of M. ikaheka. Fractions eluting between 8 and 12 mind and used to assess lethality andmyotoxic activity. Panel B,kaheka venom eluted between 8 and 12 min in panel A. PanelM. ikaheka venom eluted between 13 and 25 min in panel A.

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myotoxic activities of M. ikaheka venom, but exhibited poorneutralisation of PLA2 and anticoagulant activities.

3.4. Myotoxic activity is associated with the PLA2 venomfraction

Intramuscular injection of 30 and 60 μg of PLA2 fraction in thegastrocnemius induced prominent myotoxicity, reflected inincrements in plasma CK activity 3 h after injection (Fig. 5).

Fig. 5 – Inhibition of the myotoxic activity ofM. ikaheka venombychemicalmodificationwithpBPB.Groupsofmice (n = 4)wereinjected intramuscularly in the right gastrocnemius with either20 μg native venom (“venom”), 20 μg venom incubated withpBPB to abrogate PLA2 activity (“venom inh”), or PBS (“saline”).Injection of the PBS and pBPB controls did not induce anyevidence of toxicity inmice. Plasma creatine kinase (CK) activitywas quantified 3 h after injection. As shown in panel A,myotoxicity was completely abrogated upon inhibition of PLA2

activity. Panels B and C show, respectively, lightmicrographs ofhaematoxylin–eosin stained sections of skeletal muscleinjectedwith either 20 μg native venomnative venom or 20 μgvenom treated with pBPB. Notice the abundance of necroticmuscle fibres in (B) (arrows) whereas fibres with normalappearance are observed in (C). Bars represent 100 μm.

Mice injected with PBS, 30 μg PLA2 and 60 μg PLA2 showed CKactivities of 240 ± 30 U/L, 8029 ± 1255 U/L, and 13,572 ± 1552 U/L,respectively. On the other hand, judging from both the plasmaCK activity (Fig. 5A) and the histological morphology of injectedmuscle (compare panels B and C of Fig. 5), the incubation ofM. ikaheka venom with pBPB for 24 h completely abrogated PLA2

activity. Thus, mice receiving native venom showed widespreadareas of necrosis in the gastrocnemiusmuscle (Fig. 5B),whereas anormal histological pattern of muscle tissue was observed inmice injected with inactivated venom (Fig. 5C). Myotoxic PLA2

molecules are known components of Australian elapid venoms[66–68]. Myotoxic PLA2s have been isolated and characterizedfromM. ikaheka venom [14,15,18]. Our results indicate that theseenzymes are abundant components of M. ikaheka venom andthat myotoxicity is due to the direct action of PLA2 molecules onmuscle fibres. Myotoxic PLA2s are likely to play a key role inhuman envenoming, since rhabdomyolysis with myoglobinuriahave been described in cases ofM. ikaheka envenoming [8].

