Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: Role of neurotoxic and procoagulant effects in venom toxicity

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Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: Role of neurotoxic and procoagulant effects in venom 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,⁎ a

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica School of Medicine & Health Sciences, University of Papua New Guinea, Port Moresby, Papua New Guinea c Australian Venom Research Unit, Department of Pharmacology, University of Melbourne, Parkville, VIC, Australia d Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaume Roig 11, 46010 Valencia, Spain e Nossal Institute for Global Health, University of Melbourne, Parkville, VIC, Australia b

AR TIC LE I N FO

ABS TR ACT

Article history:

The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus

Received 14 December 2011

scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-

Accepted 8 January 2012

phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-

Available online 14 January 2012

PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following

Keywords:

seven protein families were identified in the two venoms: phospholipase A2 (PLA2), Kunitz-

Taipan

type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase,

Oxyuranus scutellatus

cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition,

Venom

C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the

Taipoxin

venom of specimens from PNG. PLA2s comprised more than 65% of the venoms of these

Proteome

two populations. Antivenoms generated against the venoms of these populations showed

Antivenom

a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization 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 a potent and very rapid mechanism for killing prey. © 2012 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Tel.: +506 2229 3135; fax: +506 2292 0485. E-mail address: [email protected] (J.M. Gutiérrez). 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2012.01.006

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1.

Introduction

Australia and New Guinea harbor a rich biodiversity of venomous snakes of the family Elapidae, which include species possessing some of the most toxic venoms in the world [1]. Among them, the taipans (Oxyuranus spp.), are distributed in Australia, Papua New Guinea and Indonesian Papua, and produce some of the world's most toxic snake venoms [2]. Combined with their agility, speed, large adult body size (up to 3 m for Oxyuranus scutellatus), high venom output (>100 mg) and nervous dispositions, taipans are extremely dangerous snakes to encounter. Snakebites by Oxyuranus spp. often result in severe neurotoxic envenoming which may be fatal in the absence of prompt antivenom treatment [3,4]. There are three species of taipans: (a) O. scutellatus, with populations in northern Australia, southern Papua New Guinea and southern Indonesian Papua [1,5,6]; (b) Oxyuranus microlepidotus, the inland taipan, which occurs in south-western Queensland and north-eastern to centralnorthern South Australia [7]; and (c) Oxyuranus temporalis, known from a handful of specimens collected in the southeastern deserts of Western Australia [7]. O. scutellatus occurs in a wide variety of habitats including sugarcane fields and woodlands in the eastern and north coast of Australia and in savannah regions of southern New Guinea [5]. Phenotypic differences between the New Guinean and Australian populations of the taipan had prompted their classification as separate subspecies, Oxyuranus scutellatus canni and Oxyuranus s. scutellatus, respectively [8]. However, recent evidence from mitochondrial DNA analysis revealed high similarities between these populations, demonstrating very recent genetic exchange among them, thus questioning their subspecific status [6,7]. Hence, further studies are required to assess the intraspecies variation between these populations. Human envenomings by O. scutellatus are not common in northern Australia [1]. However, this species inflicts many severe bites in the southern regions of Papua New Guinea (PNG) [4]. These envenomings are predominantly characterized by a neurotoxic effect of rapid onset, which often leads to respiratory paralysis in the absence of timely administration of antivenom [3,9]. In addition, coagulopathy associated with spontaneous bleeding has been described in these patients [10], together with myotoxicity and cardiac disturbances in some cases [11]. The toxins responsible for these effects have been isolated and characterized, such as the potent neurotoxic and myotoxic heterotrimeric phospholipase A2 (PLA2) complex taipoxin (named cannitoxin for the West Papuan population) [12,13], two monomeric PLA2s, named OS1 and OS2 [14], Oscutarin-C, a prothrombin activator responsible for the characteristic coagulopathy [15], post-synaptically acting α-neurotoxins [16], and taicatoxin, a blocker of voltage-dependent calcium channels [17]. In addition, cDNA analysis derived from venom gland transcripts detected various putative toxin genes, including PLA2s, neurotoxins, cysteine-rich secretory proteins (CRISPs), a venom natriuretic peptide and a nerve growth factor [18]. However, besides the proteins previously characterized from this venom, the presence in the venom of these additional components encoded by these transcripts, and their possible biological activity, remain unknown.

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The present work presents a comparative proteomic analysis of the venoms of O. scutellatus from Australia and PNG, in order to ascertain whether there are phenotypic differences in the venom composition of these populations. In parallel, the most relevant biological activities of these venoms were determined, and correlated with the venom proteome. Owing to the accelerated processes that characterize the evolution of snake venom toxins [19–21], it was of interest to assess whether the venom proteome of these very similar but allopatric populations differs, in order to predict possible variations in their biological/toxicological profiles, and in the immunologically cross-reactivity of antivenoms raised against these venoms. In addition, the potential role of neurotoxic and procoagulant components of these venoms in prey immobilization was explored in a mouse model.

2.

Materials and methods

2.1.

