Elsevier

Journal of Proteomics

Volume 75, Issue 7, 3 April 2012, Pages 2128-2140
Journal of Proteomics

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

https://doi.org/10.1016/j.jprot.2012.01.006Get rights and content

Abstract

The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A2 (PLA2), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA2s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed 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.

Graphical abstract

Highlights

► The proteomes of venoms of taipan from Australia and Papua New Guinea were compared. ► Phospholipase A2 was the most abundant component in both venoms. ► Monospecific antivenom neutralized effectively venoms from the two populations. ► Neurotoxicity and thrombosis are relevant mechanisms for killing prey.

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 central-northern South Australia [7]; and (c) Oxyuranus temporalis, known from a handful of specimens collected in the south-eastern deserts of Western Australia [7]. O. scutellatus occurs 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.

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], [20], [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.

Section snippets

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

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 Fig. 1, Fig. 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

Acknowledgments

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

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