Isolation and Characterization of A2-EPTX-Nsm1a, a Secretory Phospholipase A2 from Malaysian Spitting Cobra (Naja sumatrana) Venom

Abstract

Phospholipase A2 (PLA2) toxins are one of the main toxin families found in snake venom. PLA2 toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although Naja sumatrana venom contains substantial quantities of PLA2 components, there is limited information on the function and activities of PLA2 toxins from the venom. In this study, a secretory PLA2 from the venom of Malaysian N. sumatrana, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA2 from venoms of other Naja species. The PLA2 activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA2 isolated from Malaysian N. sumatrana venom in a human neuroblastoma cell line.

In Vitro neurotoxicity and myotoxicity of Malaysian Naja sumatrana and Naja kaouthia venoms: Neutralization by monovalent and Neuro Polyvalent Antivenoms from Thailand

“…Cytotoxicity of N. sumatrana and N. kaouthia venoms has been previously reported using tissue-based, in vivo, and cell-based assays [28][29][30][31]. However, information on the myotoxicity caused by N. sumatrana venom is relatively limited compared to N. kaouthia venom [32,33].…”

Neuroprotective effect of phospholipase A2 from Malaysian Naja sumatrana venom against H2O2-induced cell damage and apoptosis

“…Interestingly, secretory PLA 2 from Naja sumatrana venom (named A2-EPTX-NSm1a) maintained cell viability of differentiated SH-SY5Y cells (Figure 2B). This could be due to its characteristic that have less toxicity to SH-SY5Y than the crude venom as previously reported (Abdullah et al, 2021). We further evaluated the potential of PLA 2 compound as a neuroprotectant against H 2 O 2 .…”

“…This study determined the potential neuroprotective activity of a PLA 2 from Malaysian N. sumatrana venom, A2-EPTX-NSm1a, using in-vitro experiments. Our previous study found that A2-EPTX-NSm1a was less cytotoxic in SH-SY5Y as compared to N. sumatrana crude venom (Abdullah et al, 2021), suggesting A2-EPTX-NSm1a may exert other activities in neuronal cells.…”

“…All chemical reagents used in the present study were analytical grades. Items purchased for isolation and purification of secretory phospholipase A 2 are stated in a previous study (Abdullah et al, 2021). Culture medium and fetal bovine serum were purchased from Gibco ® by Life Technologies ™ , Massachusetts, US.…”

“…Cells were seeded at density 10,000 cells/cm 2 in 24 well plates (NEST ® Biotechnology Co., Ltd., Rahway, US) and allowed to differentiate until day 8. Cells with 70-80% confluency were treated with A2-EPTX-NSm1a (0-15.265 μg/ml) for 24, 48, and 72 h. The concentration range used in this study was based on a previous cytotoxicity assay of A2-EPTX-NSm1a (Abdullah et al, 2021). MTT solution with final concentration of 0.05 mg/ml was added to each well, and the cells were further incubated for 4 h at 37 °C and 5% CO 2 .…”