Venom fractionation

The venom of O. scutellatus from PNG was a pool obtained from twelve healthy, adult specimens collected in PNG's Milne Bay Province and Central Province. These snakes were maintained in a purpose-built serpentarium at the University of PNG, and venom was collected at 21 day intervals. Venom was obtained using Parafilm-covered Eppendorf tubes. Samples contaminated by blood were discarded, and all samples were handled using plastic pipettes and tubes. Venom was snap-frozen to − 80 °C, before being freeze-dried and stored away from light at −20 °C. The venom of Australian O. scutellatus was obtained from Venom Supplies Pty Limited (Tanunda, South Australia). It was stored at − 20 °C until used. For reverse-phase HPLC separations, 5 mg venom was dissolved in 200 μL of 0.1% trifluoroacetic acid (TFA) and 5% acetonitrile (buffer A). Insoluble material was removed by centrifugation at 13,000 g for 10 min at room temperature and then the supernatant was loaded on a C18 column (250 × 4.6 mm, 5 μm particle size; Agilent) using an Agilent 1100 chromatograph. For the elution of proteins, a flow of 1 mL of solvent/min was used, 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 were detected at 215 nm. Protein peaks were collected manually and dried in a Speed Vac (Savant). The relative abundances (% of the total venom proteins) of the different protein families in the venoms were estimated from the relation of the sum of the areas of the chromatographic peaks containing proteins from the same family to the total area of venom protein peaks. ChemStation v.B.04.01 (Agilent) software was used for the integration of the chromatogram. If a chromatographic peak contained two or more proteins, their relative distribution was estimated by densitometry of the SDS-PAGEseparated 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 (reducing conditions) and stained with Coomassie Blue R-250. Some isolated protein fractions were subjected to N-terminal sequence analysis (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 performed against the available databanks using the BLAST program [22]. In addition, Coomassie-stained bands were excised from gels and subjected to reduction with dithiothreitol and alkylation with iodoacetamide, followed by in-gel digestion with sequencing grade bovine trypsin on an automated digester (ProGest, Digilab), according to manufacturer's instructions. Each tryptic digest was then analyzed in a MALDI-TOF– TOF Applied Biosystems 4800-Plus mass spectrometer, after spotting 0.5 μL of α-cyano-4-hydroxycinnamic acid and 0.5 μL of each sample onto an Opti-TOF 384-well plate. For spectra acquisition, 1625 shots were accumulated in reflector positive mode, using a laser intensity of 3000. CalMix-5 (ABSciex) peptides were used as external standards. The twelve most intense ions of each MS spectrum were selected as precursors for automated collision-induced dissociation MS/MS (2KV, positive mode) using 500 shots per sample and a laser intensity of 3900. The resulting spectra were analyzed using ProteinPilot v.2.0.1 (ABSciex) to identify proteins, at a confidence level of 99%. Tryptic digests not identified by MALDI-TOF–TOF were subjected to nano-electrospray ionization (nESI)-MS/MS analysis on a Q-Trap 3200 instrument (Applied Biosystems). Doubly- or triply-charged ions of peptides selected from the MALDI-TOF mass fingerprint spectra were analyzed in Enhanced Resolution mode (250 amu/s), and the monoisotopic ions were fragmented using the Enhanced Product Ion tool with Q0 trapping. Settings for MS/MS analyses were: Q1, unit resolution; collision energy, 25–40 eV; linear ion trap Q3 fill time, 250 ms; and Q3 scan rate, 1000 amu/s. Resulting Collision-Induced Dissociation (CID) spectra were interpreted with the aid of the BioAnalyst v.1.5 (http://www.absciex. com/Products/Software/BioAnalyst-Software) manual sequencing tool, and the obtained sequences were first compared to all of the known Oxyuranus spp. sequences with the MEGA 5 alignment and analysis tool (http://www. megasoftware.net) using a genus level toxin sequence database prepared from BLAST (http://blast.ncbi.nlm.nih.gov) search data before being submitted to BLAST for a generalized search against all snake venom sequences if no genus level identity matches were found.

2.3.

Biological and enzymatic activities of venoms

2.3.1.

Lethal activity

Two types of experiments were performed concerning lethality: In the first set, the Median Lethal Dose (LD50) of venoms was assessed in CD-1 mice (18–20 g) by the intravenous (i.v.) route, in order to determine whether the differences in venom proteomes translate into variations in the systemic toxicity. Various doses of venoms were injected in the caudal vein and deaths occurring within 24 h were recorded. LD50 was estimated by the Spearman–Karber method [23]. In another set of experiments, an experimental design was used to determine the probable cause of death in mice when using low and high venom doses, in order to discern the mode of death in rodents when a large dose of venom is injected, as occurs in natural bites. In these experiments, mice were injected by the i.v. route, with specific low and high doses of either venom or purified taipoxin; the signs of

toxicity shown by mice were observed and the time of death was recorded. In some experiments, tissue samples were collected from the lung immediately after death, and placed in formalin fixative solution. After routine processing, samples were embedded in paraffin, and 5–8 μm sections were prepared and stained with hematoxylin–eosin for histological analysis. In the case of mice injected with PBS, they were sacrificed by an overdose of anesthetic (a mixture of xylazine and ketamine), and tissue samples were processed as described. The experimental protocols involving the use of animals in this study were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica.

2.3.2.

Coagulant activity in vitro

The method described by Theakston and Reid [24] was followed, with the modifications described by Vargas et al. [25]. Various amounts of venom, dissolved in 100 μL of 0.15 M NaCl, were added to aliquots of 200 μL of human citrated plasma previously incubated at 37 °C for 5 min. CaCl2 was added (15 μL of 0.2 M CaCl2 to 200 μL plasma) immediately before addition of the venom. Experiments were run in quadruplicate. Clotting times were recorded, and the Minimum Coagulant Concentration (MCC) was determined. The MCC corresponds to the concentration of venom that induced plasma clotting in 60 s.

2.3.3.

PLA2 activity

PLA2 activity was determined titrimetrically, using egg yolk phospholipids as substrates [26]. Activity was expressed as μEq fatty acid released per mg protein per min.

2.4.

Neutralization by antivenoms

In order to assess the cross-neutralization of venom lethal and coagulant activities by antivenoms raised against the venoms of O. scutellatus from PNG and Australia, monospecific taipan antivenoms produced by Instituto Clodomiro Picado (batch 4511209 ICP; expiry date November 2012) and by CSL Limited (CSL), Melbourne, Victoria, Australia (batch B054806301; expiry date March 2012) were used. The former was raised against the venom of O. scutellatus from PNG and the latter against venom from specimens collected in Australia. The production methods and physicochemical characteristics of these antivenoms were previously described [25]. For neutralization assays, a fixed dose of venom (4 LD50s for lethality and 2 MCCs for coagulant effect) was incubated with varying volumes of antivenom, at 37 °C, for either 30 min (for lethal activity) or 3 min (for coagulant activity). Controls included solutions of venom incubated with saline solution instead of antivenom. After incubation, lethal and coagulant activities were assessed as described above. Neutralization was expressed as either Effective Dose 50% (ED50), for lethality, or Effective Dose (ED), for coagulant effect, corresponding to the ratio mg venom neutralized per mL antivenom at which neutralization was achieved, as previously described [25].

2.5.

Statistical analysis

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

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

3.

Results and discussion

3.1.

Venomics

The RP-HPLC elution profiles of O. scutellatus venoms from PNG and Australia, and the SDS-PAGE migration of the proteins present in each fraction are shown in Figs. 1 and 2. Although the two chromatograms had a similar number of peaks, 23 and 25, the elution patterns revealed differences. Proteins related to seven and nine protein families were identified by mass spectrometry in the venoms of Australian and PNG O. scutellatus, respectively. These families of proteins identified 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 activator Oscutarin C (Tables 1 and 2; Fig. 3). Three sequences with identity to thrombin-like, serine proteinase enzymes (TLE) were observed in Australian O. scutellatus venom. In

