Báo cáo khoa học: Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major doc

Eur J Biochem 270, 675–686 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03419.x Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major Noriaki Iijima1, Norio Tanimoto1, Yohko Emoto1, Yohko Morita1, Kazumasa Uematsu2, Tomoya Murakami3 and Toshihiro Nakai4 Laboratory of Molecular Cell Biology, 2Laboratory of Fish Physiology and 4Laboratory of Fish Pathology, Graduate School of Biosphere Science, Hiroshima University, Japan; 3Hiroshima Fisheries Experimental Station, Ondo, Aki, Japan We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence The a-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens The three peptides were also hemolytic Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills Antimicrobial peptides are widely distributed throughout the animal and plant kingdoms [1] They display a broad spectrum of antimicrobial activity against bacteria, yeast and filamentous fungi, and are recognized as an essential component in the first line of the host defense system [2,3] There are three major sites by which bacteria enter fish: the gills, gastrointestinal tract and skin [4] Therefore, antibacterial substances are thought to exist at these sites to prevent penetration of bacteria into the circulatory system In fact, skin mucus, eggs and serum of fish contain a variety of nonspecific defense substances, such as lysozyme, complement, C-reactive protein, transferrin, lectin, and antimicrobial proteins [5–10] Furthermore, antimicrobial peptides have been purified from fish skin mucus: pardaxin from the moses sole fish Pardachirus marmoratus [11], pleurocidin from the winter flounder Pleuronectes americanus [12] and parasin I from the catfish Parasilurus asotus [13], and the gene expression of pleurocidin-like antimicrobial peptides found in the skin and intestine of the winter flounder [14] The antimicrobial peptide, misgurin, has also been purified from the whole body of the loach Misgurnus anguillicaudatus [15] These antibacterial peptides show potent antimicrobial activity against Gram-negative and Grampositive bacteria and act as nonspecific defense substances in fish skin Fish gills are constantly being flushed with water that may contain fish pathogens, but are covered with only a thin layer of protective mucus and are constructed of only a single layer of fragile cells that separate the vascular system from the external environment Thus, they are a very important site of pathogen penetration Therefore, potent antimicrobial peptides can be expected to be found in fish gills to prevent such penetration However, there is a paucity of information on nonspecific defense systems in the gills An antimicrobial peptide has been identified in the gills of only one fish species, hybrid striped bass (Morone saxatilis · M chrysops) [16–18] In this study, we therefore tried to purify antimicrobial peptides from the gills of the red sea bream Chrysophrys (formerly Pagrus) major and found that novel antimicrobial peptides, chrysophsin-1, chrysophsin-2, and chrysophsin-3, are localized in the eosinophilic granule cell-like cells of the gills They exhibited potent bactericidal activity against Gram-negative and Gram-positive pathogens of fish and crustaceans Correspondence to N Iijima, Laboratory of Molecular Cell Biology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashihiroshima 739-8528, Japan Fax: + 81 824 22 7059, Tel.: + 81 824 24 7949, E-mail: noriiij@jpc.hiroshima-u.ac.jp Abbreviations: EGC, eosinophilic granule cell; ESI-ITMS, electrospray ionization/ion trap mass spectrometry; MLC, minimal lethal concentration; mcKLH, Imject maleimide-activated mariculture keyhole limpet hemocyanin; NaCl/Pi, 50 mM phosphate buffer (pH 7.4) containing 0.14 M NaCl; Tris/NaCl, 20 mM Tris/HCl (pH 7.4) containing 150 mM NaCl (Received August 2002, revised 29 November 2002, accepted December 2002) Keywords: antimicrobial peptide; chrysophsin; gills; red sea bream; synthetic peptide 676 N Iijima et al (Eur J Biochem 270) Ó FEBS 2003 Materials and methods Chemicals Fmoc-L-amino acids, Fmoc-L-amino acid resins and TentaGelÒ S (TGS)-RAM were purchased from Shimadzu (Kyoto, Japan) Chemicals for peptide synthesis, trifluoroacetic acid, ethylmethylsulfide, ethanedithiol, thiophenol, 2-methylindole, thioanisole, phenol, and anisole were obtained from commercial sources and were of the highest purity available Lysyl endopeptidase, a silver staining II kit and 2,2,2-trifluoroethanol were purchased from Wako Pure Chemicals (Tokyo, Japan) A TSKgel G2000SW column was obtained from Tosoh (Tokyo, Japan), an Inertsil C8-3 column from GL Science (Tokyo, Japan), and a Capcellpak C18 column from Shiseido (Tokyo, Japan) SP-Sephadex C-25 was purchased from Pharmacia Biotech (Uppsala, Sweden), and PolySulfoethyl Aspartamide column was from Poly LC Inc (Columbia, MD, USA) Melittin and BSA were obtained from ICN Biomedicals Inc (Aurora, OH, USA), Imject maleimide