Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus Kosuke Kasai1,2, Takashi Ishikawa1, Takafumi Komata3, Kaori Fukuchi4, Mitsuru Chiba5, Hiroyuki Nozaka1,2, Toshiya Nakamura1,2, Tatsusuke Sato1,2 and Tomisato Miura1,2 Division of Medical Life Sciences, Hirosaki University Graduate School of Health Sciences, Japan Research Center for Biomedical Sciences, Hirosaki University, Japan Clinical Laboratory, Shichinohe Hospital, Japan Clinical Laboratory, Suzuki Lady’s Hospital, Kanazawa, Japan Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan Keywords antibacterial protein; methicillin-resistant Staphylococcus aureus (MRSA); Platichthys stellatus; L-amino acid oxidase; mucus Correspondence T Miura, Division of Medical Life Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan Fax: +81 172 39 5966 Tel: +81 172 39 5966 E-mail: tomisato@cc.hirosaki-u.ac.jp (Received 26 August 2009, revised 26 October 2009, accepted 16 November 2009) doi:10.1111/j.1742-4658.2009.07497.x 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 stellatusderived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs Introduction The mucus layer covering the body surface of many animal species plays a defensive role as both a physical and chemical barrier against bacterial and viral infection The mucus components are reported to vary widely and to have a number of biological functions for host defense [1–4] Fish also produce such mucus Abbreviations CFU, colony-forming units; GSP, gene-specific primer; HIO4 ⁄ Schiff, periodic acid ⁄ Schiff’s reagent; LAAO, L-amino acid oxidase; MRSA, methicillin-resistant Staphylococcus aureus; PSEM, Platichthys stellatus-derived epidermal mucus; psLAAO, LAAO sequence of Platichthys stellatus; PVDF, poly(vinylidene difluoride); TSA, trypticase soy agar; M urea ⁄ PAGE, PAGE in the presence of M urea FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS 453 A flounder LAAO-like antibacterial protein K Kasai et al substances for defense, as their environment is rich in microorganisms [5] Skin and gill mucus secretions of fish are known to contain many substances that are active against bacteria and viruses, including peptides, lysozymes, lectins and proteases These also play an important role in innate immunity [6,7] Antibacterial peptides isolated from the epidermal mucus of several species of fish have already been characterized One type, the cathelicidins, act by disrupting the bacterial cell membrane and are considered to be important effectors of eukaryotic immunity [8] Recently, it has been shown that infection with fish pathogens causes up-regulation of cathelicidin mRNA in various tissues such as the gill, spleen and head kidney [9] A 22-residue antibacterial peptide, moronecidin, isolated from the skin and gill of hybrid striped bass, exhibits a broad spectrum of antibacterial activity [10] A lysozyme-like peptide from rainbow trout (Oncorhynchus mykiss) demonstrates antibacterial activity against gram-positive bacteria [11] Also, an antibacterial protein with ion channel activity against both gram-negative and gram-positive bacteria has been found in mucus extract from carp (Cyprinus carpio) [12] Pleurocidin, found in skin mucus secretions of the winter flounder (Pleuronectes americanus), has been shown to exhibit antibacterial activity against both gram-negative and gram-positive bacteria [13] In recent years, some reports have documented details of high-molecular-mass antibacterial proteins in fish mucus, such as that of the rockfish (Sebastes schegeli), which demonstrates selective antibacterial activity against gram-negative bacteria [14] A pore-forming 65-kDa glycoprotein isolated from the rainbow trout (O mykiss, formerly Salmo gairdneri), has also been found to have strong antibacterial properties [15] Glycosylated proteins from the hydrophobic supernatant of mucus from tench (Tinca tinca), eel (Anguilla anguilla) and rainbow trout (O mykiss) show strong activity against both gram-negative and gram-positive bacteria [16] In the present study, we found an antibacterial protein in the epidermal mucus of the flounder Platichthys stellatus This species, which has a rich covering of mucus on its body surface, inhabits brackish water at the mouths of rivers This mucus protein was shown to exert antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillinresistant S aureus (MRSA) Moreover, we identified this antibacterial protein as a novel l-amino acid oxidase (LAAO; EC.1.4.3.2) LAAOs catalyze the oxidative deamination of an l-amino acid substrate and have been reported to exert antibacterial activity in a variety of animal fluids, such as snake venom [17] 454 The present communication describes the isolation and cloning of this LAAO-like antibacterial protein from P stellatus Results Antibacterial activity of mucus It is assumed that Platichthys stellatus-derived epidermal mucus (PSEM) includes antibacterial substances, because the body surface, which is exposed to the external environment, functions as the first barrier to invasion by bacteria Therefore, we analyzed the antibacterial activity of PSEM against 19 different grampositive and gram-negative clinically pathogenic bacteria using a growth-inhibition plate assay (Table 1) The PSEM inhibited the growth of all Staphylococcus spp (antibacterial score: 2+ to 3+) Proliferation of S epidermidis in particular was strongly suppressed, the effect being most marked among all the bacteria we studied (Fig 1A) The PSEM had intermediate Table Antibacterial activity spectra of Platichthys stellatus-derived epidermal mucus Species and strains Diameter of clear zone (mm) Scorea Gram-positive bacteria Staphylococcus aureus NIHJ JC-1 8.5 Staphylococcus aureus ATCC25923 6.3 Staphylococcus epidermidis 18.1 Methicillin-resistant Staphylococcus 8.2 aureus 87-7920 Methicillin-resistant Staphylococcus 8.3 aureus 87-7927 Methicillin-resistant Staphylococcus 8.1 aureus 87-7928 Methicillin-resistant Staphylococcus 8.2 aureus 87-7931 Methicillin-resistant Staphylococcus 8.1 aureus 87-7958 Streptococcus pyogenes 5.5 Streptococcus agalactiae 2.8 Enterococcus faecalis ATCC33186 2.8 Enterococcus faecium ATCC19434 2.8 Enterococcus faecium BM4147 (VanA+) 2.8 Enterococcus