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addition, C-type lectin/lectin-like proteins, and venom natriuretic peptides were identified in the venom of PNG O. scutellatus. Phospholipase A2 species, including taipoxin, were the most abundant proteins in both venoms (Fig. 3). Proteomic analysis highlights the abundance of the neurotoxic heterotrimer taipoxin in both venoms, albeit with quantitative differences, since a significantly higher concentration (56% vs 20%) was observed in the venom of PNG O. scutellatus (Tables 1 and 2). The higher proportion of this very potent toxin may explain the observed lower LD50 of PNG O. scutellatus venom in murine testing (Table 3). This PLA2 oligomer constitutes one of the most toxic components yet isolated from snake venoms [12,27]. It induces presynaptic neurotoxicity associated with the depletion of transmitter from the motor nerve terminals and other ultrastructural manifestations of terminal nerve damage [12,28,29], caused by the ability of this toxin to hydrolyze phospholipids in the plasma membrane of nerve terminals [30]. In addition, taipoxin induces myotoxicity owing to its ability to disrupt the integrity of skeletal muscle plasma membrane [31]. Taipoxin was originally isolated from the venom of Australian O. scutellatus [12]. More recently, an analogous toxin, named

Fig. 1 – Elution profiles of venom proteins of O. scutellatus from Papua New Guinea by RP-HPLC (A), and electrophoretic separation of proteins in each peak (B). Five milligram venom was fractionated on a C18 column, as described in Materials and methods. Fractions were analyzed by SDS-PAGE under reducing conditions and characterized by N-terminal sequencing, MALDI-TOF/TOF or nESI-MS/MS tryptic peptide MS/MS de novo sequencing, as summarized in Table 1. Molecular mass markers are shown in the left lanes of each gel.

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Fig. 2 – Elution profiles of venom proteins of O. scutellatus from Australia by RP-HPLC (A), and electrophoretic separation of proteins in each peak (B). Five milligram venom was fractionated on a C18 column, as described in Materials and methods. Fractions were analyzed by SDS-PAGE under reducing conditions and characterized by N-terminal sequencing, MALDI-TOF/TOF or nESI-MS/MS tryptic peptide MS/MS de novo sequencing, as summarized in Table 2. Molecular mass markers are shown in the left lanes of each gel.

cannitoxin, was characterized from the venom of Papuan O. scutellatus [13], although this can be regarded as taipoxin given that the PNG and Australian populations belong to the same species, and indeed, we identified the N-terminal sequence for taipoxin (NLLQFGFMIR), rather than cannitoxin (NLLQFGYMIR), in venom of our PNG O. scutellatus suggesting that both isoforms are likely to exist in this venom. In both populations, the trimers are comprised of subunit α, which is an active neurotoxic PLA2, and by subunits β and γ, which are PLA2 homologues without enzymatic activity [12,13,32–34]. Despite the lack of catalytic and toxic activities of subunits β and γ, they significantly potentiate the toxicity of subunit α [27,35]. As in the case of other oligomeric neurotoxic PLA2s, the nontoxic subunits play the role of chaperone molecules, preventing the binding of the toxic subunit to non-specific binding sites in cell membranes, thus allowing the complex to reach highaffinity binding sites on the membranes of nerve terminals and skeletal muscle cells, where the α subunit exerts its toxicity by hydrolyzing phospholipids in these membranes [27,30]. This represents a highly successful strategy to enhance the toxicity of neurotoxic and myotoxic PLA2s. In addition to this powerful presynaptic neurotoxic mechanism, the venoms of the two populations of O. scutellatus contain small quantities of post-synaptic long- and short-

chain ‘three-finger’ α-neurotoxins which block the nicotinic cholinergic receptor at the motor end-plate, inducing a flaccid paralysis [16,36]. Interestingly, pharmacological studies on nerve-muscle preparations have demonstrated differences between some of the post-synaptic neurotoxins of these two populations of O. scutellatus [16]. The adaptive implications of this difference are unclear, but it is likely they are minor, owing to the high content and potency of taipoxin, which represents a powerful immobilizing mechanism, regardless of the effects of post-synaptic neurotoxins. The presence of various neurotoxic mechanisms in the same venom is likely to constitute a redundant strategy for prey immobilization, although, in the case of taipan venom, presynaptic neurotoxicity largely dominates as the mechanism of paralysis. Besides taipoxin, the venoms of Australian and PNG populations of O. scutellatus contain a number of additional PLA2 components in different proportions, with Australian O. scutellatus having a higher overall PLA2 composition (79.7% vs. 68.3%) than the PNG population, despite having much less taipoxin. Some of these other PLA2s included sequences with identity to the monomeric enzymes OS1, OS2, OS3 and OS4 previously isolated from this venom [14,37]. OS1 and OS2 have been used in the characterization of the most thoroughly studied 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-terminal sequencing, MALDI-TOF–TOF mass spectrometry and nESI-MS/MS of selected peptide ions from in-gel digested protein bands. Peak ID

Mass kDa

Peptide ion m/z a

2.8 0.9 0.5 2.9

6787.6 ~7 7910.3 a ~7

6 0.7 2.6

7222.6 a 6677.8 a ~7

0.2

~7

0.6

~7

0.2 2.7 1.9 22.7 1.1 4.9 4.9 2 11.8 5.1

~10 ~15 ~4 ~13 ~16 ~16 ~14 ~10 ~14 ~17

3.1 0.6 2.9 0.9 0.3

~15 ~30 ~15 ~30 ~15

3.8 0.3 1.4

~15 ~100 ~30

0.2 0.4 0.2 1.3 0.8

~26 ~23 ~20 ~30 ~30 ~15 ~20 ~50 ~30

0.6 1.9 7

1338.4 1055.3 1465.5

1055.3 1465.5 1055.3 1465.5 1055.3 1465.5 996 1201.6 1352.7 1930.7 1876.9 1490.5 1238.5 1238.5 1238.5 965.6 857.5 1839.8 1839.8 1406.4 1238.7 1486.5 2859.8 1851.2 964.5 1839.8 536.2 1238.6 2859.8 1419.7 1182.6 1182.6 1839.8 1986.7 2225.8 1396.7 2265.1

z N-ter 1 N-ter 1 1 N-ter N-ter 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1

MS/MS-derived or N-terminal (Nt) sequence

Type

Related protein

Protein description

MTCYNQQSSEAKTTT CPPGBEVCYTK RRCFTTPSVRSERCP FYYNPHSK FCHXPPBPGPCR KDRPKFCHLPPKPGP KDRPKFCHLPPKPGP FYYNPHSK FCHXPPBPGPCR FYYNPHSK FCHXPPBPGPCR FYYNPHSK FCHXPPBPGPCR YYXAAEEVXWDYSP (291.1) GGSGTPVDBXDR XGNGCFGFPXDR HGCYPSXTTYTWECR TBCEVFVCACDFAAAK CAPoxYWTXYSWK NXXBFGFMXR NXXBFGFMXR NXXBFGFMXR (201.2)XNFANXXECANHGTR (228.2)VBFGFFXECAXR THDECYAEAGBXSACK THDECYAEAGBXSACK THDECYAEAGK NXXBFGFMXR TNCPASCFCBNK TAVTCFAGAPYNDDXYNXGMXECHK RPAXDFMNYGCYCGK CBTEWXK THDECYAEAGBXSACK CPATCFCR EXVDKHNDXR TAVTCFAGAPYNDDXYNXGMXECHK AEVDDVXEVRFK QDFGXVSGFGR QDFGXVSGFGR THDECYAEAGBXSACK DCPSDWSSYDXYCYK XHSWVECESGECCEBCR XGXFVDHGMoxYTK TSHDHABXXTATXFNGNVXGR

