Báo cáo khoa học: Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli pot

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli Yoichiro Kitani 1 , Chihiro Tsukamoto 1 , GuoHua Zhang 1 , Hiroshi Nagai 2 , Masami Ishida 2 , Shoichiro Ishizaki 1 , Kuniyoshi Shimakura 1 , Kazuo Shiomi 1 and Yuji Nagashima 1 1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Japan 2 Department of Ocean Science, Tokyo University of Marine Science and Technology, Japan Fish have humoral factors elaborated in a nonspecific defense system against infectious agents [1–3]. The mucus layer covering the surface of fish has a mechan- ical protective function and also contains a variety of biologically active substances, such as complements, immunoglobulins, lectins, protease inhibitors and lytic enzymes including lysozyme, that may act as defense substances [4–6]. Antimicrobial agents are thought to play an especially important role in the innate immu- nity of fish, as fish are in intimate contact with the aquatic environment, which is rich in pathogenic viru- lence. Indeed, the skin and skin mucus of fish have been shown to contain antibacterial peptides, including pardaxin (a 33-residue peptide of Moses sole fish Pardachirus marmoratus) [7], pleurocidin (a 25-residue peptide of winter flounder Pleuronectes americanus) [8,9], grammistins (12–28-residue peptides of soapfishes Grammistes sexlineatus and Pogonoperca punctata) [10,11] and moronecidin (a 22-residue peptide of hybrid striped bass Morone chrysops · Morone saxatri- lis) [12]. Although the antibacterial peptides described above are highly heterogeneous with respect to their Keywords antibacterial protein; innate immunity; L-amino acid oxidase; rockfish Sebastes schlegeli; skin mucus Correspondence Y. Nagashima, Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 108-8477, Japan Fax: +81 35463 0614 Tel: +81 35463 0604 E-mail: yujicd@kaiyodai.ac.jp (Received 30 August 2006, revised 19 October, accepted 6 November 2006) doi:10.1111/j.1742-4658.2006.05570.x Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibac- terial 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 chromato- graphy, 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 -genera- tion assay and substrate specificity for only l-Lys with a K m of 0.19 mm.It showed potent antibacterial activity against fish pathogens such as Aero- monas 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. Abbreviations AIP, apoptosis-inducing protein; ConA, concanavalin A; LAO, L-amino acid oxidase; MIC, minimum inhibitory concentration; SSAP, Sebastes schlegeli antibacterial protein. FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS 125 primary structure, they are all positively charged, are mostly amphipathic, and can form a-helical or b-sheet structures in membrane-like environments, leading to membrane destabilization and channel formation in bacterial cells. Recently, the following histones and derived peptides have also been identified as antimicro- bial polypeptides in fish skin secretion: histone H2A and oncorhycin II (histone H1 C-terminal fragment, a 69-residue peptide) from rainbow trout, Oncorhyn- chus mykiss [13,14], histone H2B from Atlantic cod, Gadus morhua [15], histone H2B-like protein from channel catfish, Ictalurus punctatus [16], parasin I (N- terminus of histone H2A, a 19-residue peptide) from catfish, Parasilurus asotus [17], hipposin (histone H2A fragment, a 51-residue peptide) from Atlantic halibut, Hippoglossus hippoglossus [18] and SAMP H1 (N-ter- minus of histone H1, a 30-residue peptide) from Atlan- tic salmon, Salmo salar [19]. In addition, other kinds of antibacterial peptides derived from ribosomal and chromosomal proteins have been detected in the skin secretions of rainbow trout, O. mykiss and Atlantic cod, G. morhua [15,20,21]. The antibacterial peptides from cytosolic or nuclear proteins appear to kill a wide range of Gram-positive and Gram-negative bacteria, although their mode of action is not fully understood. We have found a potent antibacterial protein with strict selectivity against Gram-negative bacteria from the skin secretion of rockfish, Sebastes schlegeli [22]. It is of particular interest that this protein is selective against water-borne pathogenic bacteria such as Aeromonas hydrophila, Aeromonas salmonicida, Photo- bacterium damselae ssp. piscicida and Vibrio parahae- molyticus but not against enteric bacteria such as Escherichia coli and Salmonella typhimurium, suggest- ing the importance of the antibacterial protein as a primary innate immune strategy in the rockfish skin. In a previous study [22], we obtained the antibacterial protein by lectin