faecalis V583 (VanB+) 2.8 Enterococcus gallinarum BM4174 (VanC1+) 2.8 Gram-negative bacteria Escherichia coli NIHJ JC-2 2.8 Serratia marcescens 2.8 Vibrio parahaemolyticus RIMD2210001 5.7 Pseudomonas aeruginosa ATCC27853 2.8 + + + + + + ++ + ++ ++ ++ ++ + – – – – – – – – + – a Clear zone £ 2.8 mm +, clear zone < 6.0 mm; + +, clear zone < 10.0 mm; + + +, clear zone ‡ 10.0 mm FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS K Kasai et al A C A flounder LAAO-like antibacterial protein B D Temperature sensitivity of PSEM for antibacterial activity Generally, proteins lose their activity when subjected to heat treatment, and complement (which is a component of blood) is inactivated by heating at 56 °C for 30 Therefore, the antibacterial activity of PSEM was investigated after incubation at various temperatures, in order to investigate the properties of the antibacterial components The antibacterial activity of PSEM for MRSA 87-7928 was lowered slightly at 45 °C, markedly at 56 °C and completely at 70 °C (Fig 1D), suggesting that the antibacterial component of PSEM is a protein Purification of antibacterial protein from PSEM Fig Antibacterial activity of PSEM against (A) Staphylococcus epidermidis, (B) Staphylococcus aureus NIHJ JC-1B and (C) MRSA, clinical isolate 87-7928 Each bacterial strain was suspended in TSA at a final concentration of · 106 CFmL)1 (c) Control buffer without PSEM and (mu) PSEM were applied to holes in the agar Antibacterial activity was measured after overnight incubation at 37 °C (D) Heat sensitivity of PSEM against MRSA, clinical isolate 87-7928 (c) Control buffer without PSEM at °C PSEM was exposed to temperatures of 0, 25, 37, 45, 56, 70 and 100 °C for h Each sample was applied to the holes in the agar, and antibacterial activity was measured after overnight incubation at 37 °C antibacterial activity for S aureus (Fig 1B), although the antibacterial activity of PSEM against two strains of S aureus was slightly different The growth of MRSA was also inhibited by PSEM (Fig 1C) and there was no marked difference in antibacterial activity among the five MRSA strains tested (Table 1) Among gram-positive cocci, except for the staphylococci, PSEM weakly suppressed the growth of S pyogenes (1+) Among gram-negative bacilli, the proliferation of Vibrio parahaemolyticus was weakly suppressed by PSEM However, PSEM showed no antibacterial activity against two strains of Streptococcus spp., five strains of Enterococcus spp [including vancomycinresistant Enterococcus (VRE)], Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa In the growth-inhibition plate assay, the agar medium in the clear zone formed in the MRSA assay was collected and cultured in trypticase soy agar (TSA) in order to confirm the bactericidal activity of PSEM It was clarified that the PSEM had bactericidal activity against MRSA because MRSA did not proliferate in TSA after 96 h of culture The antibacterial protein in PSEM was separated by ultracentrifugation and purified by hydrophobic chromatography (Fig 2A) Protein fractions were monitored by measuring the absorbance at 280 nm, and antibacterial activity was assayed using the growth-inhibition plate method Pooled antibacterial fractions were further purified by gel filtration chromatography (Fig 2B) and chromatofocusing (Fig 2C) In gel filtration chromatography and chromatofocusing steps, the antibacterial activity was eluted as a single peak SDS ⁄ PAGE of the fractions containing antibacterial activity that had been separated by chromatofocusing contained three main bands with molecular masses of 39, 40 and 52 kDa (Fig 2D) Because of irreversible denaturation of the protein, antibacterial activity was not detected in the gels after SDS ⁄ PAGE Therefore, we performed PAGE in the presence of m urea (6 m urea ⁄ PAGE) to separate the antibacterial protein as remaining bioactivity Interestingly, the purified PSEM retained its bioactivity after this step The antibacterial activity of gel extracts from the m urea ⁄ PAGE was analyzed using the growth-inhibition plate method, and the molecular mass of the antibacterial protein was confirmed by SDS ⁄ PAGE Antibacterial protein was detected only in fractions 19–22 (Fig 3A), and its molecular mass was estimated to be 52 kDa (Fig 3B) Two lower-molecularmass proteins of 39 kDa (fractions 23–24) and 40 kDa (fractions 15–16) did not show antibacterial activity Moreover, 2D gel electrophoresis revealed a single spot at 52 kDa with an isoelectric point of 5.3 (Fig 3C) cDNA cloning and sequence analysis of antibacterial protein For cloning, the antibacterial protein was blotted onto a poly(vinylidene difluoride) (PVDF) membrane after 2D gel electrophoresis, and the spot corresponding to FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS 455 A flounder LAAO-like antibacterial protein K Kasai et al B A D C Fig Purification of epidermal mucus protein (A) Chromatography using a Phenyl Sepharose Fast Flow high sub column One-hundred and thirty milliliters of PSEM was applied to the column at a flow rate of 30 mLỈh)1 The protein content of each fraction was monitored by measuring the absorbance at 280 nm (s) and antibacterial activity (d) was assayed using the growth-inhibition plate method Pooled fractions indicated by the bar (I) were used for gel filtration chromatography (B) Gel filtration chromatography using a Sephacryl S-100 HR column The fraction volume was 2.5 mL and the flow rate was 8.0 mLỈh)1 The protein content of each fraction was monitored by measuring the absorbance at 280 nm (s) and antibacterial activity (d) was assayed using the growth-inhibition plate method Pooled fractions indicated by bar (II) were used for chromatofocusing (C) The antibacterial protein was further purified by chromatofocusing on a PBE94 column at pH 7–4 The fraction volume was 2.5 mL and the flow rate was 30 mLỈh)1 The protein content of each fraction was monitored by measuring the absorbance at 280 nm (s) and antibacterial activity (d) was assayed using the growth-inhibition plate method The pH of each fraction is indicated by a diamond Pooled fractions indicated by bar (III) were used for M urea ⁄ PAGE (D) SDS ⁄ PAGE of the antibacterial fractions at each