Intestinal phospholipase A2 from Sparidae species: Functional properties and cytotoxic potential evaluation

Abstract

Marine species have gained significant attention as potential source for a broad spectrum of bioactive proteins. Fish phospholipases A₂ (PLA₂) have attracted renewed interest due to their excellent properties in lipid digestion. Herein, we report for the first time the catalytic properties of two intestinal secreted PLA₂ (sPLA₂) identified from Diplodus sargus (IDsPLA₂) and Sparus aurata (ISaPLA₂). The highest sequence identity was obtained with recently isolated Sparidae digestive PLA₂ (45%) and Human pancreatic PLA₂ (42%). IDsPLA₂ and ISaPLA₂ were overexpressed in E. coli as inclusion bodies, refolded and purified. Both enzymes have improved thermostability compared to mammalian pancreatic sPLA₂ since they are active and stable at 55 °C, with specific activities of 320 and 190 U mg^-1 measured on phosphatidylcholine, respectively. Interestingly, IDsPLA₂, but not ISaPLA₂, revealed weak toxicity towards macrophages and suggests its involvement in cell membrane degradation. ISaPLA₂ was found to be more active than IDsPLA₂ when using the monolayer technique at 20 mN m^-1. Structural models of both enzymes revealed their differences. In silico docking of phospholipids with both models allowed proposing key amino-acids in substrate binding and selectivity. Overall, these results provide insight into the enzymatic and structural properties of two novel sPLA₂ with potential for future applications.

Keywords: Cytotoxicity activity; Intestinal sPLA(2); Isolation and expression; Sparidae; Structural properties; Substrate specificity; Thermostability.