affinity column chromatography and gel filtration HPLC and reported it to be a glycopro- tein with a molecular mass of 150 kDa. However, dur- ing subsequent purification, the antibacterial activity was found only in the later part of the symmetrical peak obtained by gel filtration HPLC. Also, the con- tent of the antibacterial protein in the symmetrical peak was found to be very low compared with that of the 150-kDa protein, leading to our previous misidenti- fication of the 150-kDa protein as an antibacterial pro- tein. Therefore, the antibacterial protein was again purified by a revised method to clarify its structure and functional features in detail. We report here that the antibacterial protein purified from the skin mucus of S. schlegeli (SSAP) is a 120-kDa glycoprotein, being a new member of the l-amino acid oxidase (LAO) family, and that its antibacterial action is elicited by H 2 O 2 generated from l-Lys as substrate. Results Purification and physicochemical properties of SSAP SSAP was purified by three steps of column chroma- tography. It was retained on the concanavalin A (ConA)–Sepharose column and recovered in the manno- pyranoside eluate fraction as reported previously [22] 0 0.1 0.2 0.3 0.4 0.5 0 20 40 60 80 100 120 0 1020304050607080 )UA( ytivitca lairetcabitnA M)( lCaN fo noitartnecnoC Retention time (min) 0 1020304050607080 0 0.1 0.2 0.3 0.4 0 5 10 15 20 ) U A( y tivi t ca lairetca b itnA M) ( et ah p s o hp f o n oi tartn e c noC Retention time (min) A B 40 10 20 30 0 )Um( mn 082 ta ecnabrosbA )Um( mn 082 ta ecnabrosbA 400 100 200 300 0 500 Fig. 1. Purification of antibacterial protein, SSAP, by Mono Q HR 5 ⁄ 5 anion-exchange HPLC (A) and CHT5-I hydroxyapatite HPLC (B). (A) Antibacterial fractions obtained by ConA–Sepharose column chromatography were applied to a Mono Q HR 5 ⁄ 5 column, which was developed with 0.01 M Tris ⁄ HCl buffer (pH 7.4) for 10 min and then with a linear gradient of NaCl (0–0.5 M in 60 min) in 0.01 M Tris ⁄ HCl buffer (pH 7.4) at a flow rate of 0.5 mLÆmin )1 . (B) The active fraction from a Mono Q HR 5 ⁄ 5 column was applied to a CHT5-I column, which was developed with 0.01 M phosphate buf- fer (pH 6.8) for 10 min and then with a linear gradient method to 0.4 M phosphate buffer (pH 6.8) in 60 min at a flow rate of 0.5 mLÆ min )1 . The eluate was monitored by recording A 280 , collected every minute and used to measure antibacterial activity. Antibacterial LAO of rockfish skin mucus Y. Kitani et al. 126 FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS (data not shown). In anion-exchange HPLC on a Mono Q HR5 ⁄ 5 column, SSAP was eluted between retention times of 47 and 52 min (Fig. 1A). Finally, it was purified by HPLC using a CHT5-I hydroxyapatite column, in which it appeared between retention times of 47 and 49 min (Fig. 1B). Purified SSAP afforded a single peak at a retention time of 17.0 min as analyzed by gel filtration HPLC on a TSKgel G3000SW column (Fig. 2A). In RP-HPLC, it gave a major peak at a retention time of 47.3 min along with a minor one at 23.1 min (Fig. 2B). The absorbance spectrum, in which the major peak exhibited absorption maxima at 213 and 279 nm and the minor peak at 223, 267, 371 and 447 nm (data not shown), suggested the major peak to be a proteinaceous component, and the minor peak a flavin-like chromophore. In native and SDS ⁄ PAGE, purified SSAP afforded only one band (Fig. 3A,B). These results demonstrate that the purified SSAP was homogeneous. As summarized in Table 1, 1.7 mg SSAP was obtained from 150 mL crude skin mucus extract containing 3890 mg protein. The recovery of antibacterial activity was 28%, and 645-fold purifica- tion was achieved on the basis of the minimum inhibi- tory concentration (MIC). Judging from the chromatography results, SSAP is likely to be an acidic glycoprotein with a molecular 1234 0 30 60 90 Retention time (min) 0 20406080 0102030 Retention time (min) A B 200 300 0 100 100 0 50 Absorbance at 280 nm (mU) Absorbance at 220 nm (mU) Concentration of acetonitrile (%) Fig. 2. HPLC of antibacterial protein, SSAP, on a TSKgel G3000SW column (A) and a Chromolith Performance RP-18e column (B). (A) SSAP was subjected to gel filtration HPLC on a TSKgel G3000SW column, elut- ed with 0.5 M NaCl ⁄ 0.01 M Tris ⁄ HCl buffer (pH 8.0) at a flow rate of 0.5 mLÆmin )1 and monitored by recording A 280 . The following proteins were used as a reference; 1, ferr- itin (440 kDa); 2, aldolase (158 kDa); 3, albu- min (67 kDa); 4, ovalbumin (43 kDa). (B) SSAP was subjected to RP-HPLC on a Chromolith Performance RP-18e column, eluted with a linear gradient of acetonitrile (0–90% in 60 min) in 0.1% trifluoroacetic acid