3FTX b 3FTX 3FTX BPTI/Kunitz BPTI/Kunitz BPTI/Kunitz BPTI/Kunitz BPTI/Kunitz BPTI/Kunitz BPTI/Kunitz BPTI/Kunitz BPTI/Kunitz BPTI/Kunitz Oscutarin C PLA2 Natriuretic PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 PLA2 CRISP PLA2 PLA2 CRISP PLA2 CRISP CRISP PLA2 Oscutarin C Oscutarin C Oscutarin C PLA2 C-type Lectin SVMP SVMP SVMP

GI|123910874 GI|254772668 GI|254772668 GI:239977265 GI|263546 GI|263546 GI|263546 GI:239977265 GI|263546 GI:239977265 GI|263546 GI:239977265 GI|263546 GI|82073021 GI|66475092 GI|32363246 GI|129435 GI|129435 GI|71066728 GI|129413 GI|129413 GI|129413 GI|913013 GI|71066724 GI|129446 GI|129446 GI|129446 GI|129413 ACE73574 GI|129446 GI|129435 GI|123916495 GI|129446 GI|123916495 GI|123916495 GI|129446 GI|82073021 GI|82073037 GI|82073037 GI|129446 GI|332278153 GI|145982762 GI|118643 GI|469190

SNTX-1 (O. scutellatus) LNTX-1 (O. scutellatus) LNTX-1 (O. scutellatus) Venom protease inhibitor 1 (O. scutellatus) Taicatoxin serine protease inhibitor (O. scutellatus) Taicatoxin serine protease inhibitor (O. scutellatus) Taicatoxin serine protease inhibitor (O. scutellatus) Venom protease inhibitor 1 (O. scutellatus) Taicatoxin serine protease inhibitor (O. scutellatus) Venom protease inhibitor 1 (O. scutellatus) Taicatoxin serine protease inhibitor (O. scutellatus) Venom protease inhibitor 1 (O. scutellatus) Taicatoxin serine protease inhibitor (O. scutellatus) Oscutarin C non-catalytic subunit (O. scutellatus) OS7 precursor (O. scutellatus) Oxs SNP c (O. scutellatus) Taipoxin beta chain (O. scutellatus) Taipoxin beta chain (O. scutellatus) PLA-6 precursor (O. scutellatus) Taipoxin alpha chain (O. scutellatus) Taipoxin alpha chain (O. scutellatus) Taipoxin alpha chain (O. scutellatus) OS1 PLA2 (O. scutellatus) PLA-4 precursor (O. scutellatus) Gamma-taipoxin (O. scutellatus) Gamma-taipoxin (O. scutellatus) Gamma-taipoxin (O. scutellatus) Taipoxin alpha chain (O. scutellatus) CRISP (Viridovipera stejnegeri) Gamma-taipoxin (O. scutellatus) Taipoxin beta chain (O. scutellatus) Pseudechetoxin-like protein (O. scutellatus) Gamma-taipoxin (O. scutellatus) Pseudechetoxin-like protein (O. scutellatus) Pseudechetoxin-like protein(O. scutellatus) Gamma-taipoxin (O. scutellatus) Oscutarin C non-catalytic subunit (O. scutellatus) Oscutarin C FVa-like subunit (O. scutellatus) Oscutarin C FVa-like subunit (O. scutellatus) Gamma-taipoxin (O. scutellatus) CTL (Trimeresurus albolabris) Scutellatease-1 (O. scutellatus) SVMP (Trimeresurus gramineus) SVMP (Gloydius halys)

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OsPG1 OsPG2 OsPG3 OsPG4a OsPG4b OsPG5 OsPG6 OsPG7a OsPG7b OsPG8a OsPG8b OsPG9a OsPG9b OsPG13 OsPG14a OsPG14b OsPG15a OsPG15b OsPG16a OsPG16b OsPG16c OsPG17a OsPG17b.1 OsPG17b.2 OsPG17c OsPG18a OsPG18b OsPG19a OsPG19b.1 OsPG19b.2 OsPG19c OsPG20a OsPG20b.1 OsPG20b.2 OsPG20c OsPG20d OsPG20e OsPG21a OsPG21b.1 OsPG21b.2 OsPG21c OsPG23a OsPG23b.1 OsPG23b.2

%

a

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

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[14,38]. OS2 is also a highly toxic component which induces various effects, such as myotoxicity and neurotoxicity [14,39], whereas OS1 displays lower toxicity [37]. Other sequences with broad identity to previously characterized O. scutellatus PLA2 were also found, and it is clear that there are many PLA2s in both of these venoms that are yet to be fully sequenced and described. The presence of additional toxic PLA2s 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 process of gene duplication and accelerated evolution described for this family of venom toxins [19]. The existence of abundant PLA2 isoforms fits into the redundant toxin paradigm of venom composition described above. In addition, monomeric PLA2s of low toxicity but high enzymatic activity may play a predominantly digestive role. The adaptive role of myotoxicity in taipoxin [31] and other myotoxic PLA2s, such as OS2 [39], deserves consideration. Although it may be a by-product of the acquisition of strong neurotoxicity (i.e. a consequence of the ability of these toxins to interact with binding sites in the plasma membranes of nerve terminals and other excitable cells, such as myocytes), myotoxicity is likely to have a relevant adaptive role associated with the digestion of muscle mass. Since O. scutellatus feeds largely on mammals [40], characterized by a large mass to surface ratio, i.e. type III prey [41], this poses a problem for the digestion of prey characterized by a large mass of muscle tissue. In the absence of significant proteinase content in these venoms, the acquisition of myotoxicity is likely to contribute to muscle protein digestion. The action of myotoxic PLA2s on the plasma membrane of muscle fibers facilitates the release of cytosolic proteins and the access to myofilament proteins [42]. In addition, myonecrosis, with the associated increments of cytosolic Ca2 + levels following the disruption of the permeability barrier of the plasma membrane, results in activation of intracellular proteinases, such as m- and μ-calpains, with the consequent hydrolysis of cytoskeletal proteins present in myofibrils [43,44]. This, in turn, may facilitate the further digestion of the predominant myofilament proteins, i.e. actin and myosin, by proteinases present in the gastric and pancreatic secretions. It is likely that one of the adaptive roles of myotoxic PLA2s in snake venoms may be associated with digestion of muscle mass [42,45]. O. scutellatus venom contains potent Group C prothrombin activators [46]. These activators require a phospholipid substrate and free Ca2 + ions as cofactors. Our proteomic data revealed four peaks in PNG taipan venom with constituents that had identity with the Factor Va-Xa-like prothrombin activator Oscutarin C, comprising 2.5% of the venom, compared to just two peaks making up 1.3% of Australian taipan venom [15]. The percentage of the prothrombin activator in taipan venom has previously been estimated to be at most 5% [15]. However, despite its relatively low concentration, the coagulant activity of taipan venoms is very high (see below) and disturbances of hemostasis are a common feature of taipan envenoming. The higher concentration of Oscutarin C in PNG O. scutellatus venom helps to explain why antivenom raised against Australian O. scutellatus venom is less effective against this toxin than antivenom raised using venom from PNG O. scutellatus (Table 3). It may also explain why clinical