3.5. Pharmacological effects in rat isolated cardiac andvascular tissues

Previous in vivo findings showed that venom-induced vaso-relaxation correlates with systemic hypotension in pigletsintravenously injected with M. ikaheka venom [11]. In ratsmall mesenteric arteries (327 ± 5 μm internal diameter), M.ikaheka venom (9–270 μg/mL) did not cause contraction, evenwhen vessels were pre-contracted to 10–20% of the maximumcontraction to KPSS (n = 4; data not shown); this small pre-contraction is used to detect activity of weak constrictor agents.To examine vasorelaxation, arteries were pre-contracted withendothelin-1 (1–3 nM) to 70–80% of KPSSmaximum contraction.Venomcaused a significant concentration-dependent relaxation(from 9 to 270 μg/mL; P < 0.05, RM ANOVA; Fig. 6A); the maxi-mum relaxationwas −61 ± 4% KPSS (n = 5). Acetylcholine 10 μMwasaddedat the endof the experiments (on topof themaximumvenom concentration of 270 μg/mL) and relaxed vesselsfurther to −79 ± 4% KPSS – comparable to the acetylcholine-induced maximum relaxation in vessels treated only withvehicle (−80 ± 6% KPSS; n = 5; data not shown) – indicating afully functional vascular endothelium and lack of effect ofvenom on responses to this endothelium-dependent agonist.Pre-treatment with the muscarinic antagonist atropine or thecyclooxygenase inhibitor indomethacin did not cause a signifi-cant rightward shift of the overall relaxation curve to venom, butdid attenuate themaximum relaxation (to 270 μg/mL) to −34 ± 5(p = 0.011) and −33 ± 5% KPSS (p = 0.0005), respectively (n = 3each; Fig. 6A). In separate treatment groups, pre-treatment withthe nitric oxide synthase inhibitor L-NAME (n = 4) or, anecdotal-ly, the KATP channel inhibitor glibenclamide (n = 1) did not affectthe vasorelaxant response curve to venom (p > 0.05; data notshown). The initial pre-tone was the same in all treatmentgroups (p > 0.05, 1-way ANOVA).

To examine the effects of M. ikaheka venom on sympatheticnerve-induced vascular contractions, electrical field stimulationwas applied (6.25–25 Hz) to ratmesenteric arteries. In the absenceof venom, this caused frequency-dependent arterial contractionsto amaximumof 48 ± 5%KPSSat 25 Hz (n = 6; p < 0.0001; Fig. 6B).Venom at 90 and 270 μg/mL did not affect contractile responsesto electrical stimulation (data not shown), however at 360 μg/mL

Fig. 6 – Effects ofM. ikaheka venom on arterial vascular tone and nerve-induced contraction. A: Rat isolated mesenteric arterieswere pre-treated for 30 min with vehicle (PSS; venom control group), the cyclooxygenase inhibitor indomethacin (3 μM) ormuscarinic receptor antagonist atropine (3 μM) before endothelin-1 pre-contraction (ET-1 tone) and M. ikaheka venom (0.9–270 μg/mL) application. Initial pre-tone was compared between groups using one-way ANOVAwith Dunnett's post hoc test formultiple comparisons (P > 0.05). Relaxationwithin each groupwas analysedwith RMANOVA; a Dunnett post hoc test was usedto compare each venom concentration with its respective baseline (*P < 0.05). B: Electrical nerve stimulation (6.25–25 Hz)-induced contraction of rat isolated mesenteric arteries in the absence (no venom control group) or presence (30 minpre-treatment) of M. ikaheka venom (360 or 450 μg/mL). The highest venom concentration (450 μg/mL) attenuated thecontractile responses to stimulation at 25 Hz (*P = 0.0025; RM ANOVA and Dunnett post hoc test). Vascular tone is expressed as% KPSS maximum contraction. n, Number of arteries per group (each taken from separate rats). Error bars are ± average SEMfrom RM ANOVA (see Materials and methods).

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responses tended to be decreased. With the highest concentra-tion of venom tested, 450 μg/mL, the maximum contraction to25 Hz was attenuated to 26 ± 7% KPSS (n = 6; p = 0.0025; Fig. 6B).Following venom washout, the frequency–contractile responsecurve to electrical stimulation returned to (no venom) controllevels (data not shown), indicating that the effects of this highconcentration of venomwere reversible.

Fig. 7 – Effects ofM. ikaheka venom and pre-treatment with antagatria and rate of spontaneously beating right atria isolated from th(Δ) in right atrial rate (beats/min). Atria were incubated for 30 micalcitonin gene-related peptide (CGRP) antagonist CGRP8-37 (1 μMthe anti-PLA2 groups, M. ikaheka venom was incubated with pBPNumber of atria. Vertical error bars are ± average SEM from RM Aapplicable) ±1 SEM.