at a flow rate of 1.0 mLÆmin )1 and monitored by recording A 220 . AB Fig. 3. SSAP on native PAGE (A) and SDS/PAGE (B) (A) SSAP was subjected to native PAGE using a PhastGel homogeneous 20. (B) SSAP was denatured by heating in 0.01 M Tris ⁄ HCl buffer (pH 6.8) containing 1% SDS and 6% 2-mercaptoethanol and subjected to SDS ⁄ PAGE using a PhastGel gradient 8–25. After development, SSAP was detected using a PhastGel protein silver staining kit. Table 1. Purification of SSAP. MIC is defined as the lowest con- centration that inhibited the growth of P. damselae ssp. piscicida ATCC 51736. Step Protein (mg) MIC (lgÆmL )1 ) Total activity (protein ⁄ MIC) Yield (%) Crude extract 3890 20 195 000 100 ConA–Sepharose 343 2.7 127 000 65 Mono Q HR 5 ⁄ 5 16.1 0.22 73 200 38 CHT5-I 1.7 0.031 54 800 28 Y. Kitani et al. Antibacterial LAO of rockfish skin mucus FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS 127 mass of 120 kDa. SDS ⁄ PAGE analysis revealed a molecular mass of 53 kDa (Fig. 3B), suggesting that SSAP has a dimeric conformation. It was found to contain 1.6% (w ⁄ w) of d-mannose and 0.6% (w ⁄ w) of N-acetyl-d-glucosamine by HPLC after derivatization with 4-aminobenzoic acid ethyl ester. A sugar moiety was effectively liberated by digestion of SSAP with gly- copeptidase F. As seen in Fig. 4, the digest migrated ahead of the intact SSAP and gave no detectable band when the ECL glycoprotein detection module was used, indicating the presence of an N-linked carbohy- drate side chain in SSAP. Purified SSAP was yellow-colored, suggesting the involvement of flavin in the molecule, as supported by the RP-HPLC result. As illustrated in Fig. 5A, the chromophore dissociated from SSAP on heating with SDS showed absorption maxima at 263, 371 and 447 nm, consistent with those of the FAD standard. Figure 5B shows the mass spectrum of the chromo- phore from SSAP. ESI-TOF-MS in the positive mode gave the main peak at m ⁄ z 786.32 and the ion peaks at m ⁄ z 439.60 and 348.63. The former corresponded to the parent ion peak of [M +H] + of FAD (molecular mass 785.56 Da), and the latter ion peaks at m ⁄ z 439.60 and 348.63 were assignable to fragment A (a dehydro ion of adenosine monophosphate, C 10 H 13 N 5 O 7 P) and fragment B (a dehydroxy ion of flavin mononucleotide, C 17 H 20 N 4 O 8 P), respectively, accord- ing to a rule of mass shift in fragmentation [23]. These results provide evidence that FAD is the chromophore of SSAP. The CD spectrum of SSAP is shown in Fig. 6. SSAP clearly gave two negative peaks at  208 and 222 nm in 0.5 m NaCl ⁄ 0.01 m Tris ⁄ HCl buffer (pH 8.0), indicating B 50 37 150 100 75 12 12M (kDa) A Fig. 4. SSAP on SDS ⁄ PAGE (A) and poly(vinylidene difluoride) membrane (B) before and after treatment with glycopeptidase F. (A) Protein was detected using a PhastGel protein silver staining kit. (B) Glycoprotein was visualized using an ECL glycoprotein detection module. Lane M, molecular mass marker; lane 1, SSAP; lane 2, SSAP treated with glycopeptidase F. B A 250 300 350 400 450 500 ecnabrosbA Wave length (nm) FAD standard Chromophore from SSAP A B FAD standard 348.62 439.60 786.30 100 100 348.63 439.60 786.32 100 200 300 400 500 600 700 800 900 m/z 100 200 300 400 500 600 700 800 900 m/z Chromophore from SSAP )%( ytisnetnI )%( ytisnetnI 0 0 Fig. 5. Absorption spectra (A) and mass spectra (B) of FAD stand- ard and the chromophore derived from SSAP. The chromophore was obtained by boiling SSAP in 1% SDS for 10 min, ultrafiltration using an Ultrafree-MC, and RP-HPLC on a Chromolith Performance RP-18e column with a linear gradient of acetonitrile (5–25% in 10 min) in 0.1% trifluoroacetic acid at a flow rate of 1.0 mLÆmin )1 . FAD standard was obtained by application of FAD sodium salt hydrate to RP-HPLC under the same conditions as the chromo- phore. The inset in (B) illustrates the structure of FAD. Antibacterial LAO of rockfish skin mucus Y. Kitani et al. 128 FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS a-helical conformation. The a-helical content of SSAP in the buffer was found to be 20% and increased to 28% on the addition of SDS irrespective of concentration in the range 0.1–2.5%. cDNA cloning and sequence analysis of SSAP The N-terminal amino-acid sequence of SSAP was determined to be ISLRDNLAD. Of the peptide frag- ments isolated from the lysyl endopeptidase digest by RP-HPLC, three (fragments 1–3) were randomly selec- ted and sequenced as follows: SADELLQHALQK for fragment 1, EGWYAELGAMRIPS for fragment 2, and SYTWSDDSLLFLGASDED for fragment 3. A cDNA fragment was successfully amplified by 3¢ RACE using the degenerate primers designed from the amino-acid sequence of fragment 