bleeding after taipan bites is more commonly reported in the victims of snakebite in Papua New Guinea [10] than it is among taipan bite patients in Australia. Oscutarin C is a multimeric protein comprised of a factor Xa-like proteinase, a factor Va-like protein, and additional subunits [15]. From the clinical standpoint, these procoagulants activate prothrombin, thus leading to defibrinogenation, blood factor depletion and blood incoagulability, which induce bleeding in a number of patients [10]. From the biological standpoint, however, the role of these potent procoagulants in the natural prey of taipans may be associated with intravascular thrombosis, an effect that provokes rapid death (see below). The venoms of the two populations of taipan also contained additional venom components, whose biological roles remain largely unknown (Tables 1 and 2; Fig. 3). After PLA2s, the two most abundant components in both venoms are Kunitz-type inhibitors and SVMPs. A number of peaks in both venoms had sequence identity to BPTI/Kunitz-type inhibitors previously identified in O. scutellatus venom, including a subunit of taicatoxin, which is an oligomer of three different proteins: an α-neurotoxin-like peptide, a neurotoxic PLA2 and a Kunitz-type inhibitor of 7 kDa, linked by noncovalent bonds at an approximate stoichiometry of 1:1:4 [17]. Originally, taicatoxin was shown to block the high threshold Ca2 + channel current of excitable membranes from the heart [17], inducing a series of alterations in cardiac myocyte cell functions [47], although it was later shown also to interact with apamin-sensitive, small conductance, Ca2 +-activated potassium channels [48]. The potential toxic role of taicatoxin in taipan envenomings is not known, although it has been suggested that it may be associated with the electrocardiographic abnormalities described in human patients envenomed by taipans in PNG [11]. SVMPs comprise between 5 and 9% of the proteomes of these two venoms. Elapid venoms typically contain a relatively low abundance of SVMPs [49–52], whose toxicity has not been well characterized. SVMPs isolated from elapid venoms belong to the class P-III, i.e. the mature proteins are comprised by metalloproteinase, disintegrin-like and cysteine-rich domains [53,54]. Some SVMPs isolated from elapid venoms of Naja species induce hydrolysis of the complement system, von Willebrand factor and pro-TNF-α [55–58]. However, these activities have only been demonstrated in vitro and the actual biological role of these effects remains unknown. A hemorrhagic SVMP of high molecular mass was characterized from the venom of Ophiophagus hannah which induces hemorrhage in some species only [59]. A P-III SVMP, Mikarin, in the venom of Micropechis ikaheka, the New Guinea small-eyed snake, was found to be a Group I (Ca2 +-independent) prothrombin activator [60]. In the case of O. scutellatus venoms, the role of SVMPs has not been elucidated, and they do not seem to be associated with the predominant toxic activities of the venom, i.e. neurotoxicity, myotoxicity and coagulopathy and, consequently, may play a predominantly digestive role. Several bands in taipan venoms corresponded to CRISPs having identity to a pseudechetoxin-like protein (GI|123916495) from O. scutellatus venom and the related protein pseudecin, previously characterized from the venom of the Australian red-bellied blacksnake Pseudechis porphyriacus [18,61]. These CRISPs 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.

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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 secretory proteins (CRISP), factor V-like protein (FV-like).

ion channels, occluding the entrance of the pore and inhibiting the flow of current [62]. These proteins have proved to be valuable tools in the study of these channels, although their physiological/toxicological role in the venom remains unknown. In addition, our analysis revealed the presence of other minor components, such as serine proteinases, C-type lectin/lectin-

like components and venom natriuretic peptides (Tables 1 and 2, Fig. 3). A toxicologically-relevant serine proteinase in this venom corresponds to the factor Xa-like subunit of the prothrombin activator [15]. The possible biological role in this venom of the C-type lectin/lectin-like proteins and venom natriuretic peptides, previously characterized in the venoms of

Table 3 – Lethality and coagulant activity of venoms of O. scutellatus from Papua New Guinea and Australia, and neutralization by monospecific antivenoms. Lethality Neutralization (mg venom/mL antivenom) a Venom O. scutellatus (PNG) O. scutellatus (AUS)

LD50 (μg per mouse) b Antivenom against O. scutellatus (PNG) Antivenom against O. scutellatus (Australia) 0.08 ± 0.01 0.50 ± 0.10

5.6 ± 1.7 11.0 ± 2.8

5.9 ± 0.13 16.4 ± 2.1

Coagulant activity Neutralization (mg venom/mL antivenom) a Venom O. scutellatus (PNG) O. scutellatus (AUS) a

MCC (μg/mL) c 0.33 ± 0.13 1.30 ± 0.60

Antivenom against O. scutellatus (PNG) 2.37 ± 0.08 10.0 ± 2.5

Antivenom against O. scutellatus (Australia) 0.45 ± 0.17 4.1 ± 1.4

Neutralization is expressed as the amount of mg of venom neutralized by 1 mL of antivenom. For neutralization experiments, the challenge doses used corresponded to 4 LD50s (for lethality) and 2 MCC (for coagulant activity). b The Median Lethal Dose (LD50) was determined in CD-1 mice (16–18 g) by the i.v. route. It corresponds to the dose of venom that induces lethality in 50% of the injected mice within 24 h of injection. c The Minimum Coagulant Concentration (MCC) was estimated in citrated human plasma with the addition of calcium (see Materials and methods). It corresponds to the venom concentration that induces clotting of plasma in 60 s.

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

3.3. Assessing the toxicity of taipan venom from a biological perspective: which is the predominant mechanism of immobilization and death of mice?

