Purification, characterization and the hemolytic mechanism of the hemolysin NnTX-45 from the jellyfish Nemopilema nomurai

Abstract

Nemopilema nomurai, a large venomous jellyfish, caused numerous stinging incidents and even many fatal cases. The venom of N. nomurai contains various toxins, with hemolysin being one of the major components that play a crucial role in stinging. However, until now, the hemolysin has not been successfully isolated from N. nomurai, and its mechanism remains unclear. In this study, we established an improved method for preparing jellyfish toxins to facilitate subsequent purification. The SDS-PAGE profiles indicated that the crude toxin extracted using this improved method contained significantly fewer components than the traditional method. Moreover, the proteomic and toxicity analysis revealed that the refined extract still contained most of the key toxins including hemolysin, phospholipase, and other toxins. The hemolysin was then isolated from the refined jellyfish crude toxins using a two-step purification of gel filtration chromatography followed by anion-exchange chromatography. The molecular mass of this hemolysin was 45 kDa (NnTX-45) with an HC₅₀ of approximately 30 μg/mL. Transmission electron microscopy observations revealed that NnTX-45 formed numerous pores, each with an inner diameter of 5.65 nm and an outer diameter of 13 nm approximately, on the erythrocyte membranes. Overall, our study successfully isolated and elucidated the preliminary hemolytic mechanism of the NnTX-45 from N. nomurai, which provides a highly purified toxin antigens for the development of jellyfish antivenom to treat this jellyfish sting in the future.