1. On the basis of the nucleotide sequence of this cDNA fragment, the remaining 5¢ region sequence was determined by 5¢ RACE. Thus, the nucleotide sequence of the full-length SSAP cDNA (2037 bp) was elucidated (DDBJ accession number AB218876). The accuracy of this sequence was verified by recloning experiments. The 5¢-untranslated region contains stop codons TAA and TGA at nucleotides 4–6 and 31–33, respectively, upstream of the initial codon ATG present at nucleotides 46–48. In the 3¢-untrans- lated region, a polyadenylation signal AATAAA is observed at nucleotides 1997–2002, and a poly(A) tail at nucleotide 2027. An ORF is composed of 1662 bp, encoding a precursor protein of 554 amino-acid residues from the putative initiating Met to the putative last Leu. The amino-acid sequences of the N-terminal portion and the lysyl endopeptidase fragments 1–3 determined by protein sequencing are all found at positions 59–67 (N-terminal portion), 138–151 (fragment 2), 210–221 (fragment 1) and 436–453 (fragment 3) of the deduced molecule (Fig. 7). signalp version 3.0 (http://www.cbs.dtu.dk/services/ SignalP/) predicted that SSAP consists of a signal pep- tide (Met1–Ala58) and a mature protein starting with Ile59, in accord with the result of N-terminal protein sequence analysis. It is likely that the mature protein is composed of 496 amino acids with a calculated molecular mass of 55 260.63 Da. A blast homology search showed the similarity of the deduced amino- acid sequence of SSAP to LAOs. Amino-acid sequence alignment analysis using clustalw (version 1.83) revealed that SSAP shows a weak homology to anti- bacterial LAOs, such as aplysianin A (11%), achacin (12%) and escapin (12%), and a moderate homology to Pseudechis australis snake venom LAO (42%). The highest identity (76%) was observed with the apopto- sis-inducing protein (AIP) derived from the viscera of mackerel infected with the nematode, Anisakis simplex [24] (Fig. 7). LAO activity of SSAP SSAP exhibited high LAO activity, with a specific activity of 10.2 UÆmg )1 by the H 2 O 2 -generation method. SSAP catalyzed oxidation of only l-Lys and was ineffective with any of the other proteinaceous amino acids tested. No LAO activity was detected when l-Lys was replaced with d-Lys (Fig. 8). From a Lineweaver–Burk plot of LAO activity of SSAP (data not shown), K m and k cat were evaluated to be 0.19 mm and 20.4 s )1 , respectively. Antibacterial action of SSAP As shown in Table 2, SSAP specifically inhibited the growth of Gram-negative bacteria, being most active against A. salmonicida with an MIC of 0.078 lgÆmL )1 , followed by P. damselae ssp. piscicida, A. hydrophila and V. parahaemolyticus with MIC of 0.16, 0.31 and 0.63 lgÆmL )1 , respectively. LAOs have been reported to show antibacterial activity through H 2 O 2 generated from amino-acid substrates, which is markedly diminished in the presence of H 2 O 2 scaven- gers such as catalase and peroxidase [25–27]. In the present study therefore the inhibitory effect of Fig. 6. CD spectra of SSAP. CD analyses were performed using a quartz optical cell with 10-mm path length at 25 °C. Each spectrum represents the mean of three measurements in the range 200– 250 nm at 0.5-nm intervals. Solvent: (m) 0.5 M NaCl ⁄ 0.01 M Tris ⁄ HCl buffer (pH 8.0) and (n) 0.1% SDS, (s) 0.5% SDS and (d) 2.5% SDS in 0.5 M NaCl ⁄ 0.01 M Tris ⁄ HCl buffer (pH 8.0). Y. Kitani et al. Antibacterial LAO of rockfish skin mucus FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS 129 catalase on the antibacterial activity of SSAP was examined. The addition of catalase almost completely abolished the antibacterial activity of SSAP, indica- ting that H 2 O 2 is the mediator of the activity of SSAP. Discussion This study is the first to discover an LAO with anti- bacterial activity in the skin mucus of a teleost and to reveal the involvement of LAO in the innate immunity Fig. 7. Amino-acid sequence alignment of SSAP (DDBJ accession number AB218876) with AIP from mackerel Scomber japonicus (DDBJ accession number AJ400871), PA-LAO from Pseudechis australis snake venom (DDBJ accession number DQ088992), aplysianin A from Aplysia kurodai (DDBJ accession number D83255), escapin from Aplysia californica (DDBJ accession number AY615888) and achacin from Acatina fulica (DDBJ accession number X64584). Identical amino acids are shaded. Potential N-glycosylation sites are boxed. Gaps intro- duced into the sequences to optimize the alignment are represented by dashes. The N-terminal sequence (59–67) and intrapeptide frag- ments 1 (210–221), 2 (138–151) and 3 (436–453) of SSAP are thick underlined. Antibacterial LAO of rockfish skin mucus Y. Kitani et