Elsevier

Journal of Proteomics

Volume 89, 26 August 2013, Pages 15-23
Journal of Proteomics

Venom proteomic characterization and relative antivenom neutralization of two medically important Pakistani elapid snakes (Bungarus sindanus and Naja naja)

https://doi.org/10.1016/j.jprot.2013.05.015Get rights and content

Highlights

  • Pakistan Bungarus sindanus and Naja naja venoms are differentially complex.

  • Naja naja is potently myotoxic.

  • Neither venom is neutralized by Indian antivenom.

Abstract

Intra- and interspecific variation in venom composition has been shown to have a major effect upon the efficacy of antivenoms. Due to the absence of domestically produced antivenoms, Pakistan is wholly reliant upon antivenoms produced in other countries, such as India. However, the efficacy of these antivenoms in neutralising the venoms of Pakistani snakes has not been ascertained. This is symptomatic of the general state of toxicological research in this country, which has a myriad of highly toxic and medically important venomous animals. Thus, there is a dire need for knowledge regarding the fundamental proteomics of these venoms and applied knowledge of the relative efficacy of foreign antivenoms. Here we present the results of our proteomic research on two medically important snakes of Pakistan: Bungarus sindanus and Naja naja. Indian Polyvalent Antivenom (Bharat Serums and Vaccines Ltd), which is currently marketed for use in Pakistan, was completely ineffective against either Pakistani species. In addition to the expected pre- and post-synaptic neurotoxic activity, the venom of the Pakistan population of N. naja was shown to be quite divergent from other populations of this species in being potently myotoxic. These results highlight the importance of studying divergent species and isolated populations, where the same data not only elucidates clinical problems in need of immediate attention, but also uncovers sources for novel toxins with potentially useful activities.

Biological significance

Pakistan Bungarus sindanus and Naja naja venoms are differentially complex. Naja naja is potently myotoxic. Neither venom is neutralized by Indian antivenom. These results have direct implications for the treatment of envenomed patients in Pakistan. The unusually myotoxic effects of Naja naja demonstrates the value of studying remote populations for biodiscovery.

Introduction

The snakebite burden in Pakistan is difficult to determine accurately due to poor epidemiological record keeping and the fact that many victims prefer to rely on traditional remedies rather than going to hospital [1], [2]. However, it is readily apparent that snakebite is of major medical concern in this developing country [3] and that four snakes dominate the clinical landscape: two from the family Elapidae (Bungarus sindanus and Naja naja) and two from the family Viperidae (Daboia russelii and Echis carinatus sochureki). While each of these (or a close relative) has been well studied in other regions, it is a well-established general principle that geographical variation in venom profile may have a dramatic effect upon relative neutralization by antivenom [4], [5]. This is of particular concern for Pakistan, which relies upon foreign antivenoms such as those produced by India, despite their efficacy not having been ascertained for snakes in Pakistan.

Bungarus venoms have been shown to be rich in kunitz peptides, 3FTx (three finger toxins), PLA2 (phospholipase A2) and acetylcholinesterase. The 3FTx in Bungarus venoms are kappa-neurotoxins, which are disulphide-linked dimers that specifically target neuronal nicotinic acetylcholine receptors (cf. [2]). A different type of disulphide-linked dimer in the same venom is formed by the kunitz peptides and PLA2 (cf. [6]). These toxins are presynaptically neurotoxic with both the kunitz peptide (blockage of L-type calcium channels) and the PLA2 (destruction of membrane phospholipids) contributing to this toxicity (cf. [7]). The role of acetylcholinesterase in Bungarus venoms (cf. [8], [9]) is likely to further reduce the amount of available neurotransmitter [10].

Like Bungarus, Naja venoms have been shown to be rich in 3FTx and PLA2 but the specific pharmacology of the subclasses differs considerably. 3FTx in Naja venoms have been shown to be monomers that either block post-synaptic nicotinic acetylcholine receptors or are cytotoxic, while the PLA2 are presynaptically neurotoxic (cf. [10], [11]). Naja venoms are also rich in CVF (cobra venom factor), which is a mutated form of C3 (complement protein 3) that has been implicated in anaphylactic responses (cf. [12], [13]).

The polyvalent snake antivenom from India (Bharat Polyvalent Antivenom (BPAV)) has been previously revealed to have extremely poor cross-reactivity against the following non-Indian snakes [14]: Bungarus candidus (geographical locality not given), Bungarus fasciatus (geographical locality not given), Naja kaouthia (Malaysia and Thailand), N. naja (Sri Lanka), Naja siamensis (geographical locality not given), Naja philippinensis (geographical locality not given), Naja sputatrix (Thailand), Naja sumatrana (Malaysia) and Ophiophagus hannah (geographical locality not given). BPAV was only moderately effective against N. kaouthia (Thailand) and N. sumatrana venom but not against any of the other venoms tested. This antivenom even performed extremely poorly against the Indian population of N. naja. However, despite these conspicuously ineffective results, even against Indian snakes, this antivenom continues to be marketed to countries outside of India as a treatment for snakebite. Pakistan is one such targeted country.

In this study we compare the proteomic profiles of B. sindanus and N. naja venoms from Pakistan and determine the relative neutralization of these venoms by the Indian antivenin BPAV, as it is the most commonly available antivenom in Pakistan. The results not only contribute to the theoretical body of knowledge regarding venom diversification, but also have immediate implications for care of the envenomed patient.

Section snippets

Snake venoms and antivenom

B. sindanus and N. naja were collected from the Pakistan province of Sindh (districts Tharpakar and Sajawal, respectively). Venom was milked into sterile containers and stored at − 20 °C until use. The Indian polyvalent antivenom “BPAV” (i.e. serum globulins), raised against the venoms of the four most medically-significant snakes (listed as “cobra”, “common krait”, “Russell's viper” and “saw-scaled viper”) in the region (Reg. No: 053882, Mfg. Lic. No: KD-4, Batch No: A5311023, Mfg Date: 06/11,

Results and discussion

B. sindanus and N. naja venoms were quite different proteomically (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Table 1, Supplementary Table S1, Supplementary Table S2, Supplementary Table S3, Supplementary Table S4), both in types of toxins present and relative diversity within each class. Both venoms contained 3FTx, kunitz, PLA2, CRiSP and L-amino oxidase. Consistent with previous investigations of venoms from each genus, both venoms additionally contained large molecular weight proteins:

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 (28)

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