Identification of an antibacterial protein as L‐amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Abstract

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 H2O2‐generation assay and substrate specificity for only l‐Lys with a Km of 0.19 mm. 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 H2O2 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.

L-Amino Acid Oxidase, a Novel Antibacterial Protein in the Rockfish <i>Sebastes schlegeli</i>

“…LAO with a potent antibacterial activity was first isolated from skin mucus of rockfish (Kitani et al 2007a). Thereafter, fish LAOs were isolated not only from skin mucus of great sculpin Myoxocephalus polyacanthocephalus (Nagashima et al 2009) and starry flounder Platichthys stellatus (Kasai et al 2010), but also from serum of rockfish (Kitani et al 2010) and white-spotted rabbitfish Siganus oramin (Wang et al 2011).…”

“…However, it remains to be clarified how SSAP recognizes the bacteria of interest. It is unlikely that the glycochain of SSAP is involved in the binding to cells, because SSAP deglycosylated did not decrease antibacterial activity of SSAP (Kitani et al 2007a). …”

“…Interestingly, LAOs from sea hares such as Aplysianin A, APIT and escapin also preferred basic amino acids, L-lysine and L-arginine as substrates (Jimbo et al 2003;Butzke et al 2005;Ko et al 2008). From a LineweaverBurk plot of LAO activity of SSAP, Michaelis-Menten constant (K m ), turnover number (k cat ) and specificity constant (k cat /K m ) were evaluated to be 0.19 mM, 20.4 s -1 and 1.07 ¥ 10 5 s -1 M -1 , respectively (Kitani et al 2007a).…”

“…In order to analyze internal amino acid sequences, peptide fragments of SSAP were produced using lysil endopeptidase and separated by reversed phase HPLC on a Chromolith Performance RP 18e column. The three peptides (fragments 1-3) were assigned to SADELLQHALQK for fragment 1, EGWYAELGAMRIPS for fragment 2, and SYTWSDDSLLFLGASDED for fragment 3 (Kitani et al 2007a).…”

“…Furthermore, we found a novel antibacterial protein from the skin mucus of the rockfish Sebastes schlegeli, termed it as SSAP (S. schlegeli antibacterial protein). The antimicrobial protein was identified as a new member of the L-amino acid oxidase (LAO) family with target selectivity against Gram-negative bacteria (Kitani et al 2007a). In this monograph, we describe the isolation, structural character, enzymatic character and antibacterial mechanism of the first antibacterial LAO from fish skin mucus.…”