Elsevier

Journal of Proteomics

Volume 91, 8 October 2013, Pages 338-343
Journal of Proteomics

Proteomic comparison of Hypnale hypnale (Hump-Nosed Pit-Viper) and Calloselasma rhodostoma (Malayan Pit-Viper) venoms

https://doi.org/10.1016/j.jprot.2013.07.020Get rights and content

Highlights

  • Calloselasma rhodostoma and Hypnale hypnales venoms are differentially complex.

  • H. hypnale is dominated by PLA2.

  • This explains the failure of C. rhodostoma antivenom to neutralise H. hypnale venom.

Abstract

Treatment of Hypnale hypnale bites with commercial antivenoms, even those raised against its sister taxon Calloselasma rhodostoma, has never been clinically successful. As these two genera have been separated for 20 million years, we tested to see whether significant variations in venom had accumulated during this long period of evolutionary divergence, and thus could be responsible for the failure of antivenom. Proteomic analyses of C. rhodostoma and H. hypnale venom were performed using 1D and 2D PAGE as well as 2D-DIGE. C. rhodostoma venom was diverse containing large amounts of Disintegrin, Kallikrein, l-amino acid oxidase, Lectin, phospholipase A2 (acidic, basic and neutral) and Snake Venom Metalloprotease. In contrast, while H. hypnale also contained a wide range of toxin types, the venom was overwhelmingly dominated by two molecular weight forms of basic PLA2. 2D-DIGE (2-D Fluorescence Difference Gel Electrophoresis analysis) showed that even when a particular toxin class was shared between the two venoms, there were significant molecular weights or isoelectric point differences. This proteomic difference explains the past treatment failures with C. rhodostoma antivenom and highlights the need for a H. hypnale specific antivenom.

Biological significance

These results have direct implications for the treatment of envenomed patients in Sri Lanka. The unusual venom profile of Hypnale hypnale underscores the biodiscovery potential of novel snake venoms.

Introduction

The Hump-Nosed Pit-Viper (Hypnale hypnale) inhabits Sri Lanka and southern India and is classified by the World Health Organisation (WHO) as a snake of the highest medical importance in Southern Asia [1]. Contact between humans and H. hypnale is common due to its abundance and tolerance of disturbed habitat in both wet and dry deciduous zones. Its small size (rarely exceeding 0.5 m) and effective camouflage make the snake difficult to detect, and bites most often result from victims accidentally stepping on coming into contact with an unseen snake [2]. H. hypnale has been estimated to be responsible for 35% of venomous snake bites in Sri Lanka and envenomation causes local necrosis, pain and haemorrhagic blisters in most patients [3]. While H. hypnale bites are rarely lethal (fatality rate 1.8%), up to 40% of patients experience systemic effects including haemostatic dysfunction, thrombocytopenia, spontaneous haemorrhage and acute kidney injury [2], [3]. These effects have been replicated in mouse models, in which it has been demonstrated that H. hypnale venom has procoagulant, fibrinolytic, oedema-triggering and platelet aggregation activities [4], [5]. Recently, thrombotic microangiopathy has also been described in a number of H. hypnale bites, which may contribute to tissue damage at the bite site and the development of renal complications [6].

Despite the medical importance of this snake, effective venom neutralisation has not been achieved in humans. Treatment with Bharat polyvalent antivenom (ASVS) and Haffkine polyvalent antivenom, both raised against the “big four” Asian venomous snakes (Naja naja, Bungarus caeruleus, Daboia russelii and Echis carinatus), has been found to be completely ineffective against H. hypnale envenomation both in rodent assays and clinical envenomations [7]. Mitochondrial DNA analysis suggests that H. hypnale and the Malayan Pit-Viper Calloselasma rhodostoma form a phylogenetic clade, although they have been diverging from one another for approximately 20 million years [8]. As their envenomations show similar clinical features, the efficacy of Thai Red Cross Malayan Pit Viper (MPV) monovalent antivenom in neutralising H. hypnale venom has also been investigated. There is controversy in the literature regarding the effectiveness of MPV in supressing the haemorrhagic, procoagulant and necrotic activies of H. hypnale venom in rodent models [9], and it has never been successful in a clinical setting [2]. The Hemato polyvalent antivenom (HPA) produced against C. rhodostoma and two other haemotoxic Thai snakes, Cryptelytrops albolabris and Dixonius siamensis, has been demonstrated to abrogate the lethality of H. hypnale venom in rats, but has not yet been clinically trialled [9].

