Molecular characterization of laao in large yellow croaker and its functional response to Cryptocaryon irritans infection.

L-amino acid oxidase (laao) is a key immune factor capable of producing reactive oxygen species (ROS) and has been demonstrated to possess significant antibacterial and immunomodulatory functions in a variety of organisms. In recent years, increasing attention has been paid to the role of laao in the immune defense of fish against Cryptocaryon irritans infection. In this study, the laao of large yellow croaker (Larimichthys crocea) was successfully cloned, and its structural and functional characteristics were systematically analyzed. The full-length ORF of large yellow croaker was 1578 bp, encoding 526 amino acids. Structural prediction indicated that the protein possesses typical features of a secretory protein, including a distinct signal peptide region and three potential N-glycosylation sites. The Laao of large yellow croaker shows high conservation at key catalytic residues compared with those of Danio rerio and Bothrops pauloensis. Molecular docking further revealed a clear substrate preference, with hydrophobic amino acids exhibiting the strongest binding affinity, whereas polar substrates showed weaker interactions. Phylogenetic analysis revealed that laao is highly conserved among teleosts, showing the highest sequence similarity to Collichthys lucidus (96.57 %) and Nibea albiflora (81.57 %). The tissue expression analysis demonstrated that laao exhibits a tissue-specific expression pattern in large yellow croaker, being mainly expressed in the gills, fins, kidneys, and spleen, with the lowest expression in the brain. Following C. irritans infection, laao expression in the gills and spleen responded rapidly, reaching a peak at 24 h post-infection. However, although laao expression peaked in the skin as early as 12 h post-infection, its expression level was relatively low. The transcriptomic data before and after infection also confirmed that laao was activated upon C. irritans challenge, showing differential expression with an overall upregulation trend. The qRT-PCR results further demonstrated that overexpression of laao significantly altered the expression patterns of immune-related genes (hif1, tnf-α, il-8, il-1β and stat3) were downregulated, while jak1 was upregulated-suggesting that laao may participate in host immune regulation by suppressing inflammatory signaling and activating cytokine-mediated pathways. In summary, this study reveals the structural features of laao and its response characteristics during C. irritans infection, providing a theoretical basis for further understanding the role of large yellow croaker laao in fish immune defense and for developing novel immune prevention and control strategies.