´ e ´ ede m aper- µ Holothuria ´ eenne, CNRS- . All samples ´ editerran ´ e de Perpignan via Domitia, C. The cell pellet was then ◦ b ´ elides, CNRS-USTL, Lille, France Paracentrotus lividus ´ ecules et de l’Environnement, Universit a ´ eenne, Universit 2009 European Peptide Society and , and three echinoderms, c sucrose and then recentrifuged as above. ´ editerran sp., and M H. papillosa activity against Gram-positive and Gram-negative 2009 European Peptide Society and John Wiley & Sons, Ltd. Cucumaria and c , UPVD-EPHE, Perpignan, France Laboratoire de Neurobiologie des Ann Correspondence to: Richard Galinier, UMR 5244Ecologie CNRS Tropicale EPHE et UPVD, M Biologie et 66860 Perpignan cedex, France. E-mail: [email protected] Laboratoire de Biologie et d’Ecologie Tropicale et M Laboratoire de chimie des Biomol Perpignan via Domitia, Perpignan, France Centre de Biochimie Structurale, CNRS UMR5048; INSERM, U554; Universit Montpellier 1 et 2, Montpellier, France The cell pellet was then resuspended in 10 volumes of 5% ∗ c a b d ytes). Antimicrobial activities were detected only in the two acetic acid and homogenized (30 strokes) using Dounce apparatus Materials and Methods Species Collection Studies were performedsabatieri on two tunicatetubulosa species, resuspended in 0.34 tures filter into 50-ml tubesThe containing hemolymph 150 mg (approximately ofcentrifuged EDTA 5 ml/) at powder. 800 was g immediately for 15 min at 4 were collected in along the Catalan coast. Preparation of Hemocyte Acid Extracts Specimens were briefly bathedperibasally. in The ethanol, hemolymph dried was passed and through transected a 100- se 26 and 34 amino acid residues, respectively. Their primary s from two taxa ( and echinoderms) for the presence Pierre-Eric Sautiere, a y a combination of Edman degradation and mass spectrometry. and Guillaume Mitta . In addition, we report the isolation and characterization of two novel c ,thatsynthesizeAMP Halocynthia papillosa . We report also their activity and Emmanuel Roger, ∗ Bernard Banaigs a d hemocytes. These molecules display antibacterial Drosophila melanogaster :48–55 Copyright H. papillosa 15 Halocynthia papillosa ; innate immunity; antimicrobial peptide Microcosmus sabatieri 2009;

The interest in AMP reflects both their relevance to intrinsic host J. Pept. Sci. Like other invertebrates,immunity. Nevertheless, they marine have developed invertebrates animmune effective innate lack system acquired microorganisms. to Antimicrobial peptides defendin (AMP) themselves this play efficient a against defencecharacterized key system pathogenic from role (see hemocytes [1]taxa for in including review). arthropoda several They [2],[2,8]. were marine urochordata They [3–7], are invertebrate synthesized and evenprecursors in and absence of the challenge, peptides mature and are arthropoda, stored the peptides in can granules. exerton their In microbicidal engulfed mollusca activity bacteriaupon or/and stimulation by can microbial substances be [2,8,9]. Thisinsects, released differs especially from into the plasma Introduction rapidly after septic injury,released principally immediately into in the hemolymph the where they fatin participate a body, systemic which response [10]. are defence, and their potential development as therapeutics [11–13]. Marine invertebrate are expected toantimicrobial molecules. be The field an of marine important invertebrate AMP sourcenot was for extensively studied yet,were not and explored. To several enrich taxa datathe present in report like the this presence field, of echinoderms cationic we AMP in investigated hemocytestwo from in tunicate and three echinodermpurification species. and We characterization report of herethe the two tunicate peptides isolatedspectrum from and bactericidal effect.

We report here the screening of five marine invertebrate specie Halocynthia papillosa Andre Aumelas, from hemocytes of the solitary tunicate, Richard Galinier, of cationic antimicrobial peptidestunicates (AMP) in defence cellspeptides (hemoc from antimicrobial peptides isolated Halocyntin and papillosin, two new (www.interscience.com) DOI 10.1002/psc.1101 Research Article Received: 18 January 2008 Revised: 20 October 2008 Accepted: 26 October 2008 Published online in Wiley Interscience: bacteria. Complete peptide characterization was obtained b structure display no significant similarities with previously described AMP. Copyright The mature molecules, named halocyntin and papillosin, compri Keywords: John Wiley & Sons, Ltd.