activated mariculture keyhole limpet hemocyanin (mcKLH) and Imject Alum were from Pierce (Rockford, IL, USA), Simple Stain MAX-PO (Multi) and Simple Stain DAB solution were from Nichirei (Tokyo, Japan), peroxidase-labeled affinity-purified antibody to rabbit IgG (H + L) was from KPL (Gaithersburg, MD, USA), DC-protein assay kit was from Bio-Rad (Hercules, CA, USA), and marine broth 2216 was from DIFCO (Detroit, MI, USA) Fig Flow chart of the purification procedure of antimicrobial peptides from the gills of red sea bream Fish Red sea bream weighing 1.05–1.14 kg (n ¼ 11) were cultured by a commercial supplier (Mihara, Hiroshima Prefecture, Japan) After starvation for day, the fish were killed by stabbing the brain with a knife, and gill filaments were immediately collected, frozen in liquid nitrogen, and stored at )80 °C until use Red sea bream weighing 109–305 g (n ¼ 5) were obtained from the Hiroshima Fisheries experimental station (Ondo, Aki, Japan) The gill arches including gill lamellae were immediately fixed in Bouin’s solution and transported on ice to the laboratory for immunohistochemical examination as described below Purification of antimicrobial peptides from the gills The purification procedure is summarized in Fig Frozen gill filaments were crushed in liquid nitrogen The resulting gill powder (12 g) was boiled in 120 mL water for 10 After cooling, extractions were performed by adding 120 mL M HCl, 10% (v/v) formic acid, 2% (w/v) NaCl, and 1% (v/v) trifluoroacetic acid followed by vortex-mixing for 1–2 The homogenate was centrifuged, and the supernatant adjusted to pH by adding M Tris; it was then filtered The resulting filtrate (219 mL) was used as the acid extract and was applied to a Sep-Pak C18 cartridge (Waters, Milford, MA, USA) After a wash with 0.1% trifluoroacetic acid, the peptide was eluted with 80% acetonitrile/0.1% trifluoroacetic acid The dried eluate was dissolved in M acetic acid and then adsorbed on SP-Sephadex C-25 resin Successive elution with M acetic acid, M pyridine and M pyridine/acetic acid (pH 5.0) afforded three respective fractions of SP-1, SP-2 and SP-3 The SP-3 fraction was further lyophilized and dissolved in 40% acetonitrile containing 0.1% trifluoroacetic acid An aliquot of the solution was loaded on a TSKgel G2000SW column (7.6 · 600 mm) equipped with a Tosoh HPLC pump (CCPM) and a UV-VIS detector (UV-8010) and was eluted with 40% acetonitrile containing 0.1% trifluoroacetic acid The SP-3 fraction was repeatedly injected, and fraction A, estimated to be less than kDa, was pooled and subjected to RP-HPLC The on-line HPLC separation was performed on a Hewlett-Packard HP1100 series HPLC system equipped with an auto sampler, thermostatically controlled column compartment, UV-VIS detector, and degasser Solvent A was 5% acetonitrile containing 0.1% trifluoroacetic acid, and solvent B was 80% acetonitrile containing 0.085% trifluoroacetic acid The freeze-dried fraction A was reconstituted with solvent A and subjected to RP-HPLC on an Inertsil C8-3 column (4.6 · 150 mm) The gradient was 0–2 0% solvent B, 2–5 0–20% solvent B, 5–55 20–47% solvent B, and 55–80 47–100% solvent B The effluents were separated into two directions, and the flow rate of one direction was adjusted to 0.6 mLỈmin)1 and that of the other to 0.2 mLỈmin)1 The effluent from one direction (0.6 mLỈmin)1) was fractionated (0.6 mL each fraction) The effluent from the other direction Ó FEBS 2003 Three isoforms of chrysophsin in red sea bream gills (Eur J Biochem 270) 677 (0.2 mLỈmin)1) was introduced into a mass spectrometer as described below The resulting active fractions were lyophilized and reconstituted in mM KH2PO4/H3PO4 (pH 3.0) containing 25% acetonitrile, and further loaded on a PolySulfoethyl Aspartamide column (4.6 · 200 mm) equipped with a Tosoh HPLC pump The column was eluted with a linear gradient of KCl The gradient was 0–3 0–0.1 M KCl, 3–43 0.1–0.4 M KCl, and 43–48 0.4–1 M KCl CA, USA) by analyzing the data calibrated with 10 pmol phenylthiohydantoin amino-acid standards Tricine/SDS/PAGE The molecular mass of the sample was estimated by Tricine/ SDS/PAGE using a 16.5% separating gel, 10% spacer gel and 4% stacking gel in the presence of 2-mercaptoethanol [23], and the protein/peptide bands were stained with a silver staining II kit from Wako Mass spectrometry Determination of peptide concentration MS analysis of peptides was performed with a Finnigan LCQ ion trap mass spectrometer (ThermoQuest, San Jose, CA, USA) equipped with an electrospray ionization source (ESI-ITMS) as described previously [19] The mass scale was calibrated using Ultra-mark provided by the manufacturer Ions were detected and analyzed in the positive mode on the basis of their m/z ratio The concentrations of the samples from purification steps were measured with the DC-protein assay kit using BSA as standard The concentration of purified and synthetic chrysophsin-1 and chrysophsin-2 was obtained from the A280 [24], and that of chrysophsin-3 