al. 130 FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS of fish skin. In a previous report [22], the antibacterial protein was obtained from the skin secretion of rock- fish S. schlegeli by lectin affinity chromatography and gel filtration HPLC and misidentified as a 150-kDa glycoprotein consisting of 75-kDa subunits by SDS ⁄ PAGE, partly because of the scarcity of the protein of interest. In this study therefore we isolated the antibac- terial protein, SSAP, from the skin mucus of S. schle- geli by a combination of three different types of column chromatography and carefully examined the degree of purity by HPLC as well as PAGE. Further- more, we elucidated the primary structure of SSAP by cDNA cloning and identified SSAP to be a new mem- ber of the LAO family by physicochemical and bio- chemical analyses. LAOs (EC 1.4.3.2) catalyze the stereospecific oxidative deamination of an l-amino- acid substrate to a corresponding a-oxoacid with the production of H 2 O 2 and ammonia, via an imino-acid intermediate. It is well known that these enzymes are widely distributed across diverse phyla from bacteria to mammals. LAOs in micro-organisms appear to be involved in the utilization of ammonia as a nitrogen source, and those in animals have been characterized as showing distinct biological and physiological effects such as apoptosis, cytotoxicity, hemolysis, platelet aggregation, hemorrhage, edema and antimicrobial activity [28]. Since Skarnes [29] first found antibacterial activity in an LAO from snake (Crotalus adamanteus) venom, antibacterial LAOs have been reported from snake venoms of Ps. australis [30], Trimeresurus jerdo- nii [31] and Bothrops alternatus [32], the body surface mucus of the giant African snail, Achatina fulica Fe ´ russac (termed achacin) [26], the albumen gland of the sea hare, Aplysia kurodai (termed aplysianin A) [27], and the ink of the sea hare, A. californica (termed escapin) [33]. LAO family members possess in common flavin as a coenzyme and two motifs, a dinucleotide-binding motif comprising b-strand ⁄ a-helix ⁄ b-strand of the secondary structure, and a GG motif (R-x-G-G-R-x-x-T ⁄ S) shortly after the dinucleotide-binding motif [34]. In the case of SSAP, FAD was identified as a coenzyme. Moreover, the dinucleotide-binding motif to which FAD binds was certainly recognized at amino-acid resi- dues His93–Glu121, and the GG motif at amino-acid residues Arg125–Thr132 (Fig. 7). A blast search found that SSAP shares the highest sequence identity (76%) with AIP from Anisakis-infected mackerel. The secon- dary structures of SSAP and AIP appear to be similar to each other. The a-helix content of SSAP was deter- mined to be 28.2% in 2.5% SDS solution by CD spectrometry and calculated to be 31.6% by predator (http://bioweb.pasteur.fr/seqanal/interfaces/predator- simple.html), and that of AIP was estimated to be 26.9% by predator. In addition, both SSAP and AIP have strict specificity with respect to the substrate, cata- lyzing the oxidation of only l-Lys. These results suggest structural similarity between their substrate-binding sites. SSAP and AIP have two and five potential N-glyco- sylation sites, respectively. One site at residues 89–91 is common to both LAOs. SSAP was determined to con- tain 2 mol d-mannose and 5 mol N-acetyl-d-glucosa- mine per mol of subunit. Treatment of SSAP with glycopeptidase F resulted in deglycosylation (Fig. 4), but did not reduce antibacterial activity, suggesting no or little involvement of the sugar moiety in the antibacterial effect of SSAP. It has been reported that Fig. 8. Dependence of substrate concentration on LAO activity of SSAP. LAO activity was measured by the H 2 O 2 -generation assay. Reaction rate is defined as generation of H 2 O 2 per min. d, L-Lys; s, D-Lys. Table 2. Antibacterial spectrum of SSAP. MIC is defined as the lowest concentration that inhibited the growth of bacteria. Bacterium MIC (lgÆmL )1 ) SSAP SSAP with catalase Gram-positive B. subtilis IAM1026 > 5.0 > 5.0 M. luteus IAM1056 > 5.0 > 5.0 Staph. aureus IAM1011 > 5.0 > 5.0 Gram-negative A. hydrophila IAM12337 0.31 > 5.0 A. salmonicida JCM7874 0.078 > 5.0 E. coli JCM1649 > 5.0 > 5.0 P. damselae ssp. piscicida ATCC51736 0.16 > 5.0 S. typhimurium SH-1 > 5.0 > 5.0 V. parahaemolyticus NBRC12711 0.63 > 5.0 Y. Kitani et al. Antibacterial LAO of rockfish skin mucus FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS 131 glycosylation is not essential for the antibacterial activ- ity of escapin, which has one putative glycosylation site, as recombinant escapin expressed in bacteria is as active as the native one [33]. On the other hand, Geyer et al. [35] examined the