chromatography step C, crude mucus protein; I, pooled antibacterial fractions from Phenyl Sepharose chromatography; II, pooled antibacterial fractions from gel filtration chromatography; III, pooled antibacterial fractions from chromatofocusing The positions of the molecular mass markers are indicated 52 kDa was cut out Then, the N-terminal peptide sequence was analyzed by Edman degradation and the inner peptide sequences were determined using an amino acid sequencer This showed that the N-terminal peptide sequence was Leu-Ser-Phe-Arg-Ala-His-Leu-SerAsp and that the internal peptide sequences were Arg-Thr-Phe-Glu-Val-Asn-Ala-His-Pro-Asp-Ile-Leu, Ser-Ala-Asp-Gln-Leu-Leu-Gln-Gln-Ala-Leu and Ser-GluGly-Arg-Leu-His-Phe-Ala-Gly-Glu-His-Thr To determine the cDNA encoding the antibacterial protein of PSEM, mRNA was prepared from skin and gill PCR was performed using degenerate primers based on the N-terminal peptide sequence LSFRAHLSD and the internal peptide sequence RTFEVNAHPDIL Subsequently, the full-length cDNA was amplified by 3¢-RACE and 5¢-RACE Sequence analysis identified two genes, which completely corresponded to the peptides of antibacterial protein from the gill (Fig 4), 456 and another highly homologous gene from skin (DDBJ accession number AB495361) The full-length cDNA found in the gill, which encodes an antibacterial protein, consisted of 2002 bp plus poly (A) The N-terminal amino acid sequence of LSFRAHLSD was encoded by nucleotides 183–209 The internal amino acid sequences RTFEVNAHPDIL, SADQLLQQAL and SEGRLHFAGEHT were found at positions 567–602, 636–675 and 1524–1559, respectively (Fig 4) The initial codon, ATG, was found at positions 102–104, and the open reading frame was composed of a 1566-bp region, encoding a protein of 522 amino acid residues A BLAST search demonstrated that the encoded antibacterial protein shared identity with a number of LAAO flavoproteins The gene encoding this antibacterial protein had 71% identity with the skin mucus antibacterial LAAO of S schlegeli (NCBI accession no BAF43314) and 69% identity with the apoptosis- FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS K Kasai et al A flounder LAAO-like antibacterial protein Fig Identification of antibacterial protein by M urea ⁄ PAGE and 2D gel electrophoresis (A) M urea ⁄ PAGE after chromatofocusing The antibacterial activity of each gel extract from a 2-mm-wide strip was measured using the growth-inhibition plate method and is indicated as a diagram (B) SDS ⁄ PAGE after M urea ⁄ PAGE Each of the gel extracts (slice numbers 8–29) was subjected to determination of the molecular mass of the antibacterial protein Antibacterial fractions correspond to the upper diagram and are indicated by ‘+’ The asterisk indicates the specific band of the antibacterial protein (C) 2D gel electrophoresis shows a single spot of antibacterial protein indicated by a circle The positions of the molecular mass markers are indicated inducing protein of Scomber japonicus (NCBI accession no CAC00499) A domain search showed that the gene detected in the gill of P stellatus contained a dinucleotide-binding motif followed by a GG-motif (R-x-G-G-R-x-x-T ⁄ S), which is typical of flavoproteins [18] RT-PCR using primers for the 5¢-UTR and 3¢-UTR regions of the LAAO sequence of P stellatus (psLAAO) was performed to examine the tissue-specific expression The results suggested that the psLAAO gene was expressed in gill, but not in skin (Fig 5) Localization of psLAAO by immunohistochemistry To identify the localization of psLAAO protein in the gill of P stellatus, immunohistochemistry was performed with an anti-psLAAO IgG, obtained by immunization of a Japanese white rabbit with insoluble recombinant psLAAO purified from the E coli expression extracts The psLAAO cDNA sequence, without the predicted signal peptide, was cloned into the pET-20b vector and transformed into Rosetta2 (kDE3) E coli competent cells In L of Luria–Bertani (LB) broth, about 1.4 mg of insoluble recombinant psLAAO protein was expressed, but the protein was not detected in soluble form by SDS ⁄ PAGE or western blotting (Fig 6) The insoluble recombinant psLAAO protein was used for the preparation of antiserum Immunohistochemistry with the anti-psLAAO IgG showed a positive reaction in the undifferentiated cells surrounding the vacuolated mucus-secreting cells of the gill (Fig 7B), principally within the epithelium of the primary lamellae and secondary lamellae The mucus-secreting cells stained positively with periodic acid ⁄ Schiff’s reagent (HIO4 ⁄ Schiff), alcian blue and alcian blue-HIO4 ⁄ Schiff Neutralization of antibacterial activity with antipsLAAO IgG In order to confirm whether the antibacterial protein was psLAAO, western blot analysis and a neutralization FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS 457 A flounder LAAO-like antibacterial protein K Kasai et al Fig The cDNA and amino acid sequences of Platichthys stellatus antibacterial protein The nucleotide sequence of cDNA encoding the PSEM antibacterial protein (DDBJ accession number AB495360) and the derived amino acid sequence are shown The N-terminal and internal peptide sequences of antibacterial protein detected by amino acid sequencing analysis are indicated by boxes The predicted dinucleotide-binding motif and the GG-motif are indicated by a straight line and a broken line, respectively assay of antibacterial activity were performed using the anti-psLAAO IgG In the western blot analysis, psLAAO was detected in mucus and gill extract (Fig 8A) In the neutralization assay, an apparent distinction was not found between the anti-psLAAO IgG free control and the normal rabbit immunoglobulin control (Fig 8B) The neutralization activity of the antipsLAAO IgG increased in an antibody concentrationdependent manner Fig Tissue-specific expression of psLAAO mRNA by RT-PCR Tissues were collected from the same fish Lane 1, total RNA from gill; lane 2, total RNA from skin Discussion In the present study, we showed that the epidermal mucus of P stellatus contains a protein with activity against various pathogenic species and strains of 458 bacteria We isolated this antibacterial protein by column chromatography through three