The modulation of haemolymph arginine kinase on the extracellular ATP induced bactericidal immune responses in the Pacific oyster Crassostrea gigas

• CgAK was isolated and identified by LPS affinity chromatography and mass spectrometry.
• LPS directly bound to CgAK and exerted an ATP hydrolysis inhibitory activity, leading to the up-regulation of ATP.
• Extracellular ATP (eATP) stimulation induced intracellular Ca2+ flux, ROS production and lysosomal enzyme release in haemocytes.
• CgAK modulated eATP-induced ROS-dependent bactericidal activity in haemocytes.

Arginine kinase is an important phosphagen kinase (PK) which plays an essential role in ATP buffering systems in invertebrates. In the present study, an arginine kinase (designated CgAK) was isolated by the lipopolysaccharide (LPS) affinity chromatography from the haemolymph of Crassostrea gigas. CgAK could directly bind to LPS in a concentration-dependent manner with the dissociation constant (Kd) of 2.46 × 10−6 M. The interaction with LPS significantly decreased the ATP hydrolytic activity of CgAK, which in turn lead to the accumulation of ATP in vitro. The extracellular ATP stimulation could induce Ca2+ influx, reactive oxygen species (ROS) production, and the release of lysosomal enzyme in the cellular immune response. In addition, ATP stimulation provoked the bactericidal activity towards Escherichia coli, and the scavenging ROS with N-acetyl-l-cysteine (NAC) abrogated the bactericidal activity, indicating that ATP stimulation could induce ROS-dependent antimicrobial activity in haemocytes. Collectively, the results demonstrated that the haemolymph CgAK could serve as an important purinergic regulator to modulate extracellular ATP, which might further have an important effect on the purinergic signaling-activated innate immune response of oyster.