48 49 E. 600 Cor et al ◦ Pseu- Cfor ◦ of 0.001) (54.145 T), (103 015 T), 600 of 0.001) in (65.8 T), l aliquots from 600 µ l of a suspension C, depending of ◦ µ l were plated after µ strain and 37 Neisseria gonorrhoeae strain was purchased from Cor37 Salmonella thyphimurium ◦ α of 0.01) in PB and incubated Aerococcus viridans M. luteus 600 (82.91 T), Enterococcus faecalis DH5 l of an exponential phase culture suspension (starting OD µ Staphylococcus aureus of 0.001) in Mueller Hinton Broth Cfor C. Aliquots of 10 (A270), ◦ (30.86 T), ◦ C. Controls consisted in bacterial culture 600 suspension (starting OD ◦ www.interscience.wiley.com/journal/psc l at a concentration 10 times higher than (100 720 T), (starting OD µ M. luteus (66.20 T), l of sterile water. Cor37 µ ◦ E. coli Escherichia coli Cor37 ◦ lofa E. coli µ or lofan Klebsiella pneumoniae Micrococcus luteus µ l aliquots from each dilution were incubated in sterile 96 µ strain. C for 18 h. The lowest concentration of peptide that prevented ◦ M. luteus The minimal bactericidal concentration (MBC) was determined Invitrogen. Antimicrobial assays After each purification step, antibacterialby a activity liquid was growth inhibition monitored assay [15].each Briefly, 10 resuspended fraction werewith both 100 incubated in microtiter plates Enterobacter aerogenes (60.62 T), (103 711) were purchased from the Pasteur Institutel’Institut (Collection Pasteur). de domonas aeruginosa Bacillus megaterium Poor-Broth nutrient medium (PB: 1% Bactotrypton, 0.5%pH NaCl w/v, 7.5). Bacterial growth was assayed by measurement of OD 37 and 100 respectively at 30 after 12-h incubation at 30 the bacterial strainthe used. contents The of MBC thegrowth was first of three bacteria determined onto wells MH by showing agar plates plating no and incubating turbidity at visible 30 coli according to the method of Hancock [16].in Peptides were a dissolved solution containing 0.01% aceticalbumin acid and (BSA) 0.2% bovine and serum solution then were serial made doublingas in dilutions 10 0.01% of acetic thewell acid, stock polypropylene microtiter 0.2% plates BSA. with 100 As much of bacteria (starting OD any residual colony formation corresponded to the MBC. Bactericidal assay Purified peptide (10 the MIC) was mixedof with 90 System 4188, Applied Biosystems) as described by Kussmann [14]. Molecular masses were calculatedcharged from protonated a molecular ions. series of multiple- Sequencing Purified peptides (20 pmol) were submitteddegradation to automated Edman on(Applied a Biosystem 492 pulse-liquid cLC Protein sequencer). automatic peptide sequencer Peptide synthesis Five milligrams ofwithout any each modification by peptide the companyFrance). GENPEP were The (Prades HPLC le purities chemically Lez, of the synthesized peptidesand were 97% 95% for for halocyntin. papillosin Microorganisms Bacteria, 0, 3, 10 and 30and min, number 2, of colony-forming 6 units and wasincubation 24 counted at h after 30 overnight of incubation on nutrient agar incubated with 10 (MHB) medium (Sigma). Bacterialh growth incubation was checked under after agitation 18- at 30 lof Vac µ 18 column column column Cduring ◦ 8 18 (Millipore) 18 18 6 mm, Waters). . 4 × EDTA at 37 M C and supernatant was C. Subsequently, it was ◦ ◦ 2009 European Peptide Society and John Wiley & Sons, Ltd. c Absorbance Detector). Column λ l of the 60% Sep-Pak fractions were µ column (250 mm 8 , pH 8.5) containing 1 m :48–55Copyright 1 mm, Waters) using the acetonitrile gradient 1 mm, Waters) at a flow rate of 0.3 ml/min using . . M 6 mm,Waters).Elutionwasperformedwithalinear 2 . 15 2 lofpurifiedpeptides,corresponding to5pmol,were 4 To ascertain the purity of the peptides, one 10% acetonitrile of the previous elution acetonitrile × µ × × Aliquots of 150 Active fractions from the first step were further loaded The peptides were purified on a Symmetry C + 2009; m). Thereafter, 20 ml of 5% acetic acid was added and 10 to µ Elution was performed− with a linearpercentage, gradient in ranging acidified from water1 ml/min. over Fractions were 40 collected min andStep at treated (3): as a above. flow rate of the acetonitrile gradient described in step 2. described in step 2 at aStep flow rate of (4): 0.3 ml/min. additional step was performed