Seawater activates l-amino acid oxidase from the serum of the red-spotted grouper Epinephelusakaara.

l-amino acid oxidases (LAOs) catalyze the oxidative deamination of l-amino acid and generate α-keto acid, ammonia, and hydrogen peroxide as byproducts. LAOs showed the variety of bioactivity by the resulting hydrogen peroxide. The serum of the red-spotted grouper Epinephelus akaara contains an LAO (Ea-LAO) with the potential to kill bacterial pathogens Aeromonas salmonicida and Vibrio anguillarum via hydrogen peroxide. However, it is unknown how the grouper tolerates the harmful effects of the serum Ea-LAO byproducts. In this study, we analyzed the kinetics of fish LAOs to understand how they escape the toxicity of byproducts. The LAO activity of grouper serum was suppressed in low-salt solutions such as NaCl, CaCl2, MgCl2, and diluted seawater. The activity was non-linearly increased and fitted to the four-parameter log-logistic model. The EC50 of the seawater was calculated to have a 0.72-fold concentration. This result suggested that the Ea-LAO could be activated by mixing with seawater. The results of circular dichroism spectroscopy showed that the α helix content was estimated to be 12.1% and 5.3% in a salt-free buffer (inactive condition) and the original concentration of seawater (active condition), respectively, indicating that the secondary structure of the Ea-LAO in the active condition was randomized. In addition, the Ea-LAO showed reversible LAO activity regulation according to the salt concentration in the environment. Taken together, this indicates that the Ea-LAO is normally on standby as an inactive form, and it could activate as a host-defense molecule to avoid pathogen invasion via a wound when mixed with seawater.

Blood loss induces l-amino acid oxidase gene expression in the head kidney of the red-spotted grouper, Epinephelus akaara.

In fish, the innate immune system is more important than the adaptive immune system because it responds quickly and nonspecifically to protect against pathogens. Thus, a variety of innate immune molecules have been found in fish. Recently, l-amino acid oxidases (LAOs) were discovered as a new member of the antibacterial protein from fish skin mucus and serum. In this study, we newly found an antibacterial LAO in red-spotted grouper (Epinephelus akaara) serum. It showed a broad range of substrate specificity with aromatic and hydrophobic amino acids. The grouper LAO gene had a low expression level in the kidney under normal conditions; however, it was significantly upregulated by blood loss 1 day after bleeding. In addition, the LAO activity in the serum recovered within 3 days in the same experiment. This quick recovery may indicate that the LAO is an essential innate immune molecule in the whole grouper body.