3.2. Correlation between the proteomes and the toxicological and immunological profiles of the venoms

The high toxicity of O. scutellatus venoms, together with the characteristic biting pattern of ‘snap and release’ [40], represents adaptations for feeding on relatively large mammalian prey, which are capable of retaliation. The high concentration of taipoxin in these venoms suggests that taipoxin-mediated paralysis might be the main mechanism involved in rapid prey immobilization and death. Nevertheless, the possible role of the potent procoagulant activity of this venom also has to be considered. To approach this issue, we analyzed the time and the circumstances of death in mice injected with O. scutellatus venom and purified taipoxin, at different doses, using mice as a biologically-relevant model, since taipans mostly feed on mammals, including mice [40]. Mice injected i.v. with a lethal, but relatively low dose of venom from PNG specimens, i.e. 5 LD50s, died at 62±11 min (n=5) after injection, with evident manifestations of limb paralysis and respiratory difficulties, i.e. with neurotoxic manifestations. However, when a higher dose of venom (50 μg, corresponding to 625 LD50s) was administered i.v., mice had an immediate collapse, and death occurred in 228±163 s (n=5). Remarkably, these mice did not show paralytic manifestations, suggesting that other mechanisms of death may be at work at this high dose. In order to comparatively assess the effect of a high dose of taipoxin, 50 μg of this neurotoxin (corresponding to 1250 LD50s, [12]) were injected i.v. The time of death was 709±68 s (n=5), which was significantly more prolonged (p<0.05) when compared with mice injected with the same dose of venom; mice injected with this high dose of taipoxin died with evident signs of paralysis and respiratory distress. Since taipan venom has a potent procoagulant activity, the possibility that death at high doses is due to intravascular thrombosis was considered. A histological analysis of lung tissue was performed immediately after death of mice injected i.v. with 50 μg venom, which died rapidly upon venom injection. Abundant thrombi were observed in pulmonary vessels (Fig. 4), suggesting a rapid thrombotic effect as the most plausible cause of death in these circumstances, as has been observed after the i.v. injection of a prothrombin activator from the venom of Bothrops asper [64]. Clearly, therefore, the rapid cause of death in mice injected with a high dose of venom does not seem to be due to the action of taipoxin, but very likely to the thrombotic effect of the prothrombin activator. Since this snake is able to inject a large dose of venom in their prey, estimated to be around 20 mg in mice [65], it is likely that high doses of venom reach the bloodstream in a natural bite in mammalian prey, thus raising the possibility that the prothrombin activator plays a relevant role in rapid prey immobilization and death. From a biological standpoint, it appears that prey immobilization and death induced by taipan venom may occur by two different and highly effective mechanisms: intravascular thrombosis provoked by the prothrombin activator, Oscutarin C, and paralysis leading to respiratory failure induced by the PLA2 trimer, taipoxin. It is somehow puzzling that a venom that has a high concentration of one of the most potent neurotoxic components yet described in any snake species may use an alternative mechanism (intravascular coagulation), based on the action of procoagulants present in low relative amounts

The populations of taipan of Australia and PNG were split into subspecies on the basis of morphological differences [8]. However, more recent molecular analysis has not supported such differentiation and suggested the occurrence of gene flow between these populations as recently as the late Pleistocene [6]. Venom phenotyping constitutes an interesting area of analysis of geographical differentiation between populations. Our proteomic data showed that the proteomes of the venoms of these two 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 profiles and in the concentration of taipoxin, since higher amounts were found in the venom of PNG taipans, in agreement with its higher lethal activity (Table 3). The percentage of taipoxin in the venome of Australian O. scutellatus (20%) roughly corresponds to the previously described yield of this toxin [12]. In contrast, the percentage of taipoxin (56%) in PNG O. scutellatus deduced by proteomic analysis is much higher than the 16% yield of “cannitoxin” obtained from Papuan O. scutellatus [13]. This discrepancy might suggest additional variation between the venoms of the eastern PNG and western New Guinea (Western Province of PNG and Indonesian West Papua) populations which are physically disjoint from one another, an issue that deserves further investigation. The functional implications of the variations in the venom proteomes were assessed by determining the main toxic and enzymatic activities of these venoms. In terms of lethality, the venom of PNG taipans is 6.25 times more potent than the venom of Australian taipans (Table 3), in agreement with the higher concentration of taipoxin in the former. Thus, the differential expression of taipoxin in these venoms represents a mechanism to regulate the overall toxicity, owing to the potency of this toxin. On the other hand, coagulant activity in vitro is also slightly higher in the venom from PNG specimens (Table 3), and this again may be due to the higher percentage of this toxin in PNG venom. In addition, PLA2 activity was 240 and 660 μEq/mg/min fatty acid for the venoms of specimens from PNG and Australia, respectively. The higher activity of Australian venom correlates with the higher proportion of ‘non-taipoxin’ PLA2s in this venom. In agreement with the proteomic similarities observed between these venoms, antivenoms raised in horses against venoms from these populations showed a noteworthy pattern of cross-neutralization (Table 3). Both antivenoms were effective in the neutralization of lethal and coagulant activities of the venoms from both populations of taipans, thus revealing a similar antigenic makeup of these venoms regarding neurotoxic and coagulant components. Interestingly, both antivenoms had a higher neutralizing potency against lethality of the venom of Australian specimens than against the venom from PNG, probably reflecting the higher content of the potent neurotoxin taipoxin in the latter, thus making neutralization more difficult to achieve.

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venom. From a clinical perspective, on the other hand, the main cause of death in humans is neurotoxicity leading to respiratory paralysis [3], whereas the action of prothrombin activators largely results in defibrinogenation, factor depletion and incoagulable blood, in some cases with active bleeding, instead of thrombosis [10]. At the same time, however, up to 23% of patients bitten by PNG taipans in one study reported sudden collapse and transient loss of consciousness in the first minutes after the bite [3]. In Australia similar events have been reported in rapidly fatal cases of brown snake (genus Pseudonaja) envenoming [66], another species with Group C prothrombin activators. It could be suggested, based on our rodent experiments, that at very high venom doses the rapid formation of intravascular clots may have a role in these collapses, a hypothesis that deserves further consideration. In conclusion, the venom proteomes of O. scutellatus from Australia and PNG are highly similar, although differences in the amounts of taipoxin occur. The venom of PNG taipans has a higher concentration of this neurotoxin, in agreement with its higher lethal activity in mice. These venoms also present immunological similarities, revealed by the ability of monospecific antivenoms to neutralize venoms of the two populations. It is suggested that the cause of death in mice injected with O. scutellatus venom depends on the dose of venom injected; at low doses, neurotoxic paralysis predominates, whereas at high doses, such as those injected in natural bites, intravascular thrombosis occurs, thus representing a rapid and effective mechanism for killing rodents.