on a Symmetry C gradient of 5–65% acetonitrile in acidified watera over flow 90 rate min of at 1 ml/min. Fractionspeaks corresponding to absorbance were collectedreconstituted in in polypropylene tubes, 0.2activity ml as freeze described UPW, below. dried, andStep (2): tested foronto antimicrobial a Symmetry C Step (1): subjected to reverse-phase HPLC on a Symmetry C (250 mm (150 mm (150 mm Marine invertebrate antibacterial peptide Tris Buffer (25 m Peptide Primary Structure Determination Enzymatic digestion As much as 20endoproteinase Lys-C pmoles (sequencing of grade, peptide Boerhinger) in were 10 submitted to 25 ng of HPLC Purification All HPLC purification stepssystem were carried (Waters out 1525, on Binarydetector a HPLC (Waters Waters Pump) Breeze 2487, equipped Dual with a UV cartridges (Waters Associates)[0.05% TFA equilibrated in in ultrapurewith water UPW/TFA, acidified (UPW): three UPW/TFA]. water 10, successive After 60, elutions washing and werewere performed 80% with acetonitrile lyophilized in andantimicrobial UPW/TFA. dissolved activity The in as eluted described 0.6with fractions ml 60% below. acetonitrile were UPW Only active and fractions and submittedHPLC to eluted separation. tested reverse-phase for effluent was monitored by UV absorption at 224 and 280 nm. J. Pept. Sci. the mixture was stirred overnight at 4 (100 12 h. Desalting of peptides was performed on ZipTip C Mass spectrometry Asmuchas1 dissolved in UPW and analyzed byassisted Sandwich Laser method Desorption/Ionization on mass a spectrometer Matrix- (Voyager following the protocol supplied byproducts were the directly manufacturer. analyzed by The mass spectrometry digest anddegradation Edman as described below. Solid Phase Extraction After washingwere with performed acidified with 5Hemocyte water, and 40% acid two acetonitrile extracts successive in acidified were elutions water. loaded onto Sep-Pak C processed immediately. centrifuged at 20 000 g for 30 min at 4 . H. sp. et al :48–55 dbutnon 15 Cucumaria and five for 2009; R. Galinier were assayed all ,23 E. coli J. Pept. Sci. column of halocyntin (c) and and 8 xtraction, the material present in H. tubulosa dicate fractions with antimicrobial PLC purification was performed with ,6 M. luteus Microcosmus sabatieri cmg/Demo/wheel/wheelApp.html. ∼ H. papillosa specimens. After a first fractionation step on ,30 , ranged between 19 and 40% acetonitrile, and between P. lividus M. sabatieri After prepurification by solid phase e (b) are represented. Black squares in column. Elution for this first step H Helical wheel representation of an amino acid sequence The amino acidaxis side of chains an areideal alpha projected alpha helix, down orthogonal helix thethe to consists axis angle the (the between of paperand two 3.6 plane). residues thus residues As is per thereresidues. an chosen complete to exists The be turn, a figureby 100 degrees is periodicity Edward K. a afterVirginia in O’Neil snaphot five Charlottesville, and of turnshttp://cti.itc.Virginia.EDU/ Virginia). Charles The a and applet M. Java 18 is Grisham Applet accessible (University at written of papillosa Results Isolation of Antimicrobial Peptides from Hemocytes Acid extracts were17 prepared with 160 ml of hemolymphSep-Pak from cartridges (see Section onfractionselutedwith60%acetonitrilewereactiveandsubmittedto Materials and Methods), only reverse-phase HPLC separation. For the three echinoderm species, no antimicrobial activityfrom was the detected first round insubsequent of purification the reverse-phase steps were HPLC eluted performed. purification,species, For fractions and both several tunicate no activephase fractions HPLC. Activities against were obtained after reversed- and 24 along the elution gradient (5–65%) revealing several fractionsantibacterial with activities (Figure 1(a) and (b)).fractions Nineteen were antibacterial obtained for 18 flow rate 1 ml/min. Absorbance peaks were monitored at 224 nm (solidfull n antimicrobial peptides (a), whereas fraction M2 contains the isolate )and 1 / − l M 2009 European Peptide Society and John Wiley & Sons, Ltd. . 