Mini-αA-Crystallin Stifled Melittin-Induced Haemolysis and Lymphocyte Lysis

Abstract

Melittin, the most potent pharmacological ingredient of honey bee venom, induces haemolysis, lymphocyte lysis, long-term pain, localised inflammation, and hyperalgesia. In this study, efforts were made to subdue the melittin’s ill effects using a chaperone peptide called ‘mini-αA-crystallin’ (MAC) derived from eye lens αA-crystallin. Haemolytic test on human red blood cells, percentage viability, and DNA diffusion assay on Human peripheral blood lymphocytes (HPBLs) were performed with melittin in the presence or absence of MAC. Propidium iodide and Annexin V-FITC dual staining were performed to analyse quantitative levels of necrotic and apoptotic induction by melittin in the presence or absence of MAC on HPBLs using a flow cytometer. A computational study to find out the interactions between MAC and melittin was undertaken by modelling the structure of MAC using a PEP-FOLD server. The result showed that MAC inhibited melittin-induced lysis in nucleated (lymphocytes) and enucleated (RBC) cells. Flow cytometric analysis revealed a substantial increase in the necrotic and late apoptotic cells after treating HPBLs with melittin (4 µg/ml) for 24 h. Treatment with MAC at a 2:1 molar ratio prevented HPBLs from developing melittin-induced necrosis and late apoptosis. In the docking study, hydrogen, van der Waals, π-π stacking, and salt bridges were observed between the MAC and melittin complex, confirming a strong interaction between them. The MAC-melittin complex was stable during molecular dynamics simulation. These findings may be beneficial in developing a medication for treating severe cases of honeybee stings.