A Neurotoxic Snake Venom without Phospholipase A2: Proteomics and Cross-Neutralization of the Venom from Senegalese Cobra, Naja senegalensis (Subgenus: Uraeus)

Abstract

The Senegalese cobra, Naja senegalensis, is a non-spitting cobra species newly erected from the Naja haje complex. Naja senegalensis causes neurotoxic envenomation in Western Africa but its venom properties remain underexplored. Applying a protein decomplexation proteomic approach, this study unveiled the unique complexity of the venom composition. Three-finger toxins constituted the major component, accounting for 75.91% of total venom proteins. Of these, cardiotoxin/cytotoxin (~53%) and alpha-neurotoxins (~23%) predominated in the venom proteome. Phospholipase A₂, however, was not present in the venom, suggesting a unique snake venom phenotype found in this species. The venom, despite the absence of PLA₂, is highly lethal with an intravenous LD₅₀ of 0.39 µg/g in mice, consistent with the high abundance of alpha-neurotoxins (predominating long neurotoxins) in the venom. The hetero-specific VINS African Polyvalent Antivenom (VAPAV) was immunoreactive to the venom, implying conserved protein antigenicity in the venoms of N. senegalensis and N. haje. Furthermore, VAPAV was able to cross-neutralize the lethal effect of N. senegalensis venom but the potency was limited (0.59 mg venom completely neutralized per mL antivenom, or ~82 LD₅₀ per ml of antivenom). The efficacy of antivenom should be further improved to optimize the treatment of cobra bite envenomation in Africa.

Keywords: Naja (Uraeus) senegalensis; Naja haje complex; antivenom neutralization; immunoreactivity; snakebite envenomation; venomics.

Figure 1

Geographical distribution of Naja senegalensis in Western Africa, encompassing Senegal, Gambia, Mali, Burkino Faso, Ghana, Benin, Guinea-Bissau, Nigeria, and the Ivory Coast (yellow region) [5]. Insets: Naja senegalensis from the subgenus of Uraeus, elevated as a new species from the Naja haje complex. Note the distinct hood marking which is uncommon in African but Asiatic cobras. Snake photograph was courtesy of Laurent Chirio with copyright © Magnelia Press 2009 [12].

Figure 2

Reverse-phase high-performance liquid chromatography of Naja senegalensis venom. Chromatogram of the venom fractionation (top panel) and separation of venom fractions using SDS-PAGE under reducing condition (bottom panel). M, protein ladder for estimating the molecular weight of sample proteins.

Figure 3

Snake venom proteome of Naja senegalensis. Proteomic profiling was accomplished with QTOF nano-ESI-LCMS/MS of reverse-phase HPLC-fractionated venom proteins. All protein samples were subjected to in-solution tryptic digestion. Abbreviations: CTX, cytotoxin/cardiotoxin; SNTX, short neurotoxin; LNTX, long neurotoxin; WTX, weak neurotoxin; CRISP, cysteine-rich secretory protein; CVF, cobra venom factor; KSPI, Kunitz-type serine protease inhibitor; PDE, phosphodiesterase; SVMP, snake venom metalloproteinase; 5′-NUC, 5′-nucleotidase. CTX, SNTX, LNTX, and WTX constituted three-finger toxins (3FTX) superfamily.

Figure 4

Immunological binding activity of VINS African Polyvalent Antivenom (VAPAV) toward N. senegalensis and N. haje venoms. The immunological binding activity was represented as half the maximum concentration (EC₅₀). Values were means ± S.E.M. of triplicates.