4 c − Microcosmus sabatieri 100 × ons containing the antimicrobial peptide of interest. (b). Chromatograms from last reverse-phase purification steps on C C and centrifuged ◦ (a) and Copyright negative control) . The percentages of the ). Spectra were recorded l of a solution containing 1 1 µ − − l 600 M. sabatieri A M C on a Chirascan dichrograph 4 ◦ − − rile was loaded onto a reverse-phase C negative control) for halocyntin (0.74 10 1 positive control 600 − sample A l of a 10% Triton X-100 in PBS were used Halocynthia papillosa 600 µ 600 − (A (A = l of a 0.01% acetic acid and 0.2% BSA solution. µ for papillosin (1.0 10 1 − Reverse-phase HPLC of acidic extracts obtained from tunicate hemocytes. . Serial doubling dilutions were made from these peptide M %hemolysis l of each dilution were added to 180 µ µ the fraction eluted with 60% acetonit for 3 min at 10 000their g. absorbance The measured supernatants at 600 were nm finally (ADThe collected 340, hemolytic and Beckman percentage Coulter). was calculated as follow: The tests were then incubated for 30 min at 37 stock solutions in 0.01%20 acetic acid and2.5% 0.2% v/v BSA. of As a muchcontrol sheep of as erythrocytes hemolysis, 20 suspension ininstead PBS. of As positive peptideconsists dilution. of The 20 negative control of hemolyze papillosin (d) are also presented. Arrows indicate fracti varioussecondarystructureswerecalculatedwiththeDICHROWEB software [17]. www.interscience.wiley.com/journal/psc Figure 1. Hemolytical assay Synthetic peptides were dissolved in aacetic solution acid containing 0.01% and 0.2%500 BSA in order to reach a concentration of 0.33 mg ml both in water and in presence70%. of The increasing spectra amounts are of an average TFE ofto up 260 three to nm scans with recorded a from scan 180 speed of 0.7 nm s Circular dichroïsm spectroscopy analysis All CD spectra were recorded at 20 (Applied Photophysics) inof a 0.5 mm. quartz Theconcentration cell of peptides 0.2 mg with were ml initially an dissolved optical in path water at a line). Chromatograms from a linear gradient (dotted line) from 5 to 65% acetonitrile over 90 min at a characterized antimicrobial peptide from activities. Fraction H5 contain the future purified halocyntin and papillosi

50 51 P. < ) ). In M M µ µ MBC ,except 13 < M ), whereas . ). Activities µ 3 M M. luteus, B. M M M. luteus, B. µ µ µ < ), except for ). M 39 56 56 . M µ . . 0 µ 1 MBC 56 < . < 13 1 . < 3 . M < M µ < MBC MBC µ ). ) Papillosin MBC ( M < 56 M < . MBC µ µ M MBC M µ < µ 25 . < M 6 05 were also found (1 39 . M µ . µ < (0 (0 39 . . ND is used for Not Done. 56 . M www.interscience.wiley.com/journal/psc MBC µ (1 100 25–50 ND 3.13–6.25 E. fæcalis < > Native Synthetic Native Synthetic 25–50 ND ND ND 25–50 6.25–12.50 ND 0.78–1.56 50–100 12.50–25 ND 0.78–1.56 12.50–25 1.56–3.13 ND 0.39–0.78 0.75–1.56 0.39–0.78 ND 0.05–0.10 1.56–3.13 0.39–0.78 0.13–0.253.13–6.25 0.19–0.39 0.78–1.56 ND 0.19–0.39 M 6.25–12.50 1.56–3.13 ND 0.78–1.56 6.25–12.50 1.56–3.13 0.5–1.00 1.56–3.13 6.25–12.50 6.25–12.50 0.25–0.50 0.78–1.56 A. viridans A. viridans µ and 13 . and and Minimal bactericidal concentration (MBC) of halocyntin and (3 α growthwaslessaffected(1 K. pneumoniae S. aureus , reaching 22% hemolysis at 50 ). Halocyntin activity against Gram-negative bacteria was DH5 M M µ µ In summary, papillosin possess potent activities against Gram- In liquid growth inhibition, halocyntin had marked activity Papillosinshowedimportantantibacterialactivitiesagainstboth 13 13 Table 1. papillosin BacteriaGram-positive bacteria HalocyntinM. MBC luteus ( S. aureus B. megaterium A. viridans E. faecalis Gram-negative bacteria E. coli MBC were determinednative and by synthetic peptides testing in liquid growth differentdifferent inhibition assays concentrations against bacterial of strainsvalues both are according expressed in to the Hancock method. MBC S. typhimutium P. aeruginosa E. aerogenes K. pneumoniae N. gonorrhoeae . . positive and Gram-negative bacteria. Halocyntinall is the active Gram-positive against bacteria tested butnegative its bacteria activity is against effective Gram- but much lessall potent. strains In addition, tested, for papillosin presentthan a halocyntin. higher antibacterial activity Hemolytical Activity Peptides hemolytical activityblood cells was (Figure 2). assessed Integrity ofby toward red papillosin blood at cells sheep all was concentrations not red