with the DC-protein assay kit Solid-phase peptide synthesis Circular dichroism The protected peptide chain was assembled with a Shimadzu peptide synthesizer (PSSM8) according to standard Fmoc chemistry [20] Non-amidated peptides were prepared with Fmoc-L-His (Trt)-resin, and amidated peptides with TGS-RAM as described previously [21] Magainin [22] was prepared with Fmoc-L-Ser (tBu)resin, and peptide-Cys, to which cysteine was added at the carboxy end, was prepared with Fmoc-L-Cys (Trt)-resin At the end of the synthesis, the peptides were freed from the resin by cleavage with cocktail A (82.5% trifluoroacetic acid, 3% ethylmethylsulfide, 5% H2O, 2.5% ethanedithiol, 3% thiophenol, and mgỈmL)1 2-methylindole) for peptides with Trp and Arg, cocktail B (82% trifluoroacetic acid, 2% ethylmethylsulfide, 5% H2O, 5% thioanisole, 3% ethanedithiol, and 2% phenol) for peptides with Arg, or cocktail C (94% trifluoroacetic acid, 5% anisole, and 1% ethanedithiol) for peptides without Arg and Trp The resulting peptides were precipitated with cold diethyl ether, lyophilized, and then purified using a Tosoh HPLC apparatus, equipped with a Capcellpak C18 column (10 · 250 mm), eluted with a linear gradient of acetonitrile/0.1% trifluoroacetic acid Aliquots of peptides purified by RP-HPLC were characterized with ESI-ITMS as described below CD spectra were recorded on a Jasco J-500CH instrument (Tokyo, Japan) at room temperature in a 1-mm path length cell Synthetic crysophsins 1, and were dissolved in 20 mM potassium phosphate buffer (pH 7.25) containing mM EDTA or 20 mM potassium phosphate buffer (pH 7.25) containing mM EDTA and 50% trifluoroethanol The concentration of the chrysophsins was 20 lM Curves were smoothed by the algorithm provided by Jasco, and data analysis was performed as described previously [25] CD measurements were reported as Q (degreesỈ cm2Ỉdmol)1) The relative helix content was deduced as described by McLean et al [26] as follows Lysyl endopeptidase digestion The three purified peptides, P-1 (2.9 lg), P-2 (0.6 lg) and P-3 (0.46 lg), dissolved in 20 lL 20 mM Tris/HCl (pH 8.0) were mixed with lysyl endopeptidase and incubated for 2–4 h at 37 °C The ratio of enzyme to peptide was : 100 for P-1 and : for P-2 and P-3 After digestion, the reaction was stopped by adding 0.1% trifluoroacetic acid at a final volume of 0.1 mL Amino-acid sequence analysis The amino-acid sequence of the three purified peptides, P-1, P-2 and P-3 (1.5–2.9 lg), was determined on a Hewlett– Packard G1005A protein sequencing system (Palo Alto, %helix ẳ 100H222 ỵ 3000ị=33000 where Q222 is the CD at 222 nm Bactericidal assay Bactericidal activity was routinely tested using Bacillus subtilis ATCC6633 or Escherichia coli WP-2 After growth in tryptic soy broth at 37 °C to exponential phase, bacteria were washed twice with 0.85% NaCl and diluted in 50 mM Hepes/NaOH buffer (pH 7.4) to give % · 105 colonyforming unitsỈmL)1 (CFmL)1) Aliquots (25–100 lL) of the fractions at each purification step from the acid extract of the gills were lyophilized and dissolved in 0.1 mL mM citric acid/sodium citrate buffer (pH 4.0) Solutions were mixed with an equal volume of bacterial suspension and incubated at 37 °C for 60 In a control experiment, the cells were incubated with the same solvent as used for the preparation of each fraction After appropriate dilution of the mixture with 50 mM Hepes/NaOH buffer (pH 7.4), 0.1 mL aliquots were spread on tryptic soy agar plates and incubated for 14–18 h to measure the number of colonies formed The bactericidal activity was expressed as killing (%), using the formula [27]: Killing %ị ẳ ẵ1 À CFU in the sampleÞ= CFU in the controlŠ  100 Ó FEBS 2003 678 N Iijima et al (Eur J Biochem 270) Serial doubling dilutions of the three native and synthetic chrysophsins were carried out following the protocol described above, and the minimal lethal concentration (MLC) against E coli and B subtilis was defined as the lowest peptide concentration that caused % 99% killing of the bacteria [28] All assays were performed in duplicate Fish and crustacean pathogens, Lactococcus garvieae YT-3, Streptococcus iniae F-8502, Aeromonas hydrophila ET-4, Edwardsiella tarda ET-82016, Vibrio anguillarum ATCC 19264, Vibrio vulnificus ET-7617 and Pseudomonas putida ATCC 12633 were grown in tryptic soy broth at 25 °C, and Aeromonas salmonicida NCMB 1102 was grown in tryptic soy broth at 20 °C Vibrio harveyi HUFP 9111 and Vibrio penaeicida KH-1 were grown in marine broth 2216 at 25 °C After growing, exponential phase bacterial cultures were washed and diluted in 50 mM Hepes/NaOH buffer (pH 7.4) containing 2% NaCl to give % · 105 CFmL)1 The bacterial suspensions (each 0.1 mL) were incubated at 20–25 °C for h with equal volumes of twofold serial dilutions of the three synthetic chrysophsins in mM citric acid/sodium citrate buffer (pH 4.0) containing 2% NaCl After 100-fold dilution of the mixture, 0.1 mL aliquots were spread on tryptic soy agar plates and incubated