structure of the glycan moiety of the LAO from the Malayan pit viper, Calloselas- ma rhodostoma, and assumed that putative binding of the LAO to sialic acid-binding Ig superfamily lectins via its sialylated glycan moiety results in the produc- tion of locally high concentrations of H 2 O 2 in or near the binding interface. The role of the glycosyl substitu- ents of LAOs in biological activities remains to be elucidated. It should be noted that SSAP only has potent antibac- terial activity against specific Gram-negative bacteria, including fish pathogens (A. hydrophila, A. salmonicida and P. damselae ssp. piscicida) and a marine bacterium (V. parahaemolyticus), but not against enteric Gram- negative bacteria (E. coli and S. typhimurium). In contrast, other antibacterial LAOs, such as achacin, aplysianin A and escapin, broadly show an inhibitory effect on both Gram-negative (E. coli ) and Gram-posit- ive bacteria (Bacillus subtilis and Staphylococcus aure- us). The antibacterial activities of these LAOs, including SSAP, are significantly decreased in the presence of cat- alase, confirming that H 2 O 2 generation by LAOs brings about an oxidative burst in cells that is responsible for cell death. Shur & Kim [36] reported that the LAO from snake (Agkistrodon halys) venom attaches to the cell surface of mouse lymphocytic leukemia L1210, inducing apoptosis in a cell-selective manner. Similarly, achacin from the giant African snail binds to the cell wall of E. coli, and its LAO activity is inhibited by N-acetylneu- raminic acid [26,37]. Therefore, it is possible that the cell-specific antibacterial activity of SSAP is associated with its binding to the bacterial cell surface. Further investigation is in progress to elucidate the bacterium selectivity and the mode of action of SSAP. Finally, SSAP is likely to be important in the innate host defense on the skin of rockfish and might be useful as a chemo- therapeutic agent, especially in the field of aquaculture because of its selective cytotoxicity against water-borne virulent pathogens. Experimental procedures Materials Specimens of S. schlegeli ranging from 28.0 to 32.5 cm in body length were obtained from Minami-Sanriku Marine Center, Motoyoshi, Miyagi Prefecture, Japan, and trans- ported in oxygen-saturated water to our laboratory. Purification of SSAP The rockfish skin mucus was gently scraped off with a spat- ula, combined, and centrifuged at 18 800 g for 30 min using an angle rotor (SCR18B; Hitachi, Tokyo, Japan). The result- ing supernatant was applied to a ConA–Sepharose column (2.0 · 32.0 cm; GE Healthcare Bio-Science, Piscataway, NJ, USA), which was washed with 1 mm CaCl 2 ⁄ 1mm MnCl 2 ⁄ 0.5 m NaCl ⁄ 0.02 m Tris ⁄ HCl buffer (pH 7.4) and then eluted with 0.5 m methyl-a-d-mannopyranoside ⁄ 0.5 m NaCl ⁄ 0.02 m Tris ⁄ HCl buffer (pH 7.4) as reported previ- ously [22]. Antibacterial fractions were pooled and subjected to HPLC on a Mono Q HR5 ⁄ 5 column (0.5 · 5.0 cm; GE Healthcare Bio-Science). The column was developed by a linear gradient of NaCl (0–0.5 m in 60 min) in 0.01 m Tris ⁄ HCl buffer (pH 7.4) at a flow rate of 0.5 mLÆmin )1 . Finally, the antibacterial protein was purified by HPLC on a CHT5-I hydroxyapatite column (1.0 · 6.4 cm; Bio-Rad Laboratories, Hercules, CA, USA) with a linear gradient elution of 0.01–0.4 m phosphate buffer (pH 6.8) in 60 min at a flow rate of 0.5 mLÆmin )1 . At each chromatographic step, proteins were monitored by recording A 280 and the antibac- terial protein by growth inhibition against P. damselae ssp. piscicida. The purified antibacterial protein was SSAP. For purity determination, SSAP was subjected to either gel filtration HPLC on a TSKgel G3000SW column (0.78 · 30 cm; Tosoh, Tokyo, Japan) with 0.5 m NaCl ⁄ 0.01 m Tris ⁄ HCl buffer (pH 8.0) at a flow rate of 0.5 mLÆ min )1 or RP-HPLC on a Chromolith Performance RP-18e column (0.46 · 10 cm; Merck, Darmstadt, Germany) with a linear gradient of acetonitrile (0–90% in 60 min) in 0.1% trifluoroacetic acid at a flow rate of 1.0 mLÆmin )1 . SSAP was monitored by recording A 220 or A 280 with a photodiode array detector. SSAP was analyzed for its homogeneity by PAGE with a PhastSystem apparatus (GE Healthcare Bio-Science) according to the manufacturer’s instructions. Native PAGE was carried out on a PhastGel homogeneous 20 with PhastGel native buffer strips, and SDS ⁄ PAGE on a PhastGel gradient 8–25 with PhastGel SDS buffer strips. Before SDS ⁄ PAGE, SSAP was dissolved in 0.01 m Tris ⁄ HCl buffer (pH 6.8) containing 1% SDS and 6% 2-mercaptoeth- anol and heated in a boiling-water bath for 5 min. After being run, the gel was stained with a PhastGel protein silver