Acetylcholine-binding protein 5 regulates extracellular apoptosis in the wolf spider as a sacrificial ROS scavenger

Apoptosis is regulated by a conserved gene network across species for maintaining homeostasis and stress response. While intracellular apoptotic pathways are well characterized, the extracellular mechanisms governing apoptosis remain largely unexplored, probably due to divergence in species-specific gene sets involved in extracellular regulation. Here, we uncover a novel extracellular apoptotic mechanism mediated by acetylcholine-binding protein 5 (AChBP5) in the wolf spider Pardosa pseudoannulata. AChBP5 is highly expressed in the spider fat-body (midgut diverticula), while other four AChBP genes are abundantly expressed in the tissue brain. Among five AChBP genes, only AChBP5 exhibited broad transcriptional induction upon exposure to various insecticides, including neonicotinoids and other classes. AChBP5 expression was also upregulated by reactive oxygen species (ROS) including H₂O₂, key triggers of apoptosis. Functional assays demonstrated that, in cultured cells, AChBP5 acted as an extracellular sacrifice buffer against oxidative stress to maintain cell viability, becoming progressively inactivated by ROS in a concentration-dependent manner. RNAi-mediated silencing of AChBP5 significantly increased spider susceptibility to both-induced and direct oxidative stress, underscoring its critical protective function. Collectively, these findings support a model in which lineage-specific genes, AChBP5 may contribute to extracellular modulation of apoptosis and provide a mechanism by which spiders could respond to chemical stressors.