Venom Proteome of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia: Toxicity Correlation, Immunoprofiling and Cross-Neutralization by Sea Snake Antivenom

Abstract

The venom proteome of Hydrophis curtus (synonym: Lapemis hardwickii) from Penang, Malaysia was investigated with nano-electrospray ionization-liquid chromatography tandem mass spectrometry (ESI-LCMS/MS) of the reverse-phase high-performance liquid chromatography (HPLC) venom fractions. Thirty distinct protein forms were identified as toxins from ten families. The three major protein families were phospholipase A₂ (PLA₂, 62.0% of total venom proteins), three-finger toxin (3FTX, 26.33%) and cysteine-rich secretory protein (CRiSP, 9.00%). PLA₂ comprises diverse homologues (11 forms), predominantly the acidic subtypes (48.26%). 3FTX composed of one short alpha-neurotoxin (SNTX, 22.89%) and four long alpha-neurotoxins (LNTX, 3.44%). Both SNTX and LNTX were lethal in mice (intravenous LD₅₀ = 0.10 and 0.24 μg/g, respectively) but the PLA₂ were non-lethal (LD₅₀ >1 μg/g). The more abundant and toxic SNTX appeared to be the main driver of venom lethality (holovenom LD₅₀ = 0.20 μg/g). The heterologous Sea Snake Antivenom (SSAV, Australia) effectively cross-neutralized the venom (normalized potency = 9.35 mg venom neutralized per g antivenom) and the two neurotoxins in vivo, with the LNTX being neutralized more effectively (normalized potency = 3.5 mg toxin/g antivenom) than SNTX (normalized potency = 1.57 mg/g). SSAV immunorecognition was strong toward PLA₂ but moderate-to-weak toward the alpha-neurotoxins, indicating that neutralization of the alpha-neurotoxins should be further improved.

Keywords: Lapemis hardwickii; alpha-neurotoxins; envenomation; immunoreactivity; neutralization; phospholipase A2; three-finger toxins.

Figure 1

(A) Spine-bellied sea snake, Hydrophis curtus from Penang Island, Malaysia. (B) Protruding spine-like structures on the ventral scales of the snake. (C) Lyophilized H. curtus venom in whitish crystalline form. (D) Decomplexation of H. curtus (Penang, Malaysia) venom with C18 reverse-phase HPLC. (E) 15% SDS-PAGE of H. curtus venom and its HPLC fractions under reducing conditions. L: Protein markers; C: Whole venom. Numbers indicate HPLC fractions.

Figure 2

Venom proteome of the spine-bellied sea snake, Hydrophis curtus from Penang Island, Malaysia. Percentages indicate relative protein abundance by total venom proteins.

Figure 3

Immunoprofiling of H. curtus venom and protein fractions by the heterologous Sea Snake Antivenom. F1–F10: Reverse-phase HPLC of H. curtus venom; HC: H. curtus venom; NS: Notechis scutatus venom; HS: Hydrophis schistosus venom; CR: Calloselasma rhodostoma venom.