at 20–25 °C for 18–24 h Then, MLC was determined as described above Hemolytic assay Before use, freshly collected human blood was washed with 50 mM phosphate buffer (pH 7.4) containing 0.14 M NaCl (NaCl/Pi) until the supernatant was colorless A suspension was made of 1% packed cells in NaCl/Pi containing 2% glucose Synthetic chrysophsin 1, or 3, melittin or synthetic magainin was dissolved in 50% dimethyl sulfoxide at a concentration of mM and was serially diluted with NaCl/ Pi From this suspension, 90 lL aliquots were incubated with 10 lL synthetic chrysophsin 1, or 3, melittin or synthetic magainin at different concentrations As a positive control (100% lysis), a 0.1% solution of SDS was used After incubation for 30 at 37 °C, the sample was centrifuged at 900 g for 10 The supernatant was diluted 20-fold with NaCl/Pi, and the absorbance was determined at 405 nm in a Shimadzu UV-VIS spectrophotometer (UV mini 1240) To control for dimethyl sulfoxide in the peptide, erythrocyte suspensions were incubated with 50% dimethyl sulfoxide was determined by ELISA using chrysophsin-1-Cys as an antigen and peroxidase-labeled affinity-purified antibody to rabbit IgG (H + L) as a secondary antibody Chrysophsin-1-Toyopearl was used for the purification of anti-(chrysophsin-1) IgG Chrysophsin-1-Cys (1 mg) was coupled to Toyoperal AF-Epoxy-650M (1 g) according to the manufacturer’s protocol Anti-(chrysophsin-1) serum (3 mL) was filtered through a 0.45-lm filter (Dismic-13, Advantec, Tokyo, Japan) and applied to the chrysophsin1-Toyopearl column The column was washed with 20 mM Tris/HCl (pH 7.4) containing 150 mM NaCl (Tris/NaCl) and 20 mM Tris/HCl (pH 7.5) containing M NaCl and 1% Triton X-100, respectively, and specific antibody was eluted from the column with 0.1 M glycine/HCl (pH 2.5) The eluted antibody was immediately neutralized with M Tris and stored at )80 °C Immunohistochemistry Gill arches were fixed in Bouin’s solution for 24 h at room temperature After dehydration, tissues were embedded in paraffin, cut into 5-lm sections, and mounted on gelatincoated glass slides Immunohistochemical staining was performed using Simple Stain MAX-PO (Multi) as a secondary antibody [29] Briefly, sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol After a brief wash with NaCl/Pi, sections were exposed to 3% H2O2 in 90% methanol to block endogenous peroxidase and washed with NaCl/Pi They were then blocked with 10% normal goat serum in NaCl/Pi for h at room temperature, incubated with the chrysophsin-1-specific antibody (0.11 lgỈmL)1) in NaCl/Pi containing 1% BSA and mM NaN3 overnight at room temperature, and washed times with NaCl/Pi The sections were incubated with the secondary antibody for 30 at room temperature and washed with NaCl/Pi The color was developed in a Simple Stain DAB solution To determine the type of immunoreactive cells in the gills, neighboring 5-lm serial sections of the gills were stained with chrysophsin-1-specific antibody or hematoxylin and eosin As a negative control, chrysophsin-1-specific antibody preabsorbed with chrysophsin-1 (mol ratio of antibody to chrysophsin-1 ¼ : 5000) was used as a primary antibody Results Preparation of anti-(chrysophsin 1) IgG Peptide purification and primary structure Synthetic chrysophsin-1-Cys (2.3 mg) purified by RP-HPLC was dissolved in 60% dimethyl sulfoxide and conjugated with 2.3 mg mcKLH according to the manufacturer’s directions The resulting chrysophsin-1-CysmcKLH conjugate (chrysophsin-1-KLH) was dialyzed against NaCl/Pi and used as described below For initial immunization, 600 lg chrysophsin-1-KLH in 500 lL NaCl/Pi was mixed with an equal volume of Imject Alum and administered to Japanese white rabbits by multiple subcutaneous injections Ten, 21 and 33 days later, the rabbit received 600 lg chrysophsin-1-KLH in 500 lL NaCl/Pi by subcutaneous injection At days after the final injection, blood was collected, and the resulting antiserum was stored at )80 °C The titer of the antisera After fractionation of the acid-extracted gill powder on SPsephadex C-25, fraction SP-3 showed the most bactericidal activity (MLC 1.2 lgỈmL)1) and was further separated by gel-filtration HPLC (Fig 2) Bactericidal activities against B subtilis were detected in almost all of the fractions eluted from the column The molecular mass of fractions eluted between 48 and 81min, showing high antibacterial activity, was determined by Tricine/SDS/PAGE As this study focuses exclusively on bactericidal peptides of less than kDa which were mainly detected in fraction A (data not shown), it was subjected to RP-HPLC followed by ESIITMS (Fig 3) The deduced mean ± SD molecular masses of the three peaks, P1, P2 and P3, showing strong bactericidal activity were 2891.2 ± 0.2, 2919.3 ± 0.4 and Ó FEBS 2003 Three isoforms of chrysophsin in red sea bream gills (Eur J Biochem 270) 679 Fig Gel-filtration HPLC of the SP-3 fraction obtained from the acid extract of red sea bream gills The SP-3 fraction eluted with M