In rat isolated left and right atria, M. ikaheka venom (0.009–90 μg/mL) caused concentration-dependent positive ionotropy(EC50 2.2 ± 0.5 μg/mL; n = 6) (i.e., strengthens the force of theheartbeat) and tachycardia (EC50 1.6 ± 0.9 μg/mL; n = 4), respec-tively (p < 0.0001; Fig. 7A and B). Pre-treatment with the β-adrenoceptor antagonist propranolol to inhibit sympatheticnerve-mediated ionotropic and chronotropic responses had

onists or abrogation of PLA2 activity on contractile force of lefte rat. A: Change (Δ) in left atrial contractile force (g). B: Changen with the b-adrenoceptor antagonist propranolol (1 μM) or) prior to M. ikaheka venom (0.009–90 μg/mL) application. In

B to abrogate PLA2 activity prior to application to the atria. n,NOVA and horizontal error bars represent the EC50 (where

226 J O U R N A L O F P R O T E O M I C S 1 1 0 ( 2 0 1 4 ) 2 0 9 – 2 2 9

no effect on responses to the venom in either left or rightatria. Further, inhibition of the sensory nerve neurotrans-mitter, calcitonin gene-related peptide, with the antagonistCGRP8–37 did not affect venom-induced increases in contrac-tility or rate (Fig. 7A and B). However, when venom wastreated overnight with pBPB to abrogate PLA2 activity, thepositive ionotropic and chronotropic atrial responses werecompletely abolished (n = 3 each; Fig. 7). The PLA2 activity ofM. ikaheka venompre- and post-pBPB treatment was 4593 and0.4 μmol/min/mg, respectively. Separate untreated aliquotsof venom were left at room temperature overnight and thentested in left and right atria (n = 2 each); this venom retainedits full potency in both sets of tissues (data not shown).

In summary, these in vitro pharmacological studies suggestthatM. ikaheka venom is cardiotoxic inducing tachycardia andpositive ionotropy, together with significant vascular smoothmuscle relaxation (which has been correlatated with systemichypotension [11]) and, at high concentrations, reversible inhibi-tion of sympathetic nerve-induced vascular contraction. Inarteries, arachidonic acid metabolites and muscarinic receptoractivation contributed only in small part to theM. ikaheka venomvasorelaxation; further, NO synthase and ATP-sensitive K+

channels did not appear to be involved. Additional studies arerequired to determine the mechanism of action of M. ikahekavenom in vascular smooth muscle. In cardiac tissues, muchlower concentrations (approximately 10-fold less) of venomwere required to elicit major increases in atrial rate and forceof contraction compared with relaxant effects in vascularsmooth muscle. Inhibition of the sympathetic nervous system(β1-adrenoceptor signalling) or the sensory neurotransmitterCGRP had no effect on either the tachycardia or positiveinotropy. However, when PLA2 activity was abrogated, theright and left atrial effects of the venom were completelyprevented, indicating that PLA2s are responsible for thesecardiotoxic effects.

4. Concluding remarks

Our joint venom gland cDNA sequencing and proteomicsapproach revealed peptide evidence for ~50 venom proteinsincluding 25 of the 27 full-length toxin sequences identified inthe novel sequences of M. ikaheka venom gland cDNA clones.Several toxins sequenced in this study shared close identity toproteins in Asian elapid venoms, notably Naja spp. andBungarus spp., an observation in broad agreement with thewidely accepted concept of an Asian (perhaps Bungarus →Laticauda) origin leading to colonization by rapidly diversify-ing ancestral species that split from a Laticauda-like predeces-sor between 13 and 19 MYBP and then diversified into thepresent day Australo-Papuan snake fauna [2,69–71]. At thesame time, the remarkable homology of the venom toxinsequences of M. ikaheka to venom proteins from terrestrialand marine Australian Hydrophiinae and viviparous sea snakesalso supports the hypothesis of a sister-group relationshipbetween the monotypic Melanesian Micropechis genus and thesubfamily Oxyuraninae within the family Hydrophiidae [4]. Inconcordance with this view, an initial investigation of thevenoms of some small Australian elapids has shown them tobe equally as complex as those of their larger, better-investigated

