RESUMO
BACKGROUND AND OBJECTIVE: Snakebite envenoming is a serious public health issue causing more than 135,000 annual deaths worldwide. Naja naja oxiana is one of the most clinically important venomous snakes in Iran and Central Asia. Conventional animal-derived polyclonal antibodies are the major treatment of snakebite envenoming. Characterization of venom components helps to pinpoint the toxic protein responsible for clinical manifestations in victims, which aids us in developing efficient antivenoms with minimal side effects. Therefore, the present study aimed to identify the major lethal protein of Naja naja oxiana by top-down proteomics. METHODS: Venom proteomic profiling was performed using gel filtration (GF), reversed-phase (RP) chromatography, and intact mass spectrometry. The toxicity of GF-, and RP-eluted fractions was analyzed in BALB/c mice. The rabbit polyclonal antisera were produced against crude venom, GF fraction V (FV), and RP peak 1 (CTXP) and applied in neutralization assays. RESULTS: Toxicity studies in BALB/c identified FV as the major toxic fraction of venom. Subsequently, RP separation of FV resulted in eight peaks, of which peak 1, referred to as "CTXP" (cobra toxin peptide), was identified as the major lethal protein. In vivo neutralization assays using rabbit antisera showed that polyclonal antibodies raised against FV and CTXP are capable of neutralizing at least 2-LD50s of crude venom, FV, and CTXP in all tested mice. CONCLUSION: Surprisingly, the Anti-CTXP antibody could neutralize 8-LD50 of the CTXP peptide. These results identified CTXP (a 7 kDa peptide) as a potential target for the development of novel efficient antivenom agents.
RESUMO
Hemoscorpius lepturus is the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation are remarkably similar to those reported for brown spiders, including dermonecrosis, hematuria, renal failure and even death. The lethality and toxicity of brown spiders' venom have been attributed to its phospholipase D activity. This study aims to identify a phospholipase D with possible lethality and dermonecrotic activity in H. lepturus venom. In this study, a cDNA library of the venom glands was generated by Illumina RNA sequencing. Phospholipase D (PLD) from H. lepturus was characterized according to its significant similarity with PLDs from brown spiders. The main chain designated as Hl-RecPLD1 (the first recombinant isoform of H. lepturus PLD) was cloned, expressed and purified. Sphingomyelinase, dermonecrotic and lethal activities were examined. Hl-PLD1 showed remarkable sequence similarity and structural homology with PLDs of brown spiders. The conformation of Hl-PLD1 was predicted as a "TIM beta/alpha-barrel". The lethal dose 50 (LD50) and dermonecrotic activities of Hl-RecPLD1 were determined as 3.1 µg/mouse and 0.7 cm2 at 1 µg respectively. It is the first report indicating that a similar molecular evolutionary mechanism has occurred in both American brown spiders and this Iranian scorpion. In conclusion, Hl-RecPLD1 is a highly active phospholipase D, which would be considered as the lethal dermonecrotic toxin in H. lepturus venom.