pyridine/acetic acid (pH 5.0) from SP-sephadex C-25 was loaded on a TSKgel G2000SW column pre-equilibrated with 40% acetonitrile/ 0.1% trifluoroacetic acid The flow rate was 0.25 mLỈmin)1, and absorbance was monitored at 220 nm (solid line) The fraction volume was 0.5 mL The bactericidal activity against B subtilis of a 100-lL aliquot of each fraction was measured and expressed as killing (%) (open bar) as described in Materials and methods Fraction A, indicated by the bar, was collected 2285.7 ± 0.2 Da, respectively By RP-HPLC, P1, P2 and P3 were pooled As a broad but single band was observed by Tricine/SDS/PAGE (data not shown), aliquots of P1, P2 and P3 were directly sequenced by Edman degradation The amino-acid sequences were then partially determined (Table 1) As the underlined residues could not be determined, they were digested with lysyl endopeptidase, and the resulting peptide fragments subjected to RP-HPLC followed by ESI-ITMS From the difference between the deduced mean molecular mass of the peptide fragment by ESI-ITMS and the theoretical mean molecular mass of the identified amino-acid sequence of the peptide fragment, the unidentified amino acid was determined as His For example, three peptide fragments, (1) FFGWLIK, (2) GAIXAGK, and (3) AIXGLIXRRRX, were released from P1 by lysyl endopeptidase The mean molecular mass (909.6 ± 0.2 Da) of fragment determined by ESI-ITMS was very similar to that of fragment calculated from the identified amino-acid sequence (910.10 Da) The difference in the mean molecular mass of fragment determined by ESI-ITMS (652.5 ± 0.0 Da) and that of fragment calculated from the identified amino-acid sequence (515.61 Da) was 137.1 Da, which agreed with the average molecular mass of His (Table 1) On replacing the unknown amino acids of fragment with His, the theoretical average molecular mass (1365.62 Da) of fragment calculated from the amino-acid sequence was almost the same as that of fragment deduced by ESI-ITMS (1364.8 Da) Finally, amino-acid sequences of P1, P2 and P3 were determined as follows P1, FFGWLIKGAIHAGKAIHGLIHRRRH; P2, FFGWLIRGAIHAGKAIHGLIHRRRH; P3, FIG LLISAGKAIHDLIRRRH The theoretical average molecular masses of P1 (2892.43 Da), P2 (2920.45 Da) and P3 (2286.76 Da) matched the deduced average molecular masses with a difference of Da This discrepancy (1 Da) suggests amidation of the C-terminal histidine Therefore, elution profiles of synthetic nonamidated peptides named P1-COOH, P2-COOH and P3-COOH, and synthetic amidated peptides named P1-CONH2, P2-CONH2 and P3-CONH2 were superimposed on those of native P1, P2 and P3 by HPLC Native P1 and P3 were recognized as single peaks by RP-HPLC (data not shown) and also by anion-exchange HPLC (Fig 4A,C) P1-COOH and P1-CONH2 could not be separated by RP-HPLC; however, they were clearly separated into two peaks at retention times of 18 and 22 (Fig 5A), and the mixture of native P1and P1-CONH2 showed a single peak on ion-exchange HPLC (Fig 5B) Thus, the C-terminal amino acid His of native P1 was found to be Fig RP-HPLC of fraction A obtained by gel-filtration HPLC Fraction A obtained by gel-filtration HPLC was loaded on an Inertsil C8-3 column and eluted with a linear gradient of acetonitrile in aqueous trifluoroacetic acid (dotted line) The flow rate was 0.8 mLỈmin)1, and the absorbance was monitored at 220 nm (solid line) The effluent was separated into two directions, and the effluent from one direction was collected The bactericidal activity of each fraction was expressed as killing (%) (open bar) as described in Materials and methods The effluent from the other direction was directly introduced into an electrospray ionization/mass spectrometer (ESI/ITMS) Ó FEBS 2003 680 N Iijima et al (Eur J Biochem 270) Table Mass measurement of peptide fragments of P1, P2 and P3 Partial sequence PI ESI-ITMS Mr FFGWLIKGAIXAGKAIXGLIXRRRX 2891.2 P2 FFGWLIRGAIXAGKAIXGLIXRRRX 2919.3 P3 FIGLLISAGKAIXDLIRRRX 2285.7 Peptide fragment ESI-ITMS Mr Calc Mr His (·) FFGWLIK GAIXAGK AIXGLIXRRRX FFGWLIRGAIXAGK AIXGLIXRRRX FIGLLISAGK AIXDLIRRRX 909.6 652.5 1364.8 1573.0 1364.8 1017.8 1285.3 910.10 515.61 954.20 1435.71 954.20 1018.26 1012.24 137.1 ·137.1 137.1 ·137.1 – · 137.1 P2-1 fraction From these results, the primary structures of three novel bactericidal peptides, P1, P2-2 and P3, were determined, and we decided to call them chrysophsin-1, chrysophsin-2 and chrysophsin-3 based on the genus of red sea bream, C major An alignment of these three peptides with other antimicrobial peptides is shown in Fig Chrysophsin-1, chrysophsin-2 and chrysophsin-3 are C-terminally amidated, 25, 25 and 20 amino acids in length, and rich in cationic residues (9/25, 9/25 and 6/20, respectively) Chrysophsin-2 corresponds to the chrysophsin-1 isoform differing by a single residue at position (lysine or arginine) Interestingly, the characteristic C-terminal cationic tetrapeptide, RRRH, is conserved in chrysophsins, in addition to the C-terminal amidation Secondary structure of