Deep venomics of the Pseudonaja genus reveals inter- and intra-specific variation

Abstract

Australian elapid venom remains an under-investigated resource of novel bioactive peptides. In this study, the venom gland transcriptomes and proteomes of the Australian western brown snakes, Pseudonaja aspidorhyncha and Pseudonaja nuchalis, were compared to Pseudonaja textilis. A deep venomics strategy incorporating high throughput 454 pyrosequencing gave a total of 200,911 raw reads for the three venoms. Subsequent annotation identified 5716 transcripts from 20 different toxin families with inter-specific variation between species observed in eight of the less abundant families. Integration of each venom proteome with the corresponding annotated reads identified 65 isoforms from six toxin families; high sequence coverage highlighted subtle differences between sequences and intra and inter-specific variation between species. High quality MS/MS data identified unusual glycoforms with natriuretic peptides from P. aspidorhyncha and P. nuchalis containing O-linked trisaccharides with high homology to the glycosylated region of TNPc. Molecular evolutionary assessments indicated the accelerated evolution of all toxin families with the exception of both natriuretic peptides and P. aspidorhyncha PLA2s that were found to be evolutionarily constrained under purifying selection pressures. This study has revealed a wide range of novel peptide sequences from six bioactive peptide families and highlights the subtle differences between toxins in these closely related species. Biological SignificanceMining Australia's vastly untapped source of toxins from its venomous creatures has been significantly advanced by employing deep venomics methodology.Technological advances in transcriptome analysis using next generation sequencing platforms and proteome analysis by highly sensitive tandem mass spectrometry allowed a more comprehensive interrogation of three underinvestigated brown snake (Pseudonaja) venoms uncovering many novel peptide sequences that are unique to these closely related species. This generic strategy will provide invaluable information when applied to other venomous snakes for a deeper understanding of venom composition, envenomation, venom evolution, as well as identifying research tools and drug leads.

Correlation between ontogenetic dietary shifts and venom variation in Australian brown snakes ( Pseudonaja )

“…The Factor Xa assay ( Figure 1A) confirmed the possession of this activity by the venom of adults of all species of Pseudonaja barring P. modesta, corroborating previous proteomic, transcriptomic, pharmacological and clinical data (White et al, 1986;Jackson et al, 2013;2016).…”

“…textilis from venom purely composed of peptide toxins in juvenile snakes to procoagulant venom including the prothrombinase toxin complex in adults (Jackson et al 2016). In contrast, P. modesta maintains a venom profile similar to the juveniles of other species into adulthood, and neither clots plasma in vitro or expresses the prothrombinase complex in its venom gland transcriptome (Jackson et al 2013;2016). As coagulopathy is typically the most significant of the clinical sequelae resulting from human envenomation by Pseudonaja, it is the adult venom of the mammal feeders that is largely responsible for the clinical importance of brown snake envenomations.…”

Trends in the Evolution of Snake Toxins Underscored by an Integrative Omics Approach to Profile the Venom of the ColubridPhalotris mertensi

“…The protein and peptide composition of the venoms from a number of dangerous snake species have been extensively studied by means of high-throughput technologies such as transcriptomics, proteomics, and peptidomics (Durban et al 2011; Tashima et al 2012; Aird et al 2013; Rokyta et al 2013; Hargreaves et al 2014; Lomonte et al 2014; Margres et al 2014; Mcgivern et al 2014, 2015; Chapeaurouge et al 2015; Junqueira-de-Azevedo et al 2015; Petras et al 2015; Viala et al 2015; Reeks et al 2016; Tan et al 2016). …”

Venoms of related mammal-eating species of taipans (Oxyuranus) and brown snakes (Pseudonaja) differ in composition of toxins involved in mammal poisoning

“…Venoms of these elapids contain proteins belonging to several protein families [21][22][23]. This project focused on expression patterns of proteins that have been shown experimentally to be toxic to rodents and are presumed to have a role in mammal prey incapacitation.…”