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Identification of an antibacterial protein as L ‐amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli
Author(s) -
Kitani Yoichiro,
Tsukamoto Chihiro,
Zhang GuoHua,
Nagai Hiroshi,
Ishida Masami,
Ishizaki Shoichiro,
Shimakura Kuniyoshi,
Shiomi Kazuo,
Nagashima Yuji
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05570.x
Subject(s) - rockfish , sebastes , mucus , chemistry , identification (biology) , fishery , biochemistry , fish <actinopterygii> , microbiology and biotechnology , biology , ecology
Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli , which showed selective antibacterial activity against Gram‐negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP ( S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion‐exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N‐linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino‐acid sequence determined, a full‐length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino‐acid residues was cloned by 3′ RACE, 5′ RACE and RT‐PCR. A blast search showed that a mature protein (496 residues) is homologous to l ‐amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H 2 O 2 ‐generation assay and substrate specificity for only l ‐Lys with a K m of 0.19 m m . It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila , Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H 2 O 2 is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.