chrysophsins Fig Cation-exchange HPLC of P1, P2 and P3 Aliquots of P1 (A), P2 (B) and P3 (C) were loaded on a PolySulfoethyl Aspartamide column and eluted with a linear gradient of KCl in mM KH2PO4/H3PO4 (pH 3.0)/25% acetonitrile at a flow rate of 0.8 mLỈmin)1 (dotted line) Absorbance was monitored at 220 nm (solid line) amidated Native P3 was also found to be amidated (Figs 4C and 5F) Native P2 was detected as a single peak by RP-HPLC (data not shown) However, native P2 separated into two peaks, P2-1 and P2-2 (Fig 4B), and P2-2 was coeluted with P2-CONH2 on anion-exchange HPLC (Fig 5C,D) P2-1 and P2-2 were separated by anionexchange HPLC and further analyzed by RP-HPLC followed by ESI-ITMS As the mean molecular mass of P2-2 (2919.3 ± 0.4 Da) was identical with that of P2CONH2, P2-2 was also an amidated peptide Two peptides, 2925 ± 0.7 Da and 2907 ± 0.3 Da, were detected in the Schiffer–Edmunson helical wheel modeling was used to predict hydrophobic and hydrophilic regions within the secondary structure of chrysophsin-1, chrysophsin-2 and chrysophsin-3 (Fig 7) All three had an amphipathic a-helix conformation, indicating hydrophilic and hydrophobic residues on opposite sides of the N-terminal 21 residues for chrysophsin-1 and chrysophsin-2, and 18 residues for chrysophsin-3 This conformation was confirmed by CD spectroscopy of the three synthetic chrysophsin peptides in the absence or presence of 50% trifluoroethanol (Fig 8) In the absence of 50% trifluoroethanol, the spectra of all three chrysophsins are typical of a disordered structure However, their ellipiticities decreased at both 208 and 222 nm and increased at 190 nm, indicating stabilization of the a-helix structure (87%, 88% and 81% helix content in chrysophsin-1, chrysophsin-2 and chrysophsin-3, respectively) in the presence of 50% trifluoroethanol Bactericidal spectrum, salt sensitivity and hemolytic activity of chrysophsins As the bactericidal activities of the three native and synthetic chrysophsins were found to be almost equal against B subtilis and E coli (Table 2), bactericidal activity against fish and crustacean pathogens was investigated with the synthetic chrysophsins (Table 3) All three were active against Gram-positive bacteria (MLC < 10 lM) Most of the Gram-negative pathogens were sensitive to less than 40 lM, except for A hydrophila and E tarda Ó FEBS 2003 Three isoforms of chrysophsin in red sea bream gills (Eur J Biochem 270) 681 Fig Cation-exchange HPLC of native P1, P2-2 and P3, and their synthetic peptides Mixtures of synthetic P1-COOH and P1-CONH2 (A), native P1 and P1-CONH2 (B), P2-COOH and P2-CONH2 (C), native P2 and P2-CONH2 (D), P3-COOH and P3-CONH2 (E), and native P3 and P3-CONH2 (F) were loaded on a PolySulfoethyl Aspartamide column and eluted with a linear gradient of KCl in mM KH2PO4/H3PO4 (pH 3.0) containing 25% acetonitrile at a flow rate of 0.8 mLỈmin)1 (dotted line) Absorbance was monitored at 220 nm (solid line) Fig Comparison of the amino-acid sequence of the chrysophsins with other antimicrobial peptides Alignment of the mature amino-acid sequences of chrysophsin-1, chrysophsin-2 and chrysophsin-3, misgurin [15], pleurocidin [12] and pleurocidin-like antimicrobial peptides/WF2-4 [14], piscidin (sb-moronecidin), (wb-moronecidin) and [16,17] and melittin [22] Gaps have been introduced to maximize sequence similarities Identical amino-acid residues are shaded, and basic amino-acid residues are shown in bold (MLC > 40 lM) P putida was sensitive to chrysophsin-1 and chrysophsin-3, but not to chrysophsin-2 (MLC > 40 lM) To determine whether the bactericidal action of chrysophsins is dependent on salt, NaCl concentration was varied with the chrysophsin concentration kept fixed (Fig 9) Chrysophsin-1 and chrysophsin-2 were bactericidal up to 0.32 M NaCl, but chrysophsin-3 was effective only up to 0.16 M NaCl Chrysophsins were hemolytic for human red blood cells, but they were less hemolytic than melittin and more hemolytic than magainin (Fig 10) Ó FEBS 2003 682 N Iijima et al (Eur J Biochem 270) Fig Schiffer–Edmunson helical wheel diagram demonstrating probable amphipathic a-helical conformation of chrysophsin-1, chrysophsin-2 and chrysophsin-3 Shaded gray indicates hydrophobic amino acids Residue numbers starting from the N-terminus are shown Localization of chrysophsin-1 in the gills of red sea bream Immunohistochemical staining showed that chrysophsin-1 was localized in the gills of red sea bream as two distinct cell populations The first type of immunopositive cells were found at the base of the secondary lamellae (Fig 11A, arrows in Fig 11C and Fig 11G), and were located adjacent to the blood capillaries in the connective tissue (Fig 11D,H) In the immunopositive cells, cytoplasmic granules were immunostained (arrows in Fig 11E,I) and were eosinophilic in nature (arrows in Fig 11F) The second type of immunoreactive cell was an epithelial cell on the secondary lamellae (Fig 11A and arrowheads in Fig 11C) No immunoreactivity was observed