staining kit (GE Healthcare Bio-Science). Analysis of amino-acid sequence Amino-acid sequence analyses of SSAP and its peptide fragments produced by digestion with lysyl endopeptidase (EC 3.4.21.50; Wako Pure Chemical Industries, Osaka, Japan) were performed with an automatic gas-phase sequencer (LF-3400D TriCart with high sensitivity chem- istry; Beckman Coulter, Fullerton, CA, USA). To produce peptide fragments, SSAP (60 lg) was denatured in 400 lL Antibacterial LAO of rockfish skin mucus Y. Kitani et al. 132 FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS 4 m urea, diluted twofold with 0.1 m Tris ⁄ HCl buffer (pH 9.3) and digested with 1 lg lysyl endopeptidase at 37 °C for 24 h. The digest was subjected to RP-HPLC on a Chromolith Performance RP-18e column (0.46 · 10 cm; Merck) with a linear gradient of acetonitrile (0–70% in 120 min) in 0.1% trifluoroacetic acid at a flow rate of 0.5 mLÆmin )1 . Peptides were monitored at 220 nm with a photodiode array detector. cDNA cloning Total RNA was extracted from the skin of a live specimen with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First- strand cDNA was synthesized from 5 lg total RNA using a 3¢ RACE System for Rapid Amplification of cDNA Ends Kit (Invitrogen) following the manufacturer’s instructions. Oligonucleotide primers were designed on the basis of the determined amino-acid sequences of peptide fragments. The degenerate primer 5¢-CIGAYGARYTIYTICARAYGCIYT IC-3¢ (forward; corresponding to 211 ADELLQHAL 219 ) and the abridged universal amplification primer (AUAP) 5¢-GG CCACGCGTCGACTAGTAC-3¢ (reverse) were used for the first 3¢ RACE, and the degenerate primer 5¢-AYGARYTIY TICARCAYGCIYTICARAA-3¢ (forward; corresponding to 212 DELLQHALQK 221 ) and the AUAP (reverse) for the sec- ond 3¢ RACE. Amplification was carried out using rTaq polymerase (Takara, Otsu, Japan) under the following condi- tions: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s; and 72 °C for 5 min. The secondary PCR products were subcloned into the pT7Blue T-vector (Novagen, Darmstadt, Germany), and their nucleotide sequences were analyzed with a Thermo Sequenase Cy5 Dye Terminator Sequencing Kit (GE Healthcare Bio-Science) and a Long-Read Tower DNA Sequencer (GE Healthcare Bio-Science). The remaining 5¢-terminal sequence was ana- lyzed by 5¢ RACE as follows. First-strand cDNA was syn- thesized from 5 l g total RNA using a 5¢ RACE System for Rapid Amplification of cDNA Ends Kit (Invitrogen) and the gene-specific primer (5¢-CCTTCTTCTTTCAGATAATCC- 3¢, corresponding to 244 KDYLKEEG 251 ). The first 5¢ RACE reaction was completed using the gene-specific primer (5¢- AGAGTAACGGTCATATTTTAGC-3¢, corresponding to 235 LLKYDRYS 242 ) and the abridged anchor primer (AAP) 5¢-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGI IG-3¢, followed by reamplification of the PCR products using the gene-specific primer (5¢-CCACCTCATCTTGCACCT TC-3¢, corresponding to 220 QKVQDEVE 227 ) and the AUAP. Amplification conditions were the same as described above. The secondary PCR products were subcloned into the pT7Blue T-vector and sequenced. The nucleotide sequence of the full-length SSAP cDNA was confirmed by RT-PCR using the forward primer 5¢-ATAACTTTGGAGACGG AGTTC-3¢ and the reverse primer 5¢-TGGAGGAACATTA GTGGTCC-3¢. Measurement of LAO activity LAO activity was assayed in a 96-well microtiter plate by the peroxidase ⁄ o-phenylenediamine method [24] with slight modifications. For the standard assay, 25 lL SSAP, 25 lL 20 mml-Lys, 25 lL peroxidase (0.2 UÆmL )1 ; Type VI; EC 1.11.1.7; Sigma-Aldrich Corp., St Louis, MO, USA) and 25 lL o-phenylenediamine (0.5 mgÆmL )1 ) were added sequentially to the well. After incubation at 37 °C for 150 min, the reaction was terminated by adding 100 lL1m H 2 SO 4 , and A 490 was measured. The substrate specificity of SSAP was examined using Gly, 19 l-amino acids and d-Lys as the substrates. To determine kinetic parameters, various concentrations (0–5 mm)ofl-Lys were used. K m was evaluated using a Lineweaver–Burk plot by measuring the rate of H 2 O 2 production. Measurement of antibacterial activity Antibacterial activity was measured by a liquid growth-inhi- bition assay in a 96-well microtiter plate, as previously repor- ted [22]. The following nine species of bacteria were used: three species of Gram-positive bacteria, B. subtilis IAM1026, Micrococcus luteus IAM1056 and Staph. aureus IAM1011; six species of Gram-negative bacteria, A. hydrophila IAM12337, A. salmonicida JCM7874, E. coli JCM1649, P. damselae ssp. piscicida ATCC51736, S. typhimurium SH-1 isolated from fish meal, and V. parahaemolyticus NBRC12711. During the isolation procedure