tested.activity affected In was contrast, detected hemolytical for3 halocyntin concentrations higher than against Circular Dichroism Study CD spectra of halocyntinand and papillosin in were presence recordedtwo of in peptides displayed water various a very percentages similarrandom of behavior. coil In TFE water, spectrum a (Figure typical spectra 3). was were obtained. The progressively modified In with presence characteristic bands of at TFE, their against S. aureus contrast to halocyntin, papillosin also hasGram-negative a bacteria strong (0 activity against against Gram-positive strains, andmegaterium, more particularly significantly lower with MBC between 6.25 and 50 aeruginosa 3 Gram-positive and Gram-negative bacteria. GrowthGram-positive inhibition bacteria of wasmegaterium, particularly strong for ,the M. sabatieri (Figure 1(a), fraction 2009 European Peptide Society and John Wiley & Sons, Ltd. -terminal sequence was ob- c (Gram-positive bacteria) and 2 H.papillosa :48–55Copyright 15 M. luteus, S. aureus 2009; (Gram-negative bacteria). All other strains were tested For the second peptide, the following 34 amino acid se- An incomplete 22 residues NH Marine invertebrate antibacterial peptide with synthetic papillosin. For comparison, the activitysynthetichalocyntinwasalsodetermined(Table spectrum of 1).Thedifferences between synthetic andapproximative native estimation peptides ofEdman native are degradation peptide probably protocol. amountalways due four given fold Indeed, to more active by synthetic thanpapillosin the native halocyntin is form, while is synthetic twodifferences in fold activity are less identical for all active bacterial strains. than the native one. These E. Coli Antimicrobial Activity SpectrumHalocyntin and and Papillosin Bactericidal Assay of Activity spectrum againstnative a and synthetic variety peptides was of investigated (Tableenough bacterial 1). Whereas native strains halocyntin of (104these both tests, nmol) only was 28 available nmolthe to of purification native steps. perform papillosin Consequently, native wasrestricted papillosin obtained tests to after were tained for the firstcomplete peptide: biochemical FWGHIWNAVKRVGANALHGAVT. characterization The wasmatic achieved digestion of after this peptide enzy- with an endoproteinase Lys-C.digestion, After the analysis by massage spectrometry products revealed of two 1257.48 cleav- andwere 1492.68 also sequenced Da. by These Edman degradation two and gave subpeptides respectively the sequences: FWGHIWNAVKcombination of and all these RVGANALHGAVTGALS. data gives thefirst The complete peptide: sequence FWGHIWNAVKRVGANALHGAVTGALS. of This this 26 amino acid sequence has aDa, which calculated is monoisotopic in good massisolated agreement peptide (2731.55 of with Da). Searches the 2731.45 in mass sequence databasesnot measured did yield for any the homology with known peptidesname and of we halocyntin propose for the this novel cationic antibacterial peptide. quence was obtained by EdmanGVKAAAKGAAVGGLNALAKHIQ. degradation: GFWKKVGSAAWG- This sequencemonoisotopic mass has of 3318.85 a Da, which is calculated alsothe in mass agreement with measured forhomology the with purified known peptide peptide (3318.44 waschosen Da). found the name in As of databases, papillosin no for we this second have cationicpeptide. antibacterial J. Pept. Sci. initial active fractions were also submittedpurification to steps, the and three one additional peptideinitial was completely fraction purified from M2sufficient (Figure 1(b)). for However, biochemical thefocuses exclusively amount characterization. on was the The antibacterial not 30–32% molecules present acetonitrile eluted fraction from around study 16 and 34% acetonitrile, respectively. Concerning Primary Structure of Isolated Antimicrobial Peptides Mass spectrometryrevealed and deduced molecular masses of analysis 2731.55 and 3318.44the Da of for two molecules the isolated fromeach wide-scan the peptide, fraction only H5 spectrum one (Figure mass 1(a)).of was For obtained, the illustrating the molecules. purity degradation. These peptides were sequenced by Edman H5). These fractions werephase submitted HPLC to purification three steps. additional Forlost reverse- other fractions, during