★ The rockfish Sebastes schlegeli skin mucus has a potent antibacterial protein, SSAP. ★ SSAP is a novel l-amino acid oxidase. ★ Localization of SSAP was examined by in situ hybridization. ★ SSAP located near the basal membrane of skin epidermis and the gill epithelia. ★ SSAP functions locally as a humoral defense factor in S. schlegeli skin and gills.
The rockfish Sebastes schlegeli skin mucus contains a potent antibacterial protein, SSAP (S. schlegeli antibacterial protein), a novel l-amino acid oxidase with strict substrate specificity that acts against water-borne Gram-negative bacteria. We previously demonstrated that SSAP distributes in the skin and gills. Here we investigated the intra-tissue localization of SSAP in the tissues by in situ hybridization. Skin and gill sections were hybridized with digoxigenin-conjugated SSAP-specific RNA probe. SSAP mRNA-positive cells located near the basal membrane of skin epidermis and the gill epithelium. Furthermore, skin section was analyzed by immunohistochemistry and reacted with anti-SSAP antiserum as a primary antibody. The mucus layer and mucous cells in the skin were immunopositive. Skin and gill extracts produced hydrogen peroxide, responsible for antibacterial activity, in the presence of l-lysine. These results suggested that SSAP functions locally as a humoral defense factor in S. schlegeli skin and gills and prevents pathogenic bacterial invasion.