in any sections stained with chrysophsin-1-specific antibody preabsorbed with chrysophsin-1 (Fig 11B) Discussion Here we report the isolation of chrysophsin-1, chrysophsin-2 and chrysophsin-3, novel peptides of 25, 25 and 20 residues with bactericidal and hemolytic activity, from the gills of the red sea bream, C major They are highly Ó FEBS 2003 Three isoforms of chrysophsin in red sea bream gills (Eur J Biochem 270) 683 Fig CD spectrum for synthetic chrysophsins The spectra of synthetic chrysophsin-1 (A), chrysophsin-2 (B) and chrysophsin-3 (C) were obtained in 20 mM potassium phosphate buffer/8 mM EDTA, pH 7.25, in the presence (solid line) or absence (dotted line) of 50% (v/v) trifluoroethanol cationic peptides without cysteine (Fig 6) Searches of sequence databases show 70% identity between chrysophsin-1 and the mature peptide sequence predicted from the nucleotide sequence of winter flounder pleurocidin-like genomic clone (WF3), which has not yet been purified as a mature peptide [14] However, chrysophsin-1 shows low identity (24–36%) with other fish antimicrobial peptides, such as piscidins [16], moronecidins [17] and pleurocidin [12,30] The C-terminal amino acid was amidated in all three chrysophsins, similarly to those in marine animals, such as solitary ascidians (styelin D) [31] and hybrid striped bass (Morone saxatilis x M chrysops) (moronecidin/piscidin) [16,17] It has been proposed that amphipathic a-helical peptides show antimicrobial activity by interacting electrostatically with the anionic bacterial membrane, adopting an amphipathic a-helical conformation that allows them to insert the hydrophobic face into the lipid bilayers and form a pore [1,8,22] The amphipathic a-helical structure of chrysophsins was predicted by the Schiffer–Edmundson wheel analysis (Fig 7) All three chrysophsins form an a-helical structure (> 80% helical content) in the structure-forming solvent, trifluoroethanol, but not in phosphate buffer (Fig 8) This suggests that random-coiled chrysophsins in the water environment will form amphipathic a-helical conformation after contacting with the bacterial membrane Thus, chrysophsins will show antimicrobial activity in a similar way to other previously studied amphipathic a-helical antimicrobial peptides Interestingly, the identity in amino-acid sequence between chrysophsin and misgurin is low (16%), but chrysophsins and misgurin have strongly cationic tetrapeptide sequences, RRRH and RRRK, at the C-terminus Bee venom melittin also has a C-terminal cationic tetrapeptide sequence, KRKRQQ, and was found to form a nonhelical hydrophilic domain that allows electrostatic binding with the polar head group of negatively charged phospholipids [32–34] Thus, the C-terminal RRRH of chrysophsins could form a nonhelical hydrophilic domain similarly to bee venom melittin All three chrysophsins showed bactericidal activity against Gram-negative and Gram-positive pathogens in the presence of 0.34 M NaCl, except for A hydrophila and E tarda The results indicate that chrysophsins show broad-spectrum bactericidal activity against pathogenic bacteria up to 0.34 M NaCl The bactericidal activity of chrysophsin-1 and chrysophsin-2 at a concentration of 0.5 lM was retained at physiological NaCl concentrations against E coli, but was lost at 0.64 M NaCl, similarly to winter flounder pleurocidin: the bactericidal activity of pleurocidin (5.6 lM) against E coli was also lost at 0.625 M NaCl [12] On the other hand, hybrid striped bass wb-moronecidin retained bacteriostatic activity against Staphylococcus aureus even in the presence of 1.28 M NaCl; however, it remains unclear whether wb-moronecidin is bactericidal or not in 1.28 M NaCl [17] The net charge of chrysophsin-1 (pI 12.64) and chrysophsin-2 (pI 12.79) is slightly higher than that of wb-moronecidin (piscidin 2) (pI 12.60) In addition, the C-terminal amino acids of chrysophsins and wb-moronecidin were both amidated These findings may indicate that the difference in salt tolerance between chrysophsins and wb-moronecidin is partly due to the difference in bacteria, Gram-negative E coli and Gram-positive S aureus used in the experiment Antimicrobial peptides must pass the lipopolysaccharide-rich external leaflet of the outer membrane to interact with the inner membrane of Gram-negative bacteria such as E coli; however, they can directly interact with the anionic cytoplasmic membrane of Gram-positive bacteria such as S aureus It is necessary to compare the bacteriostatic and bactericidal activities of chrysophsins against Gram-positive bacteria, Ó FEBS 2003 684 N Iijima et al (Eur J Biochem 270) Table Bactericidal activity (minimal lethal concentration) of native and synthetic chrysophsins compared with that of magainin-2 Minimal lethal concentrations of peptide are given in lM and are the concentration of substance required necessary to kill % 99% of the bacteria Chrysophsin-1 Chrysophsin-2 Chrysophsin-3 Strains of bacteria Native Synthetic Native Synthetic