Proteomic Deep Mining the Venom of the Red-Headed Krait, Bungarus flaviceps

Abstract

The use of -omics technologies allows for the characterization of snake venom composition at a fast rate and at high levels of detail. In the present study, we investigated the protein content of Red-headed Krait (Bungarus flaviceps) venom. This analysis revealed a high diversity of snake venom protein families, as evidenced by high-throughput mass spectrometric analysis. We found all six venom protein families previously reported in a transcriptome study of the venom gland of B. flaviceps, including phospholipases A₂ (PLA₂s), Kunitz-type serine proteinase inhibitors (KSPIs), three-finger toxins (3FTxs), cysteine-rich secretory proteins (CRISPs), snaclecs, and natriuretic peptides. A combined approach of automated database searches and de novo sequencing of tandem mass spectra, followed by sequence similarity searches, revealed the presence of 12 additional toxin families. De novo sequencing alone was able to identify 58 additional peptides, and this approach contributed significantly to the comprehensive description of the venom. Abundant protein families comprise 3FTxs (22.3%), KSPIs (19%), acetylcholinesterases (12.6%), PLA₂s (11.9%), venom endothelial growth factors (VEGFs, 8.4%), nucleotidases (4.3%), and C-type lectin-like proteins (snaclecs, 3.3%); an additional 11 toxin families are present at significantly lower concentrations, including complement depleting factors, a family not previously detected in Bungarus venoms. The utility of a multifaceted approach toward unraveling the proteome of snake venoms, employed here, allowed detection of even minor venom components. This more in-depth knowledge of the composition of B. flaviceps venom facilitates a better understanding of snake venom molecular evolution, in turn contributing to more effective treatment of krait bites.

Keywords: Bungarus flaviceps; Red-headed Krait; enzymes; proteome; snake venom; toxins.

Figure 1

Approximate distribution of the Red-headed Krait (Bungarus flaviceps). Inset A shows the extraction of venom from B. flaviceps, a low-yielding front-fanged snake. Distributions are adapted from the literature ([17], 2010, New Holland Publishers) and the map is from Google Earth.

Figure 2

One-dimensional gel of the crude venom of B. flaviceps. Prominent venom protein families (boxed) are based on their molecular masses and confirmed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) (see also Table S1). Bands were excised for further analysis. PDE—phosphodiesterases; AChE—acetylcholinesterases; LAAO—L-amino-acid oxidase; 5’-NUC—5’inucleotidase; SVMP—snake venom metalloproteinase; PLB—phospholipase B; CRISP—cysteine-rich seceretory proteins; PLA₂—phospholipase A; KUN—Kunitz-type serine proteinase inhibitor; 3FTx—three-finger toxin.

Figure 3

(A) Abundances of the venom protein families of B. flaviceps as evidenced by normalized mass spectrometric spectral count. (B) Comparison of abundances of venom protein families (B. flaviceps) by transcriptomic (red, adapted from [25], 2010, Springer Nature) and proteomic analysis. KSPI—Kunitz-type serine proteinase inhibitors; VEGF—vascular endothelial growth factor; VNGF—venom nerve growth factor; SVSP—snake venom serine proteinase; CVF—cobra venom factor.