Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin‐resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of l‐amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT‐PCR using a specific primer, and immunohistochemical analysis with anti‐LAAO IgG, demonstrated tissue‐specific expression and localization in the gill. Moreover, the anti‐LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin‐resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus‐derived epidermal mucus, is a novel LAAO‐like protein with antibacterial activity, similar to snake LAAOs.
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