Equatorial Spitting Cobra (Naja sumatrana) from Malaysia (Negeri Sembilan and Penang), Southern Thailand, and Sumatra: Comparative Venom Proteomics, Immunoreactivity and Cross-Neutralization by Antivenom

Abstract

The Equatorial Spitting Cobra (Naja sumatrana) is a medically important venomous snake species in Southeast Asia. Its wide geographical distribution implies potential intra-specific venom variation, while there is no species-specific antivenom available to treat its envenoming. Applying a protein-decomplexing proteomic approach, the study showed that three-finger toxins (3FTX), followed by phospholipases A₂ (PLA₂), were the major proteins well-conserved across N. sumatrana venoms of different locales. Variations were noted in the subtypes and relative abundances of venom proteins. Of note, alpha-neurotoxins (belonging to 3FTX) are the least in the Penang specimen (Ns-PG, 5.41% of total venom proteins), compared with geographical specimens from Negeri Sembilan (Ns-NS, 14.84%), southern Thailand (Ns-TH, 16.05%) and Sumatra (Ns-SU, 10.81%). The alpha-neurotoxin abundance, in general, correlates with the venom's lethal potency. The Thai Naja kaouthia Monovalent Antivenom (NkMAV) was found to be immunoreactive toward the N. sumatrana venoms and is capable of cross-neutralizing N. sumatrana venom lethality to varying degrees (potency = 0.49-0.92 mg/mL, interpreted as the amount of venom completely neutralized per milliliter of antivenom). The potency was lowest against NS-SU venom, implying variable antigenicity of its lethal alpha-neurotoxins. Together, the findings suggest the para-specific and geographical utility of NkMAV as treatment for N. sumatrana envenoming in Southeast Asia.

Keywords: Naja kaouthia monovalent antivenom (NkMAV); antivenom potency; monocled cobra antivenom; snakebite envenoming; venom variation.

Figure 1

Protein decomplexation profiles of Naja sumatrana venoms sourced from (A) Negeri Sembilan, Malaysia, (B) Penang, Malaysia, (C) Southern Thailand, and (D) Sumatra, Indonesia. Upper panel: chromatographic profiles of venoms by C18 reverse-phase high-performance liquid chromatography. Lower panel: electrophoretic profiles of venom proteins separated by 15% sodium dodecyl sulfate-polyacrylamide gel under reducing conditions.

Figure 2

Venom proteomes of Naja sumatrana profiled using C18 RP-HPLC followed by tandem mass spectrometry for specimens from various geographical locales: (A) Negeri Sembilan, Malaysia (Ns-NS), (B) Penang, Malaysia (Ns-PG), (C) Southern Thailand (Ns-TH), and (D) Sumatra, Indonesia (Ns-SU). Abbreviation: 3FTx, three-finger toxin; PLA₂, phospholipase A₂; vNGF, venom nerve growth factor; KSPI, Kunitz-type serine protease; SVMP, snake venom metalloproteinase; CVF, cobra venom factor; PDE, phosphodiesterase; CRiSP, cysteine-rich secretory protein; LAAO, L-amino acid oxidase; SVSP, snake venom serine protease; HYA, Hyaluronidase; AChE, acetylcholinesterase; and 5′NUC, 5′nucleotidase. Bar graphs represent the sub-proteome of three-finger toxins in the venoms. Insets: Images of N. sumatrana with adaptations: A, www.reptilefact.com/wp-content/uploads/2016/08/Equatorial-Spitting-Cobras.jpg, accessed on 21 July 2022; B, author CHT; C, Joel Sartore; D, Gernot Vogel (www.reptile-database.reptarium.cz/species?genus=Naja&species=sumatrana, accessed on 21 July 2022).

Figure 3

Concentration-dependent immunological binding activity of Naja kaouthia Monovalent Antivenom (NkMAV) toward Naja sumatrana venoms from four different geographical locales. Naja kaouthia venom was used as positive control. Values were means ± S.E.M of triplicates.