different matrices and gel electrophoresis Furthermore, we detected the FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS K Kasai et al A A flounder LAAO-like antibacterial protein B Fig Recombinant protein expression in the transfected bacteria (A) SDS ⁄ PAGE and (B) western blot analysis of the bacterial extracts Lane 1, soluble cytoplasmic fraction; lane 2, insoluble cytoplasmic fraction The positions of the molecular mass markers are indicated M, positions of the molecular mass markers N-terminal and internal peptide sequences of this protein and elucidated its complete mRNA sequence by cDNA cloning Because a BLAST search demonstrated that the encoded antibacterial protein shared identity with a number of LAAO flavoproteins, and a domain search showed that the gene contained typical flavoprotein motifs, the protein was suggested to be a new member of the LAAO family RT-PCR and immunohistochemical analysis demonstrated tissue-specific expression and localization in the gill Western blot analysis with an anti-psLAAO IgG detected the protein in mucus and gill extract Moreover, a neutralization assay of antibacterial activity against MRSA demonstrated that the clear zone was slightly reduced depending on the volume of anti-psLAAO IgG employed Thus, we confirmed that the protein present in PSEM was a novel LAAO-like antibacterial protein LAAOs are flavoenzymes that catalyze the oxidation of l-amino acids, resulting in the production of a-keto acids, ammonia and hydrogen peroxide [19] It has A B C D E F Fig Immunohistochemical analysis of Platichthys stellatus gill tissues with antipsLAAO IgG Gill sections of P stellatus were stained with (A) nonimmune control immunoglobulin, (B) anti-psLAAO IgG (C) hematoxylin & eosin, (D) HIO4 ⁄ Schiff, (E) alcian blue and (F) alcian blueHIO4 ⁄ Schiff Arrows denote the mucous cells Scale bar, 50 lm FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS 459 A flounder LAAO-like antibacterial protein K Kasai et al Fig Reaction of anti-psLAAO IgG with antibacterial protein (A) SDS ⁄ PAGE and western blot analysis Lanes and 3, PSEM; lanes and 4, gill extract The 52 kDa band is indicated by an asterisk (B) Neutralization of antibacterial activity with anti-psLAAO IgG The MRSA clinical isolate 87-7928 was suspended in TSA at a final concentration of · 106 CFmL)1 Ten microliters of PSEM (upper panel) or gill extract (lower panel) with the indicated volume (0–10 lL) of antipsLAAO IgG were applied to each hole in the agar after incubation at 37 °C for h Control immunoglobulin (10 lL) was applied with 10 lL of PSEM or gill extract to the holes, as indicated by the hole labelled ‘C’ The total volume was adjusted with NaCl ⁄ Pi to 20 lL PSEM and gill extract protein in the clear zone on the growth-inhibition plate are indicated as a diagram M, positions of the molecular mass markers been reported that LAAOs have bioactivities as antibacterial, antiviral and cytotoxic agents in a variety of animal fluids, such as snake venom [20–25], mouse 460 milk [26,27], fish epidermal mucus and extract [28,29], body surface mucus of the giant African snail [30] and the ink of the sea hare [31,32] Previous studies have suggested that the bioactivity of LAAO is elicited by hydrogen peroxide generated from l-amino acid oxidation [25,32] and the binding of LAAO to bacterial cells and viruses [33,34] Achacin, an antibacterial protein in the mucus of the giant African snail, also shows significant bacterial-binding and LAAO activity against S aureus and E coli [33] Escapin, from the ink of the sea hare, has an l-lysine-dependent antibacterial effect and a broad antimicrobial spectrum, being most effective against S aureus [32] Moreover, the antimicrobial and antiparasitic LAAO isolated from Bothrops jararaca has the highest effectiveness against S aureus [25] These findings suggest that the antibacterial effect is dependent on hydrogen peroxide production, because the antibacterial activity was abolished by catalase In the present study, PSEM also showed specific antibacterial activity against S aureus, and MRSA was significantly suppressed depending on the dose of catalase employed (data not shown) Thus, psLAAO in PSEM exerts antibacterial activity through hydrogen peroxide generated from the catalytic oxidation of l-amino acid, although details of the selective effect against bacteria are still unclear In the cloning analysis, we identified a cDNA corresponding to the peptide sequence of the antibacterial protein RT-PCR analysis suggested that psLAAO mRNA was specifically expressed in the gill, and immunohistochemistry with anti-psLAAO IgG also showed that psLAAO-positive cells were present in the gill These results suggest that psLAAO has tissuespecific expression and is localized in gill Interestingly, using cloning analysis, we identified a highly homologous gene that was expressed in the skin A domain search analysis suggested that this homologous gene also has a dinucleotide-binding motif and a GG motif, which are characteristic of the LAAO family Furthermore, a BLAST search demonstrated high identity with the antibacterial protein of S schlegeli and other members of the LAAO family Immunohistochemical staining also showed a positive reaction with antipsLAAO IgG in skin tissue (data not shown) because the anti-psLAAO IgG was cross-reactive with highly homologous LAAO extracted from skin mucus These results suggest that some types of LAAO are expressed in different tissues of fish epidermis The gill has a very important function as the main respiratory organ of fish and it also has an additional role in defense by secreting a mucus layer, which includes antibacterial proteins, as it is constantly exposed to bacteria in the external environment [6,7] FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS K Kasai et al The biological importance of the mucus interface between the body and the aqueous environment includes functions such as physiological and chemical protection In the present study, the N-terminal peptide sequence of psLAAO was found to start with a leucine residue, not a methionine residue Moreover, the complete psLAAO sequence was 1566 bp in length, encoding a protein of 522 amino acid