Both MPV and HPA are substantially more effective in neutralising C. rhodostoma venom than that of H. hypnale, suggesting that some toxins present in the latter venom may not be neutralised [9]. However, immunological profiling of H. hypnale venom using indirect ELISA with antisera revealed 90% cross-reactivity with C. rhodostoma venom [10]. C. rhodostoma venom toxins have been comprehensively described and include the metalloproteases kistomin and rhodostoxin [11]; multiple phospholipase A2 isoforms (PLA2) [12]; l-amino oxidases [13]; C-type lectins [14] and serine proteases such as Ancrod, which is being trialled as a clinical anticoagulant (under the brand name Viprinex) [15]. These toxins act in concert to produce haemostatic dysfunction: metalloproteases and serine proteases cleave or inhibit fibrinogen, while enzymatic PLA2 isoforms and C-type lectins inhibit platelet aggregation. Non-enzymatic PLA2 isoforms have been shown to be myotoxic and to cause oedema in vivo [5]. l-amino oxidases trigger deamination cascades that produce hydrogen peroxide, leading to a variety of clinical sequelae including haemorrhage, haemolysis, oedema and apoptosis of vascular endothelial cells [13]. An enzymatic comparison with C. rhodostoma venom found that H. hypnale venom had similar protease, l-amino acid oxidase, thrombin-like enzyme and hemorrhagin activities but much higher levels of PLA2 activity [9], consistent with the fact that PLA2 isoforms are readily purified from the venom [5].

Gaining a better understanding of the venom composition of H. hypnale may be invaluable in the development of more effective treatments for envenomation in addition to providing insight into divergent venom evolution subsequent to geographical separation and speciation of snakes as well as highlighting potential regional variations in H. hypnale venom. Additionally, as snake venoms are a rich source of pharmacologically active proteins and peptides, the comprehensive characterisation of venom proteomes is an important avenue of biodiscovery [16]. We have previously shown that comparative 2D-PAGE gel with in-gel protein digestion followed by MS/MS sequencing is an effective way to compare snake venom profiles [17]. In this paper we compare the proteomic profiles of C. rhodostoma and H. hypnale, providing insight into compositional differences predicted by previous paraspecific antivenom cross-neutralisation studies.

Section snippets

Venom samples

Venom samples were obtained from a single captive bred specimen each of H. hypnale from Sri Lanka and C. rhodostoma from Malaysia. After milking by BGF, samples were immediately placed in liquid nitrogen until being lyophilised and stored at − 70 °C.

1D gel, 2D gel, Shotgun sequencing, LC-MS/MS conditions were as previously described by us [17].

Differential In-Gel Analysis (2D DIGE)

For DIGE Cy-Dye labelling, venom samples were first cleaned and quantified by 2-D Clean-up and 2-D-Quant Kits (GE Healthcare) respectively. 50 μg of the two

Results and discussion

In this study we undertook the first detailed proteomic characterisation of H. hypnale venom and provided a comparison with the sister-taxon C. rhodostoma venom using 1D and 2D PAGE, as well as DIGE. The combined proteomics approaches revealed a diverse composition of venom components (Fig. 1, Fig 2, Fig. 3, Fig. 4, summarised in Table 1, full-details in Supplementary Table 1, Supplementary Table 2, Supplementary Table 3, Supplementary Table 4, Supplementary Table 5, Supplementary Table 6).

Acknowledgements

SAA was the recipient of postdoctoral fellowship (PDRF Phase II Batch-V) from Higher Education Commission (HEC Islamabad) Pakistan. BGF was funded by an Australian Research Council Future Fellowship and by the University of Queensland. TNWJ was funded by an Australian Postgraduate Award. EABU acknowledges funding from the University of Queensland (International Postgraduate Research Scholarship, UQ Centennial Scholarship, and UQ Advantage Top-Up Scholarship) and the Norwegian State Education

References (26)

Cited by (28)

  • Comparative proteomes, immunoreactivities and neutralization of procoagulant activities of Calloselasma rhodostoma (Malayan pit viper) venoms from four regions in Southeast Asia

    2019, Toxicon
    Citation Excerpt :

    Hemorrhagic SVMPs are known to disrupt the mechanical stability of vascular walls, leading to erythrocytes extravasation (Fox and Serrano, 2005; Gutiérrez et al., 2009b; Herrera et al., 2015). Some geographical variations were noted, for example, the CR-V venom has a lower SVMP content compared to CR-M, CR-I and CR-T. All classes of SVMP, i.e. P–I, P-II and P-III were detected in the four C. rhodostoma venom samples, consistent with the presence of the three different SVMP classes (P–I, P-II and P-III) reported previously in other C. rhodostoma venom samples of Malaysian origin (by qualitative detection without differential quantitiation) (Ali et al., 2013; Kunalan et al., 2018). The present proteomic study analyzed the relative protein abundances and further showed that all four venoms from Malaysia, Indonesia, Thailand and Vietnam have P–I subtype as the most dominant.

View all citing articles on Scopus
1

Shared-first authorship.

View full text