Characterization of a novel acidic phospholipase A 2 isolated from the venom of Bothrops mattogrossensis: From purification to structural modeling

• Published in: International journal of biological macromolecules 2024-12, Vol.292, p.139217
• Main Authors: de Melo Cordeiro Eulálio, Micaela, de Lima, Anderson Maciel, Brant, Rodrigo Soares Caldeira, Francisco, Aleff Ferreira, Santana, Hallison Mota, Paloschi, Mauro Valentino, da Silva Setúbal, Sulamita, da Silva, Carolina Pereira, Silva, Milena Daniela Souza, Boeno, Charles Nunes, Kayano, Anderson Makoto, Rita, Paula Helena Santa, de Azevedo Calderon, Leonardo, Soares, Andreimar Martins, Salvador, Daniela Priscila Marchi, Zuliani, Juliana Pavan
• Format: Article
• Language: English
• ISSN: 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.139217

Phospholipases A (PLA s) are highly prevalent in Bothrops snake venom and play a crucial role in inflammatory responses and immune cell activation during envenomation. Despite their significance, the specific role of PLA s from Bothrops mattogrossensis venom (BmV) in inflammation is not fully understood. This study sought to isolate and characterize a novel acidic PLA from BmV, designated BmPLA -A, and to evaluate its effects on human umbilical vein endothelial cells (HUVECs), with a specific focus on cytotoxicity, adhesion, and detachment. BmPLA -A was isolated through a multi-step chromatographic procedure, involving cation exchange (CM-Sepharose), hydrophobic interaction (n-butyl-Sepharose-HP), and reversed-phase (C-18) chromatography. SDS-PAGE analysis revealed a single protein band of approximately 15 kDa. The primary structure of BmPLA -A was determined by LC-MS/MS, while its tertiary structure was modeled using AlphaFold. Enzymatic activity was verified with the synthetic substrate 4N3OBA. Molecular dynamics simulations were conducted to further investigate the catalytic mechanism of BmPLA -A at the molecular level. In vitro assays on HUVECs revealed that BmPLA -A neither induce cytokine release (IL-6, IL-8, IL-1β, TNF) nor affected cell viability, adhesion, or detachment. The characteristics of BmPLA -A are consistent with those of acidic Asp-49 PLA enzymes, highlighting its potential involvement in the cytotoxic and inflammatory effects of the venom.