Acknowledgments

Fig. 4 – Light micrographs of sections of lung tissue injected with either PBS (A) or O. scutellatus venom from PNG (B and C) by the intravenous route. Mice were injected with either 100 μL of PBS or with 50 μg venom, dissolved in 100 μL PBS. Animals injected with venom had an immediate collapse and died within the first min of injection. A sample of lung tissue was collected immediately after death and processed for histological evaluation. Control mice were sacrificed with an overdose of anesthetic and lung tissue was collected and processed. Thrombi are observed in large and small blood vessels in mice injected with venom (arrows in B and C), whereas control mice injected with PBS did not show any intravascular thrombi, and their vessels were filled with erythrocytes (arrow, A). Magnification: 200× in A and B, and 400× in C. in the venom, for immobilizing and killing prey in natural conditions. This may constitute an example of a highly effective redundancy mechanism for prey immobilization in a snake

The authors thank Dr Andrés Hernández and B.Sc. Julissa Fonseca (Instituto Clodomiro Picado) for their collaboration, as well as Dr Bruno Lomonte for performing some of the mass spectrometry analyses. This study was supported by CONARE, Vicerrectoría de Investigación (Universidad de Costa Rica) (projects 741-A7-611 and 741-A9-506), CRUSA-CSIC (Project 2009CR0021), Ministerio de Ciencia e Innovación, Madrid, Spain (grant BFU2010-17373), Generalitat Valenciana, Valencia, Spain (grant PROMETEO/2010/005), the PNG Office of Higher Education, CTP Limited (Milne Bay Estates), and the Australian Venom Research Unit (University of Melbourne), which is funded by the Australian Government Department of Health and Ageing, the Australia Pacific Science Foundation and Snowy Nominees. Analyses performed at the Proteomics Laboratory of Instituto Clodomiro Picado were supported by CONARE and Vicerrectoría de Investigación, Universidad de Costa Rica.

REFERENCES [1] White J. Clinical toxicology of snakebite in Australia and New Guinea. In: Meier J, White J, editors. Handbook of clinical toxicology of animal venoms and poisons. Boca Raton: CRC Press; 1995. p. 595–617. [2] Broad AJ, Sutherland SK, Coulter AR. The lethality in mice of dangerous Australian and other snake venoms. Toxicon 1979;17:661–4.

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 21 2 8 –2 14 0

[3] Lalloo DG, Trevett AJ, Korinhona A, Nwokolo N, Laurenson IF, Paul M, et al. Snake bites by the Papuan taipan (Oxyuranus scutellatus canni): paralysis, hemostatic and electrocardiographic abnormalities, and effects of antivenom. 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. Venomous bites and stings in Papua New Guinea: a treatment guide for health workers and doctors. Melbourne: University of Melbourne; 2005. p. 5–32. [5] O'Shea M. A guide to the snakes of Papua New Guinea. Port Moresby: Independent Publishing; 1996. [6] Wüster W, Dumbrell AJ, Hay C, Pook CE, Williams DJ, Fry BG. Snakes across the Strait: Trans-Torresian phylogeographic relationships in three genera of Australasian snakes (Serpentes: Elapidae: Acantophis, Oxyuranus, and Pseudechis). Mol Phylogenet Evol 2005;34:1–14. [7] Doughty P, Maryan B, Donnellan SC, Hutchinson MN. A new species of taipan (Elapidae: Oxyuranus) from central Australia. Zootaxa 2007;1422:45–58. [8] Slater KR. On the New Guinea taipan. Mem Natl Mus Vic 1956;20:201–5. [9] Connolly S, Trevett AJ, Nwokolo NC, Lalloo DG, Naraqi S, Mantle D, et al. Neuromuscular effects of Papuan Taipan snake 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 patients bitten by taipans (Oxyuranus scutellatus canni) and other elapid snakes in Papua New Guinea. Trans R Soc Trop Med Hyg 1997;91:53–6. [12] Fohlman J, Eaker D, Karlsson E, Thesleff S. Taipoxin, an extremely potent presynaptic neurotoxin from the venom of the Australian snake taipan (Oxyuranus s. scutellatus). Isolation, characterization, quaternary structure and pharmacological properties. Eur J Biochem 1976;68:457–69. [13] Kuruppu S, Reeve S, Banerjee Y, Kini RM, Smith AI, Hodgson WC. Isolation and pharmacological characterization of cannitoxin, a presynaptic neurotoxin from the venom of the Papuan Taipan (Oxyuranus scutellatus canni). J Pharmacol Exp Ther 2005;315:1196–202. [14] Lambeau G, Barhanin J, Schweitz H, Qar J, Lazdunski M. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from 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 of Oxyuranus scutellatus (Taipan snake). J Biol Chem 1986;261: 13258–67. [16] Kornhauser R, Hart AJ, Reeve S, Smith AI, Fry BG, Hodgson WC. Variations in the pharmacological profile of post-synaptic neurotoxins isolated from the venoms of the Papuan (Oxyuranus scutellatus canni) and coastal (Oxyuranus scutellatus scutellatus) taipans. Neurotoxicology 2010;31: 239–43. [17] Possani LD, Martin BM, Yatani A, Mochca-Morales J, Zamudio FZ, Gurrola GB, et al. Isolation and physiological characterization of taicatoxin, a complex toxin with specific effects on calcium channels. Toxicon 1992;30:1343–64. [18] St Pierre L, Woods R, Earl S, Masci PP, Lavin MF. Identification and analysis of venom gland-specific genes from the coastal taipan (Oxyuranus scutellatus) and related species. Cell Mol Life 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 the functional diversity of snake toxins associated with a