cousins [72], suggesting that their overall venom compositionwas derived at the base of the Australian elapid snake radiation.The split between Laticauda and Oxyuraninae snake lineages isestimated at 12.6 MYBP, and the last common ancestor ofMicropechis and all living oxyuranines is dated at 11.5 MYBP[1,2]. Bayesian relaxed clock analyses indicated that the ~160species of the core Australian radiation arose recently, withinthe last 10 MYBP, with most inter-generic splits dating tobetween 10 and 6 MYBP. Analysis of the rapid evolutionaryradiation of Australasian elapids and sea snakes from avenomics perspective may shed light on the lethal mecha-nisms of elapid snake venoms. In this respect, the recentreport of the sequencing of the first genome of a venomoussnake, the king cobra, O. hannah, has revealed that toxingenes important for prey capture have massively expandedby gene duplications and evolved under positive selection,resulting in protein neofunctionalisation [73]. Gene dupli-cates exhibit varying degrees of structural resemblance totheir progenitor loci. The rate at which a new functional genecopy appears in a population depends on both the time sinceduplication (genetic drift) and the adaptive potential of theduplicated gene [74,75]. The high structural conservationexhibited by M. ikaheka and other Australo-Papuan elapidvenom toxins suggests a high conservation of protein class-specific immunologic epitopes across this diverse radiationof species.

M. ikaheka is a large, powerfully built elapid that averages1.2–1.7 m in length [5] but very large specimens of 2.1–2.3 mhave been recorded [76]. This species often holds onto tightlywhen it bites, and envenomingmay lead to significant illness orto death [8,76,77]. Although there is no known specific antiven-om available for the treatment of M. Ikaheka envenoming inPapuaNewGuinea, positive clinical outcomes and recoveryhavebeen documented through the use of bioCSL Australo-Papuanpolyvalent antivenom and bioCSL monovalent tiger snakeantivenom, if given early [8,77,78]. These methods of treatment,however, have been largely empirical, leading us to undertakefurther studies [22] to elucidate which toxins harbour immuno-logic epitopes for neutralizing bioCSL antivenom immunoglobu-lins, and while these and other bioCSL products demonstratedgood neutralisation of lethality, as well as dose-dependent at-tenuation of myotoxicity, they were poorly efficacious in theneutralisation of PLA2 and anticoagulant activity [22]. Formalstudies of envenoming by this species should in future enable usto evaluate the practical effectiveness of these antivenoms, anddetermine whether a need for an improved antivenom againstthis species is required in order to achieve the best possibleclinical outcomes for all patients.

Acknowledgements

Thanks are due toDaniela Solano, Instituto Clodomiro Picado, forher support in the laboratory work. Funding for the researchdescribed in this paper was provided by grants BFU2010-17373from theMinisterio de Ciencia e Innovación (currently,Ministeriode Economía y Competitividad), Madrid; PROMETEO/2010/005from the Generalitat Valenciana; CYTED project BIOTOX P211RT0412; project 741-B2-652 (Vicerrectoría de Investigación, UCR);and FEES-CONARE (Costa Rica). Research in PNG was supported

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by AVRU from a grant by the Australian Government's Depart-ment of Health & Aging (DoHA) and a grant [APSF 07/1] from theAustralia-Pacific Science Foundation (APSF), as well as theInternational CooperativeBiodiversityGroup (ProfessorTeatulohiMatainaho & Professor Louis Barrows). We are particularlygrateful to Prof. Martin Lavin, Dr. Geoff W. Birrell, and Dr. LiamSt Pierre (Queensland Institute of Medical Research, Brisbane,Queensland, Australia) who made it possible for Owen Paiva tocarry out much of his work in their laboratories, using primersand protocols developed by them.

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