residues and with an expected molecular mass of higher than 52 kDa The antibacterial protein we isolated was estimated to have a molecular mass of approximately 52 kDa Therefore, psLAAO may be cleaved at Ala27 to become a mature protein and secreted from the gill into the extracellular matrix, and the antibacterial protein starting at Leu28 may be a component of the mucus covering the body surface and acting as a barrier against bacteria We found that psLAAO is effective against various species of bacteria, suggesting its potential use against clinical pathogens MRSA is a major cause of hospital-acquired infections and a matter of serious publichealth concern worldwide [35], including the UK [36], Japan [37] and the USA [38] The appearance of such multidrug-resistant bacteria has made it imperative to develop effective and novel antimicrobial agents that could be used to treat infection with these pathogens We speculate that the psLAAO included in PSEM could be one such agent because it has activity against MRSA Our future work will be aimed at improving the expression of bioactive recombinant psLAAO and evaluating the mechanism of its antibacterial effect Experimental procedures Collection of epidermal mucus P stellatus was caught in the brackish-water region of Jusanko Lake, in Goshogawara City, Aomori, Japan After rinsing the body surface with distilled water, the epidermal mucus was scraped off with a rubber spatula and frozen at ) 80 °C The PSEM was then thawed and centrifuged at 105 000 g for h The supernatant was stored at ) 80 °C Bacterial species and strains Nineteen species or strains of bacteria were used to test the antibacterial activity of PSEM: the gram-positive bacteria S aureus (ATCC25923 and NIHJ JC-1), S epidermidis (community isolate), MRSA (clinical isolates 87-7920, 87-7927, 87-7928, 87-7931 and 87-7958), Streptococcus pyogenes (clinical isolate), Streptococcus agalactiae (clinical isolate), Enterococcus faecalis ATCC33186, Enterococcus faecium ATCC19434, E faecium BM4147 (VanA+, clinical isolate), E faecalis V583 (VanB+, clinical isolate) and Entero- A flounder LAAO-like antibacterial protein coccus gallinarum BM4174 (VanC1+, clinical isolate); and the gram-negative bacteria E coli NIHJ JC-2, S marcescens (clinical isolate), V parahaemolyticus RIMD2210001 and P aeruginosa ATCC27853 All clinical isolates were provided by Hirosaki University School of Medicine and Hospital Antibacterial assay The antimicrobial effects of PSEM were determined using a growth-inhibition plate assay The various bacterial species and strains were cultured in TSA (Difco, Detroit, MI, USA) for 16 h at 37 °C, except for V parahaemolyticus, which was cultured in trypticase soy broth supplemented with 0.5% NaCl The cell culture density was measured at 655 nm in a spectrophotometer and then adjusted to approximately · 108 colony-forming units (CFU)ỈmL)1 with phosphate-buffered saline (NaCl ⁄ Pi), based on the standard curve In order to prepare pour plates, bacteria were suspended in TSA at a final concentration of · 106 CFmL)1 Next, a hole of 2.8 mm in diameter was punched in the pour plate and filled with 12 lL of mucus or fractions from each of the purification steps After overnight incubation at 37 °C, the clear zone around the hole was measured To examine heat resistance, the PSEM was incubated for h at 25, 37, 45, 56, 70 and 100 °C Each PSEM sample that had been subjected to the heating treatment was then applied to each hole After incubation overnight at 37 °C, the diameter of the clear zone around each spot was then measured Purification of antibacterial protein from epidermal mucus Unless indicated otherwise, all procedures were performed at °C One-hundred and thirty milliliters of PSEM was thawed and dialyzed against m (NH4)2SO4 in 50 mm phosphate buffer (pH 7.0), then applied to a column of Phenyl Sepharose Fast Flow high sub (1.0 · 25 cm; GE Healthcare UK Ltd., Little Chalfont, Bucks, UK), equilibrated previously with the same buffer, and the column was then washed with the buffer The flow rate of the column was 30 mLỈh)1 and the fraction volume was 10 mL The protein concentration in each fraction was monitored by measuring the absorbance at 280 nm Adsorbed proteins were eluted from the column using a linear gradient of 1–0 m (NH4)2SO4 in 50 mm phosphate buffer, followed by elution with 50 mm phosphate buffer and 10 mm phosphate buffer Antibacterial activity was assayed using the growth-inhibition plate method The fractions with antibacterial activity were collected and the solution was subjected to 80% ammonium sulfate fractionation After centrifugation, the resulting precipitate was dissolved in a small quantity of 0.1 m NaCl in 20 mm Tris ⁄ HCl buffer (pH 7.5) and dialyzed against the same buffer The collected proteins were subjected to gel FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS 461 A flounder LAAO-like antibacterial protein K Kasai et al filtration chromatography on a column of Sephacryl S-100 HR (1.2 · 147cm; GE Healthcare) equilibrated with the same buffer The fraction volume was 2.5 mL and the flow rate was 8.0 mLỈh)1 Antibacterial protein was further purified by chromatofocusing at pH 7–4 The protein in the antibacterial activity fraction was concentrated by 80% ammonium sulfate fractionation, as described above The resulting precipitate was dialyzed against 25 mm imidazoleHC1 (pH 7.4) and applied to a column of PEB94 polybuffer exchanger (1.0 · 27 cm; GE Healthcare) equilibrated with 25 mm imidazole-HC1 (pH 7.4) The fraction was eluted with polybuffer 74 (pH 4.0), diluted 12-fold with de-aerated water and further eluted with 0.5 m NaCl The fraction volume was 2.5 mL and the flow rate was 30 mLỈh)1 Tissue collection and purification of antibacterial protein from gill After rinsing P stellatus in distilled water, the gill tissue was harvested and ground into powder using a mortar and pestle under liquid nitrogen Proteins were extracted in the CytoBuster Protein Extraction Reagent (Novagen, Madison, WI, USA) containing the protease inhibitor by incubation at room temperature for After centrifugation, the supernatants were collected Extracted protein from the gill was thawed and dialyzed