(791.19) Homologues Phospholipases A2 isolated from Naja naja atra snake venom exhibit different activity against intact human erythrocytes

Poster Board Number: A100

Luis Cabrera Hernández (University of Puerto Rico Rio Piedras), Juan Boyne Mascarell (University of Puerto Rico Rio Piedras), Rafael Maldonado Hernández (University of Puerto Rico Humacao), Orestes Quesada (University of Puerto Rico Rio Piedras)

Phospholipases A2 (PLA2) catalyze the hydrolysis of fatty acid esters in the 2-position of 3-sn phospholipids and are found in intracellular and secreted forms. Secretory PLA2s (sPLA2s) are small proteins (13-15 kDa) classified into five groups based on primary structure differences. Despite their structural similarity, sPLA2s show a wide range of enzymatic and pharmacological activities. Most sPLA2s have high activity toward anionic membranes due to cationic residue presence on their binding recognition site (BRS). Within the same snake venom, several PLA2s can be found, which present differences in their ability to hydrolyze phospholipids in biological bilayers.

In this study, Chinese cobra, Naja naja atra (N. n. atra) venom was purified by Size Exclusion Chromatography using a Superdex 75 and cation exchange using a column packed with Carboxymethyl Cellulose (CM-Cellulose) to pre-separate protein components from the venom. Subsequently, fractions displaying PLA2 activity were pooled, concentrated, and applied to a GE mini-CM-Cellulose column coupled to the AKTA Fast Protein Liquid Chromatography (FPLC) system. The final purification presents four peaks but only two present PLA2 activity (Band D and E). Peak purity was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE).

We have shown that hydrophobic residue presence on the PLA2 BRS is crucial for binding and hydrolytic activity toward human erythrocyte membranes. PLA2 activity was assayed against human erythrocytes and monomeric substrate to determine differences in hydrolytic activity. Primary sequence alignment shows 94% homology, however Band E presented 40% less activity against human erythrocytes, despite having 100% active site homology. Molecular modeling of both sPLA2s revealed differences at the Cys-19 and Trp-19 position and spatial orientation differences at the Trp-61, Tyr-63 Phe-64, and Lys-65 positions. Due to amino acid differences at the BRS, differences in hydrolytic activity may be linked to the anchorage of sPLA2 to the erythrocyte surface.

This research was funded by the National Institute of General Medical Sciences (NIGMS) Research Training Initiative for Student Enhancement (RISE) Program, grant number 5R25GM061151-20.