Native Synthetic Magainin2 Bacillus subtilis ATCC 6633 Escherichia coli WT-2 0.25 0.25 0.125 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.985 0.985 Table Bactericidal activity (minimal lethal concentration) of synthetic chrysophsin-1, -2 and -3 against pathogenic bacteria Minimal lethal concentrations of peptide are given in lM and are the concentration of substance required necessary to kill % 99% of the bacteria Strains were considered resistant (R) when their growth was not inhibited by peptide up to 40 lM Strains of pathogenic bacteria Gram-positive bacteria Lactococcus garvieae YT-3(ATCC 49156) Streptococcus iniae F-8502 Grain-negative bacteria Vibrio anguillarum ATCC 19264 Vibriopenaeicida KHA Vibrio harveyi HUFP911 (ATCC 14126) Vibrio vulnificus ET-7617(ATCC 33148) Aeromonas hydrophila ET-4 Aeromonas salmonicida NCMB 1102 Pseudomonas putida ATCC 12633 Edwardsiella tarda ET-82016 Chrysophsin-1 Chrysophsin-2 Chrysophsin-3 10 2.5 1.5 10 10 2.5 10 2.5 R 10 40 R 1.25 5 2.5 R R R 10 10 10 R 10 40 R in addition to the Gram-negative bacteria Chrysophsins showed hemolytic activity against human erythrocytes, in addition to bactericidal activity (Fig 10); however, they are less hemolytic than melittin, a cytotoxic peptide, but more hemolytic than magainin It is still necessary to examine whether chrysophsins are cytotoxic to the gill cells of red sea bream or not Chrysophsin-3 was the least hemolytic and bactericidal This may correlate with the lower isoelectric point (pI 12.16) than that of chrysophsin-1 and chrysophsin-2 We used polyclonal antibody against chrysophsin-1 as a primary antibody for immunohistochemistry (Fig 11) Fig Effect of NaCl on bactericidal activity of chrysophsins Bactericidal activity of synthetic chrysophsin-1 (s), chrysophsin-2 (h) and chrysophsin-3 (n) at 0.5 lM was determined with various concentrations of NaCl ranging from to 1.28 M Fig 10 Hemolytic activity of chrysophsins Synthetic chrysophsin-1 (s), chrysophsin-2 (h) and chrysophsin-3 (n), synthetic magainin (j), an antimicrobial peptide from the aquatic frog Xenopus laevis and melittin (d), a peptide from bee venom cytotoxic to human erythrocytes, were incubated with a 1% suspension of washed human erythrocytes for 30 at 37 °C Ó FEBS 2003 Three isoforms of chrysophsin in red sea bream gills (Eur J Biochem 270) 685 cells appeared to have eosinophilic granules in the cytoplasm (Fig 11F) Eosinophilic granule cells (EGCs) were located in the connective tissues of the primary lamellae of fish gills, in close association with blood vessels [35] Piscidins have been reported in the mast cells of gills of a hybrid striped bass [16], but it is not known whether histamine is released or whehter they possess other characteristics of mast cells [35] Thus, we assume that the immunopositive cells located in the connective tissue at the base of secondary lamellae are EGCs Chrysophsins also accumulated in some cells of the secondary lamellae, located on the surface of gill tissue (Fig 11C) Piscidins were very recently found to localize in the mast cells of gills, not in epithelial cells [16] Teleost gill epithelia line the external boundary and consist mainly of pavement, mucous and chloride cells [36], and the pavement cells constitute more than 80% of gill epithelium [37] It remains unclear whether chrysophsins localize in the epithelial cells, such as mucous and chloride cells, of the secondary lamellae or in EGCs that have infiltrated the epithelium Future work is required to ascertain the exact cell-type that produces chrysophsins It remains unclear which chrysophsin isoforms exist in certain cells of secondary lamellae and EGC-like cells of the primary lamellae of red sea bream gills We are currently trying to obtain cDNAs encoding chrysophsins from the gills of red sea bream, and we aim to investigate the gene expression of the chrysophsin isoforms in these cells by in situ hybridization Acknowledgements We thank Dr S Ohta (Instrument Center for Chemical Analysis, Hiroshima University) and Professor K Gekko (Graduate School of Science, Hiroshima University) for the CD analysis This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan References Fig 11 Immunohistochemical localization of chrysophsins in the gills of the red sea bream Adjacent sections in the gills were immunostained with chrysophsin-1 antibody (A, C, E and F) and with hematoxylin and eosin (D, F and H) as described in Materials and Methods Immunopositive cells were mainly observed among connective tissues at the base of 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(minimal lethal concentration) of native and synthetic chrysophsins compared with that of magainin-2 Minimal lethal concentrations of peptide are given in lM and are the concentration of substance... Immunohistochemical localization of chrysophsins in the gills of the red sea bream Adjacent sections in the gills were immunostained with chrysophsin-1 antibody (A, C, E and F) and with hematoxylin and eosin