of the antibac- terial protein, P. damselae ssp. piscicida was used because of its high sensitivity to SSAP [22]. Briefly, a mixture of 50 lL sample solution and 50 lL bacterial suspension (1 · 10 5 col- ony-forming unitsÆmL )1 ), both of which were made in Muller-Hinton broth medium (Difco Laboratories, Detroit, MI, USA), was incubated at 25 °C for 24 h. After incuba- tion, bacterial growth was observed with the unaided eye. When the culture medium was completely transparent or no precipitate was recognized, inhibition of bacteria growth was judged to have occurred. The reciprocal of the maximum inhibitory dilution was used to calculate arbitrary units (AU) per ml. The MIC was defined as the lowest concentration that inhibited the growth of bacteria. To examine the inhibi- tory effect of catalase on the antibacterial activity of SSAP, 50 U catalase (EC 1.11.1.6; from bovine liver; Wako Pure Chemical Industries) was added to the culture medium con- taining 5 lgÆmL )1 SSAP and bacterial cells, and the mixture was incubated under the same condition as described above. Determination of molecular mass The molecular mass of the native SSAP was determined by gel filtration HPLC on a TSKgel G3000SW column (0.78 · 30 cm; Tosoh) as described above. Four reference Y. Kitani et al. Antibacterial LAO of rockfish skin mucus FEBS Journal 274 (2007) 125–136 ª 2006 The Authors Journal compilation ª 2006 FEBS 133 proteins, ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa) and ovalbumin (43 kDa), were used to calibrate the column. The molecular mass of the denatured SSAP was determined by SDS ⁄ PAGE on a PhastGel gradient 8–25 with PhastGel SDS buffer strips (GE Healthcare Bio-Science) as described above. Precision Plus Pro- tein standard (Bio-Rad Laboratories) was used as a refer- ence. Analysis of FAD An aliquot of SSAP (5 lg) was boiled in 200 lL 1% SDS for 10 min and filtered with an Ultrafree-MC (molecular mass cut off 10 kDa; Millipore, Bedford, MA, USA). The filtrate was chromatographed on a Chromolith Performance RP-18e column (0.46 · 10 cm; Merck) with a linear gradi- ent of acetonitrile (5–25% in 10 min) in 0.1% trifluoroace- tic acid at a flow rate of 1.0 mLÆmin )1 , with monitoring by recording A 447 . The eluate containing the chromophore was collected and subjected to spectrophotometry and MS. The MS ⁄ MS measurement of the chromophore was performed using a Q-tof Ultimate API (Micromass, Altrincham, UK) in the positive-ion mode under the following conditions: capillary voltage, 3200 V; cone voltage, 100 V; desolvation temperature, 150 °C; source temperature, 80 °C; collision gas, argon. CD spectrometry SSAP (30 lg) was dissolved in 3 mL 0.5 m NaCl ⁄ 0.01 m Tris ⁄ HCl buffer (pH 8.0) containing 0%, 0.1%, 0.5% or 2.5% SDS and subjected to CD analysis with a CD spec- tropolarimeter (J-720; Jasco, Tokyo, Japan). The CD spec- tra were measured at 25 °C with 40 scans per min in the range 200–250 nm at 0.5-nm intervals. a-Helical contents were calculated by the method of Greenfield & Fasman [38]. Carbohydrate analysis SSAP (2 lg) was deglycosylated by incubation with glyco- peptidase F (25 · 10 )3 U; EC 3.5.1.52; Sigma-Aldrich Corp.) in 10 lL 0.05 m Tris ⁄ HCl buffer (pH 8.6) at 37 °C for 18 h and subjected to SDS ⁄ PAGE. The proteins separated by SDS ⁄ PAGE were electrotransferred from the gel to a poly(vinylidene difluoride) membrane and visualized by the chemical luminescence method using an ECL glycoprotein detection module (GE Healthcare Bio-Science) following the manufacturer’s instructions. Total sugar composition analy- sis was performed using the ABEE (4-aminobenzoic acid ethyl ester) labeling kit plus S (Seikagaku Corporation, Tokyo, Japan) and HPLC on a Honenpak C18 (0.46 · 7.5 cm; Seikagaku Corporation), as reported previ- ously [22]. Determination of protein Protein was determined by the micro bicinchoninic acid method [39] using BSA as a standard protein. Acknowledgements This study was partly supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. We thank Dr Y. Shida, Tokyo College of Pharmacy, for MS analysis for FAD, and Mr T. Katsukura and Mr H. Oikawa, Minami-Sanriku Marine Center, for provision of the rockfish specimens. References 1 Iwama G & Nakanishi T (1996) The Fish Immune System. Academic press, San Diego, CA. 2 Bayne CJ & Gerwick L (2001) The acute phase response and innate immunity of fish. Dev Comp Immunol 25, 725–743. 3 Ellis AE (2001) Innate host defense mechanisms of fish against viruses and bacteria. Dev Comp Immunol 25, 827–839. 4 Alexander JB & Ingram GA (1992) Noncellular nonspecific defense mechanism of fish. 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