Gene expression and distribution of antibacterial l-amino acid oxidase in the rockfish Sebastes schlegeli

Abstract

Antibacterial factors in the epidermal mucus of fish have a potential importance in the first line of the host defense response to bacterial pathogens. We previously isolated a novel antibacterial protein termed SSAP ( Sebastes schlegeli antibacterial protein) from the skin mucus of the rockfish S. schlegeli and identified it as a new member of the l-amino acid oxidase (LAO) family. In the present study, the localization of SSAP in S. schlegeli was investigated by reverse transcription (RT)-PCR, quantitative real time RT-PCR, Western blotting and measurements of LAO and antibacterial activities. SSAP mRNA was expressed dominantly in skin and gill and weakly in ovary or kidney as shown by RT-PCR and real time RT-PCR. The quantity of SSAP mRNA in skin varied among the individuals, ranging from 1.1 to 13.9 ng μg −1 total RNA, although no relationship was found between the size of fish and gene expression. SSAP was exclusively detected in skin and gill by Western blotting using a specific anti-SSAP antiserum. In addition, the extracts of both tissues apparently showed LAO activity and antibacterial activity against Photobacterium damselae subsp. piscicida. This study demonstrates that SSAP is predominantly synthesized in skin and gill and probably functions as an antibacterial LAO in both tissues.