against m (NH4)2SO4 in 50 mm phosphate buffer (pH 7.0), then applied to a column of HiTrap Phenyl FF high sub (1.6 · 2.5 cm; GE Healthcare) equilibrated with the same buffer, and the column was then washed with the buffer The flow rate of the column was mL ⁄ and the fraction volume was mL Proteins were eluted stepwise from the column using 1–0 m (NH4)2SO4 in 50 mm phosphate buffer, followed by elution with 50 mm phosphate buffer Antibacterial activity was assayed using growth-inhibition plates The fractions with antibacterial activity were collected Electrophoresis SDS ⁄ PAGE was performed according to the method of Laemmli [39] The samples were heated in 10% glycerol, 2% SDS, 6% 2-mercaptoethanol and 0.05 m Tris ⁄ HCl buffer (pH 6.8) for in a boiling water bath and subjected to SDS ⁄ PAGE with a 10% polyacrylamide gel Protein was stained with Coomassie Brilliant Blue R-250 The antibacterial protein fraction separated by chromatofocusing was subjected to m urea ⁄ PAGE at room temperature The lower gel consisted of 7.5% acrylamide, m urea, 0.06% ammonium persulfate, 0.15% N,N,N¢,N¢-tetramethyl ethylenediamine (TEMED) and 0.3 m acetate buffer (pH 4.8), while the upper gel consisted of 5.0% acrylamide, m urea, 0.002% riboflavin 0.015% TEMED and 0.2 m acetate buffer (pH 5.0) The reservoir buffer was composed of 0.35 m b-alanine and 0.136 m acetate buffer (pH 4.8) The upper gel was polymerized by illumination with a fluorescent 462 light After electrophoresis, the lower gel was cut into strips mm wide Then, 40 lL of 10 mm phosphate buffer was added and the gel was broken into small pieces The supernatant obtained by centrifugation was then used to measure antibacterial activity or to determine the molecular mass of antibacterial protein by SDS ⁄ PAGE 2D gel electrophoresis was performed according to the method of O’Farrell [40], as modified by Hirsch et al [41] Protein was stained with Coomassie Brilliant Blue R-250 The second-dimension electrophoresis was carried out on a 10% acrylamide gel Amino acid sequencing After 2D gel electrophoresis, proteins in the gel were blotted onto a PVDF membrane (Millipore Corp., Bedford, MA, USA) using a semidry-type blotting apparatus, and the target protein spot was cut out The N-terminal amino acid sequence was analyzed using the Edman degradation method An inner peptide amino acid sequence analysis was also performed Peptidase digestion using lysyl endpeptidase, separation of the fragments by RP-HPLC and amino acid sequence analysis were assigned to the APRO Life Science Institute Inc (Naruto, Tokushima, Japan) mRNA extraction and degenerate PCR Total RNA was extracted from the epidermis and gill tissues of P stellatus using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions Total RNA was transcribed to cDNA at 42 °C for 60 in the presence of the oligo (dT)15 Primer (Promega, Madison, WI, USA) and Primescript Reverse Transcriptase (Takara, Tokyo, Japan) Degenerate oligonucleotide primers were designed on the basis of the determined amino acid sequences of the peptide fragments The forward degenerate primers were 5¢-YTITCITTYCGIGIGCNCAY-3¢, 5¢-YTIAGYTTYCGIGCNCAY-3¢, 5¢-YTITCITTYAGRG CNCAY-3¢ and 5¢-YTIAGYTTYAGRGCNCAY-3¢ (corresponding to LSFRAHLSD) The reverse degenerate primer was 5¢-RTGIGCRTTIACYTCRAANGT-3¢ (corresponding to RTFEVNAHPDIL) Amplification was carried out using Ex Taq polymerase (Takara) under the following conditions: 95 °C for min; 35 cycles of 95 °C for min, 48 °C for and 72 °C for min; 72 °C for All PCR products were subcloned into the T-vector prepared by dT addition on EcoRV-digested blunt ends of pBluescript II SK+ (Stratagene, LA Jolla, CA, USA) DNA sequences were determined using an abi prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) 5¢-RACE and 3¢-RACE 5¢-RACE was carried out according to the procedure of the 5¢-RACE System for Rapid Amplification of cDNA Ends FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS K Kasai et al (Invitrogen, Carlsbad, CA, USA) using a gene-specific primer (GSP) (5¢-CATTCCTGTACGTCTCCACTC-3¢) and a nested GSP (5¢-GTTCTCTACTGTCTGCAGCAG-3¢) 3¢RACE was carried out using the procedure of the 3¢-RACE System for Rapid Amplification of cDNA Ends (Invitrogen) using a GSP (5¢-GGAATGAGCAAGGCTGGTAC-3¢) and a nested GSP (5¢-CTCTTCTTCGTGGAGTTACTC-3¢) The GSPs used for 5¢-RACE and 3¢-RACE were designed on the basis of the determined sequences of degenerate PCR clones Amplification was carried out using AmpliTaq Gold DNA polymerase (Applied Biosystems) under the following conditions: 95 °C for 10 min; 35 cycles of 95 °C for min, 48 °C for and 72 °C for min; 72 °C for The PCR products were subcloned into the T-vector and sequenced The nucleotide sequence of the full-length cDNA was amplified by RT-PCR using the forward primer 5¢-GAAGTTTCTCTACGGACTGC-3¢ and the reverse primer 5¢-CAACCATCGATTGTGTCCAG-3¢ The betaactin primer pair (forward primer 5¢-CATGTACGTTGC CATCCAAG-3¢ and reverse primer 5¢-TCTCAGCTGTGG TGGTGAAG-3¢) was designed on the basis of the European flounder (P flesus) beta-actin gene sequence (NCBI accession number AF135499) Amplification was carried out using AmpliTaq Gold DNA polymerase (Applied Biosystems) under the following conditions: 95 °C for 10 min; 35 cycles of 95 °C for min, 55 °C for and 72 °C for 1.5 min; 72 °C for PCR products were subcloned into the T-vector and sequenced Recombinant protein expression Primers were designed to amplify the active form without the secretory signal sequence, so that antibacterial protein could be expressed in E coli as a His-tagged fusion protein The forward primer included an NdeI restriction site (5¢-CCGCATATGCTCAGCTTCAGGGCACATCTG-3¢) and the reverse degenerate primer included a XhoI restriction site (5¢-GCACTCGAGGGTGTGTTCAACCAGCAA AG-3¢) Amplification was carried out using AmpliTaq Gold DNA polymerase under the following conditions: 95 °C for 10 min; 35 cycles of 95 °C for min, 55 °C for and 72 °C for 1.5 min; 72 °C for The PCR products were then cleaved with restriction enzymes and the gene was subcloned into the pET-20b expression vector (Novagen) using the same enzymes The DNA sequence was determined using an abi prism 310 Genetic Analyzer (Applied Biosystems) For