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

2139

mechanism of accelerated evolution? In: Moldave K, editor. Progress in nucleic acid research and molecular biology. New York: Academic Press; 1998. p. 307–64. Ohno M, Chijiwa T, Oda-Ueda N, Ogawa T, Hattori S. Molecular evolution of myotoxic phospholipases A2 from snake venom. Toxicon 2003;42:841–54. Fry B, Scheib H, van der Weerd L, Young B, McNaughtan J, Ramjan SF, et al. Evolution of an arsenal: structural and functional diversification of the venom system in the advanced snakes (Caenophidia). Mol Cell Proteomics 2008;7: 215–46. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Res 1997;25: 3389–402. World Health Organization. Progress in the characterization of venoms and standardization of antivenoms. WHO Offset Publication, No. 58. Geneva: World Health Organization; 1981. Theakston RDG, Reid HA. Development of simple standard assay procedures for the characterization of snake venoms. Bull World Health Org 1983;61:949–56. Vargas M, Segura A, Herrera M, Villalta M, Estrada R, Cerdas M, et al. Preclinical evaluation of caprylic acid-fractionated IgG antivenom for the treatment of Taipan (Oxyuranus scutellatus) envenoming in Papua New Guinea. PLoS Negl Trop Dis 2011;5:e1144. Gutiérrez JM, Lomonte B, Chaves F, Moreno E, Cerdas L. Pharmacological activities of a toxic phospholipase A isolated from the venom of the snake Bothrops asper. Comp Biochem Physiol C 1986;84:159–64. Montecucco C, Rossetto O. On the quaternary structure of taipoxin and textilotoxin: the advantage of being multiple. Toxicon 2008;51:1560–2. Cull-Candy SG, Fohlman J, Gustavsson D, Lüllmann-Rauch R, Thesleff S. The effects of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction. Neuroscience 1976;1:175–80. Harris JB, Grubb BD, Maltin CA, Dixon R. The neurotoxicity of the venom phospholipases A2, notexin and taipoxin. Exp Neurol 2000;161:517–26. Paoli M, Rigoni M, Koster G, Montecucco C, Postle AD. Mass spectrometry analysis of the phospholipase A2 activity of snake pre-synaptic neurotoxins in cultured neurons. J Neurochem 2009;111:737–44. Harris JB, Maltin CA. Myotoxic activity of the crude venom and the principal neurotoxin, taipoxin, of the Australian taipan, Oxyuranus scutellatus. Br J Pharmacol 1982:61–75. Fohlman J, Lind P, Eaker D. Taipoxin, an extremely potent presynaptic snake venom neurotoxin. Elucidation of the primary structure of the acidic carbohydrate-containing taipoxin subunit, a phospholipase homolog. FEBS Lett 1977;84:367–71. Lind P. Amino-acid sequence of the β1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan (Oxyuranus s. scutellatus). Eur J Biochem 1982;128:71–5. Lind P, Eaker D. Amino-acid sequence of the α-subunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan (Oxyuranus s. scutellatus). Eur J Biochem 1982;124:441–7. Bon C. Multicomponent neurotoxic phospholipases A2. In: Kini RM, editor. Venom phospholipase A2 enzymes. Structure, function and mechanism. Chichester: Wiley; 1997. p. 269–85. 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 nicotinic acetylcholine receptors in brain and skeletal muscle. Biochemistry 1996;35:7910–6.

2140

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 21 2 8 –21 4 0

[37] Lambeau G, Cupillard L, Lazdunski M. Membrane receptors for venom phospholipases A2. In: Kini RM, editor. Venom phospholipase A2 enzymes. Structure, function and mechanism. Chichester: Wiley; 1997. p. 389–412. [38] Lambeau G, Lazdunski M. Receptors for a growing family of secreted 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 pharmacological activities of the snake venom phospholipase A2 OS2: the N-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 tetrapod vertebratesSan 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 Coastal Taipan, Oxyuranus scutellatus (Peters, 1867). Toxicon 2006;48: 422–8. [43] Sorimachi H, Ishiura S, Suzuki K. Structure and physiological function of calpains. Biochem J 1997;328:721–32. [44] Gutiérrez JM, Ownby CL. Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity. Toxicon 2003;42:915–31. [45] Lomonte B, Angulo Y, Sasa M, Gutiérrez JM. The phospholipase A2 homologues of snake venoms: biological activities and their possible adaptive roles. Protein Pept Lett 2009;16:860–76. [46] Kini RM. The intriguing world of prothrombin activators from snake venom. Toxicon 2005;45:1133–45. [47] Fantini E, Athias P, Tirosh R, Pinson A. Effect of taicatoxin (TCX) on the electrophysiological, mechanical and biochemical characteristics of spontaneously beating ventricular cardiomyocytes. Mol Cell Biochem 1996;160–161: 61–6. [48] Doorty KB, Bevan S, Wadsworth JD, Strong PN. A novel small conductance Ca2 +-activated K+ channel blocker from Oxyuranus scutellatus taipan venom. Re-evaluation of taicatoxin as a selective Ca2 + channel probe. J Biol Chem 1997;272:19925–30. [49] Nawarak J, Sinchaukul S, Wu MY, Phutrakul S, Chen ST. Proteomics of snake venoms from Elapidae and Viperidae families 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 naja atra 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 the Thai 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 the Central American coral snake, Micrurus nigrocinctus (Elapidae). J Proteome Res 2011;10:1816–27.

[53] Fox JW, Serrano SMT. Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases. Toxicon 2005;45: 969–85. [54] Gutiérrez JM, Rucavado A, Escalante T. Snake venom metalloproteinases. Biological roles and participation in the pathophysiology of envenomation. In: Mackessy SP, editor. Handbook of venoms and toxins of reptiles. Boca Raton: CRC Press; 2010. p. 115–38. [55] Ward CM, Vinogradov DV, Andrews RK, Berndt MC. Characterization of mocarhagin, a cobra venom metalloproteinase from Naja mocambique mocambique, and related proteins from other Elapidae venoms. Toxicon 1996;34:1203–6. [56] Hamako J, Matsui T, Nishida S, Nomura S, Fujimura Y, Ito M, et al. Purification and characterization of kaouthiagin, a von Willebrand factor-binding and -cleaving metalloproteinase from Naja kaouthia cobra venom. Thromb Haemost 1998;80: 499–505. [57] Sun QY, Bao J. Purification, cloning and characterization of a metalloproteinase 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 venom metalloproteinases with distinct activities highlight the disulfide patterns in the D domain of ADAMalysin family proteins. J Struct Biol 2010;169:294–303. [59] Weissenberg S, Ovadia M, Kochva E. Species specific sensitivity towards the hemorrhagin of Ophiophagus hannah (Elapidae). Toxicon 1987;25:475–81. [60] Gao R, Kini RM, Gopalakrishnakone P. A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization. Arch Biochem Biophys 2002;408:87–92. [61] Yamazaki Y, Brown RL, Morita T. Purification and cloning of toxins from elapid venoms that target cyclic nucleotide-gated ion channels. Biochemistry 2002;41:11331–7. [62] Brown RL, Lynch LL, Haley TL, Arsanjani R. Pseudechetoxin binds to the pore turret of cyclic nucleotide-gated ion channels. 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 the venom of the inland taipan (Oxyuranus microlepidotus): isolation, chemical and biological characterization. Biochem Biophys 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’, a prothrombin-activating metalloproteinase, from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis. Arch Biochem Biophys 2003;418:13–24. [65] Morrison JJ, Peam JH, Coulter AR. The mass of venom injected by two Elapidae: the taipan (Oxyuranus scutellatus) and the Australian 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.