protein expression, the plasmid was transformed into E coli strain Rosetta (kDE3) Five liters of these bacterial cells were grown in LB broth (Difco) at 37 °C until the culture reached a D at 600 nm of approximately 0.5, and proteins were induced with 0.4 mm isopropyl thio-b-d-galactoside at 15 °C for 16 h The expressed His-tagged fusion proteins were isolated by means of Ni-nitrilotriacetic acid agarose (Qiagen), in accordance with the manufacturer’s instructions The purified A flounder LAAO-like antibacterial protein His-tagged fusion protein was digested with trypsin and the amino acid sequence was analyzed using nanoFrontier nLC-Linear-Trap-TOF MS (Hitachi, Tokyo, Japan) Antiserum preparation and IgG purification An antiserum against the antibacterial protein was obtained by injecting a Japanese white rabbit (Kitayama Labes Co Ltd., Nagano, Japan) with the insoluble recombinant protein of His-tagged psLAAO purified from the E coli expression extracts The recombinant psLAAO (300 lg) was emulsified with 1.5 mL of Freund’s complete adjuvant (Difco) and injected subcutaneously into each animal Booster injections of 100 lg of recombinant psLAAO in an emulsion of Freund’s incomplete adjuvant (Difco) were then given at 2, and weeks after the primary immunization At weeks, the antiserum was obtained Control serum was obtained from a naive Japanese white rabbit The IgG fraction was purified according to the recommended procedure for the ImmunoPure Melon Gel IgG Spin Purification kit (Pierce, Rockford, IL, USA) Western blot analysis PSEM, gill extract and His-tagged recombinant psLAAO protein were individually heated at 100 °C in 10% glycerol, 2% SDS, 6% 2-mercaptoethanol and 0.05 m Tris ⁄ HCl buffer (pH 6.8) and subjected to SDS ⁄ PAGE (10% polyacrylamide gel) After electrophoresis, the proteins were electrically transferred from the gel onto a PVDF membrane (GE Healthcare) The membrane was blocked with 20 mm Tris ⁄ HCl (pH 7.4), 125 mm NaCl, 0.2% Tween 20 and 5% skim milk (Yotsuba, Sapporo, Japan) psLAAO in the PSEM and gill extracts was detected using the antipsLAAO IgG (1 : 2000 dilution) His-tagged fusion proteins were detected using a mouse monoclonal anti-His IgG (1 : 3000 dilution; GE Healthcare) Horseradish peroxidaseconjugated secondary antibody mouse anti-rabbit IgG (1 : 5000 dilution; GE Healthcare) or sheep anti-mouse IgG (1 : 10000 dilution; GE Healthcare) was used for detection, followed by enhanced ECL Plus Western blotting detection reagents (GE Healthcare) Histology Gill tissues of P stellatus were fixed in 4% paraformaldehyde and embedded in paraffin Sections (4 lm thick) were mounted on Mac-coated slides (Matsunami Trading Co Ltd., Osaka, Japan) Deparaffinized and rehydrated sections were stained with hematoxylin and eosin Immunohistochemical staining for antibacterial protein was performed using the avidin–biotin–peroxidase complex method using a Histofine SAB-PO (MULTI) kit (Nichirei, Tokyo, Japan) in accordance with the manufacturer’s instructions Sections FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS 463 A flounder LAAO-like antibacterial protein K Kasai et al were counterstained with hematoxylin for microscopic examination Anti-psLAAO IgG or control IgG was used as the primary antibody (1 : 1000 dilution) To characterize the psLAAO-positive cells, HIO4 ⁄ Schiff, alcian blue (pH 2.6) and alcian blue-HIO4 ⁄ Schiff staining reactions were performed Neutralization assay of antibacterial activity Samples of MRSA (clinical isolates) were suspended in TSA at a final concentration of · 106 CFmL)1 Then, 1–10 lL of anti-psLAAO IgG was added to 10 lL of either mucus or gill extract and applied to holes 2.8 mm in diameter After overnight incubation at 37 °C, the diameter of the clear zone around each hole was measured Acknowledgements We are grateful to Dr M Senda for advice, to J Oikawa for technical assistance, to APRO Life Science Institute Inc (Naruto, Tokushima, Japan) for performing amino acid sequence analysis and to the Jusanko fishermen’s cooperative association (Goshogawara, Aomori, Japan) for provision of P stellatus This work was supported, in part, by a Grant for Priority Research Designated by the Japan Science and Technology Agency References Kubota Y, Watanabe Y, Otsuka H, Tamiya T, Tsuchiya T & Matsumoto JJ (1985) Purification and characterization of an antibacterial factor from snail mucus Comp Biochem Physiol C 82, 345–348 Simmaco M, Mignogna G & Barra D (1998) Antimicrobial peptides from amphibian skin: what they tell us? Biopolymers 47, 435–450 Andreu D & Rivas L (1998) Animal antimicrobial peptides: an overview Biopolymers 47, 415–433 Bulet P, Stocklin R & Menin L (2004) Anti-microbial ă peptides: from invertebrates to vertebrates Immunol Rev 198, 169–184 Hellio C, Pons AM, Beaupoil C, Bourgougnon N & Gal YL (2002) Antibacterial, antifungal and cytotoxic activities of extracts from fish epidermis and epidermal mucus Int J Antimicrob Agents 20, 214–219 Ellis AE (2001) Innate host defense mechanisms of fish against viruses and bacteria Dev Comp Immunol 25, 827–839 ´ Magnadottir B (2006) Innate immunity of fish (overview) Fish Shellfish Immunol 20, 137–151 Zanetti M, Gennaro R & Romeo D (1995) Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain FEBS Lett 374, 1–5 464 Chang CI, Zhang YA, Zou J, Nie P & Secombes CJ (2006) Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and atlantic salmon (Salmo salar) Antimicrob Agents Chemother 50, 185–195 10 Lauth X, Shike H, Burns JC, Westerman ME, Ostland VE, Carlberg JM, Van Olst JC, Nizet V, Taylor SW, Shimizu C et al (2002) Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass J Biol Chem 277, 5030–5039 11 Smith VJ, Fernandes JM, Jones SJ, Kemp GD & Tatner MF (2000) Antibacterial proteins in rainbow trout, Oncorhynchus mykiss Fish Shellfish Immunol 10, 243– 260 12 Lemaıˆ tre C, Orange N, Saglio P, Saint N, Gagnon J & Molle G (1996) Characterization and ion channel activities of novel antibacterial proteins from the skin mucosa of carp (Cyprinus carpio) Eur J Biochem 240, 143–149 13 Cole AM, Weis P & Diamond G (1997) Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder J Biol Chem 272, 12008–12013 14 Nagashima Y, Kikuchi N, Shimakura K & Shiomi K (2003) Purification and characterization of an antibacterial protein in the skin secretion of rockfish Sebastes schlegeli Comp Biochem Physiol C 136, 63–71 15 Molle V, Campagna S, Bessin Y, Ebran N, Saint N & Molle G (2008) First evidence of the pore-forming properties of a keratin from skin mucus of rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri) Biochem J 411, 33–40 16 Ebran N, Julien S, Orange N, Auperin B & Molle G (2000) Isolation and characterization of novel glycoproteins from fish epidermal mucus: correlation between their pore-forming properties and their antibacterial activities Biochim Biophys Acta 1467, 271–280 17 Du XY & Clemetson KJ (2002) Snake venom L-amino acid oxidases Toxicon 40, 659–665 18 Vallon O (2000) New sequence motifs in flavoproteins: evidence for common ancestry and tools to predict structure Proteins 38, 95–114 19 Wellner D & Meister A (1961) Studies on the mechanism of action of L-amino acid oxidase J Biol Chem 236, 2357–2364 20 Franca SC, Kashima S, Roberto PG, Marins M, Ticli ¸ ´ FK, Pereira JO, Astolfi-Filho S, Stabeli RG, Magro AJ, Fontes MR et al (2007) Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling Biochem Biophys Res Commun 355, 302–306 21 Tempone AG, Andrade HF Jr, Spencer PJ, Lourenco ¸ CO, Rogero JR & Nascimento N (2001) Bothrops moojeni venom kills Leishmania spp with hydrogen FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS K Kasai et al 22 23 24 25 26 27 28 29 30 peroxide generated by its L-amino acid oxidase Biochem Biophys Res Commun 280, 620–624 ´ Stabeli RG, Marcussi S, Carlos GB, Pietro RC, Selistre-de-Araujo HS, Giglio JR, Oliveira EB & Soares ´ AM (2004) Platelet aggregation and antibacterial effects of an l-amino acid oxidase purified from Bothrops alternatus snake venom Bioorg Med Chem 12, 2881–2886 Zhang YJ, Wang JH, Lee WH, Wang Q, Liu H, Zheng YT & Zhang Y (2003) Molecular characterization of Trimeresurus stejnegeri venom L-amino acid oxidase with potential anti-HIV activity Biochem Biophys Res Commun 309, 598–604 Sant’Ana CD, Menaldo DL, Costa TR, Godoy H, Muller VD, Aquino VH, Albuquerque S, Sampaio SV, ´ Monteiro MC, Stabeli RG et al (2008) Antiviral and antiparasite properties of an L-amino acid oxidase from the snake Bothrops jararaca: cloning and identification of a complete cDNA sequence Biochem Pharmacol 76, 279–288 Ciscotto P, Machado de Avila RA, Coelho EA, Oliveira J, Diniz CG, Farı´ as LM, de Carvalho MA, Maria WS, Sanchez EF, Borges A et al (2009) Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom Toxicon 53, 330–341 Sun Y, Nonobe E, Kobayashi Y, Kuraishi T, Aoki F, Yamamoto K & Sakai S (2002) Characterization and expression of L-amino acid oxidase of mouse milk J Biol Chem 277, 19080–19086 Nagaoka K, Aoki F, Hayashi M, Muroi Y, Sakurai T, Itoh K, Ikawa M, Okabe M, Imakawa K & Sakai S (2009) L-amino acid oxidase plays a crucial role in host defense in the mammary glands FASEB J 23, 2514– 2520 Kitani Y, Tsukamoto C, Zhang G, Nagai H, Ishida M, Ishizaki S, Shimakura K, Shiomi K & Nagashima Y (2007) Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli FEBS J 274, 125–136 Jung SK, Mai A, Iwamoto M, Arizono N, Fujimoto D, Sakamaki K & Yonehara S (2000) Purification and cloning of an apoptosis-inducing protein derived from fish infected with Anisakis simplex, a causative nematode of human anisakiasis J Immunol 165, 1491–1497 Obara K, Otsuka-Fuchino H, Sattayasai N, Nonomura Y, Tsuchiya T & Tamiya T (1992) Molecular cloning of the antibacterial protein of the giant African snail, ´ Achatina fulica Ferussac Eur J Biochem 209, 1–6 A flounder LAAO-like antibacterial protein 31 Petzelt C, Joswig G, Stammer H & Werner D (2002) Cytotoxic cyplasin of the sea hare, Aaplysia punctata, cDNA cloning, and expression of bioactive recombinants in insect cells Neoplasia 4, 49–59 32 Yang H, Johnson PM, Ko KC, Kamio M, Germann MW, Derby CD & Tai PC (2005) Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid oxidase from ink of the sea hare Aplysia californica J Exp Biol 208, 3609– 3622 33 Ehara T, Kitajima S, Kanzawa N, Tamiya T & Tsuchiya T (2002) Antimicrobial action of achacin is mediated by L-amino acid oxidase activity FEBS Lett 531, 509–512 34 Kitani Y, Kikuchi N, Zhang G, Ishizaki S, Shimakura K, Shiomi K & Nagashima Y (2008) Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii Comp Biochem Physiol B 149, 394–400 35 Boyce JM, Cookson B, Christiansen K, Hori S, Vuopio-Varkila J, Kocagoz S, Oztop AY, ă Vandenbroucke-Grauls CM, Harbarth S & Pittet D (2005) Meticillin-resistant Staphylococcus aureus Lancet Infect Dis 5, 653–663 36 Wyllie DH, Peto TE & Crook D (2005) MRSA bacteraemia in patients on arrival in hospital: a cohort study in Oxfordshire 1997-2003 BMJ 331, 992–997 37 Takeda S, Yasunaka K, Kono K & Arakawa K (2000) Methicillin-resistant Staphylococcus aureus (MRSA) isolated at Fukuoka University Hospital and hospitals and clinics in the Fukuoka city area Int J Antimicrob Agents 14, 39–43 38 National Nosocomial Infections Surveillance System (2004) National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004 Am J Infect Control 32, 470–485 39 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 40 O’Farrell PH (1995) High resolution two-dimensional electrophoresis of proteins J Biol Chem 250, 4007– 4021 41 Hirsch FW, Nall KN, Busch FN, Morris HP & Busch H (1978) Comparison of abundant cytosol proteins in rat liver, Novikoff hepatoma, and Morris hepatoma by two-dimensional gel electrophoresis Cancer Res 38, 1514–1522 FEBS Journal 277 (2010) 453–465 ª 2009 The Authors Journal compilation ª 2009 FEBS 465 ... antibacterial protein in the epidermal mucus of the flounder Platichthys stellatus This species, which has a rich covering of mucus on its body surface, inhabits brackish water at the mouths of. .. the antibacterial component of PSEM is a protein Purification of antibacterial protein from PSEM Fig Antibacterial activity of PSEM against (A) Staphylococcus epidermidis, (B) Staphylococcus aureus. .. The present communication describes the isolation and cloning of this LAAO-like antibacterial protein from P stellatus Results Antibacterial activity of mucus It is assumed that Platichthys stellatus- derived