activities subsequent were purificationwere steps. purified Finally, from twoextracts peptides (Figure the 1(c) and fraction (d)). H5 of these hemocyte acidic . . [28]. et al :48–55 respond 15 Styela clava [29]. Despite 2009; R. Galinier S. aureus . However, the small hemocytes [6,33]. The Halocynthia roretzi J. Pept. Sci. [27], but it has not been Styela plicata S.clava M. sabatieri -terminalamidation.Alsoexpressed C -helical, amphipathic, histidine-rich AMP α Cucumaria frondosa , that we named halocyntin and papillosin. Several AMP have already been isolated and characterized from AMP, also known as cationic host defense peptides are diverse in In contrast, we revealed antimicrobial activities on the basis These two small tetrapeptides are reportedactivity to against have both antimicrobial Gram-negative andbut Gram-positive also bacteria, to havecell strains. cytotoxic Another short activities AMP against was characterizednamed different : plicatamide an eucaryote octapeptide and isolated from its small size andactivity its is modest very cationic potent. It properties, formslipid its cation-selective antibacterial channels bilayers in model composedto of plicatamide bacterial exposurebegins lipids. with a within massiveis seconds potassium characterized [30]. efflux by that terminal Moreover, an residue, oxidatively like conferring proteolytic decarboxylatedwere halocyamines, resistance. aromatic also Several it C- characterized AMP A from first the group solitary of[4,31]. ascidian five The AMP named styelins styelinprepropeptides, are A–E and highly was are basic characterized Gram-positive polypeptides, effective bacteria, with encoded against lowsignificantly as Gram-negative MICs. from Styelin and D styelinsthese and molecules A–C, could E be subjected differed to and differentmodifications post-translational probably it involved has inlow-pH the been control or of shown high-salt activityclavanins that A–E under conditions was also [32]. isolated from A second groupof23aminoacidsthatexhibit of AMP, as prepropeptides, theGram-positive molecules are bacteria, broadly and effectivebacteria. also It against was shown against that their some[34]. mode of At Gram-negative action is neutral pH-dependent bacterial pH, membrane clavanins as interactand nonspecifically well form with as an the artificialconditions, ionophore the phospholipid accross membrane bilayers not the is interact membrane. still with phospholipids. Under the Theyproteins seem target, acidic that to generate but transmembrane target ion membrane clavanins gradients.suggested It do has that been clavaninsconformation could allowing interaction adopt withdisturbing transmembrane a the protein, ion flux flexible accross the membrane. hydrophobic Clavaspirinpurified was from also the same speciespresents [7]. exactly If the the preproclavaspirin same cDNA mature structure than peptide those displays of weakbeen clavanins, similarities suggested the that with clavaspirin clavanins.into nucleotides It have a has been preexisting inserted clavanin[7]. gene Such by recombination some eventsthe recombination have diversity event participated of in cathelicidins, expanding a family of AMP from the innate clavanins are a family of hemocytes of different tunicate species. Halocyamine A and B were the first, isolated from the solitary ascidian their sequence and structure. They aremore small in than size, contain 45 rarely residues,and they are generally have amphipathic with at hydrophilicresidues least and spatially hydrophobic two well-separated. Most positive AMP charges havehydrophobic more amino than acids 50% and apolar lower and proportion negatively of charged both residues. neutral of cationic peptidescationic in AMP both from hemocytes tunicate of quantity species. of We pure peptide purified didcharacterization. one not allow In for complete addition, biochemical antibacterial peptides we from the hemocytes fully ofH. the papillosa characterized solitary tunicate two new characterized. the echinoderm / -helical α 2009 European Peptide Society and John Wiley & Sons, Ltd. c negative control) 600 100. Percentage hemol- A Copyright × ´ etic/BSA solution as neg- − Triton-X100 10% was used as d sterols [25]. They are present sample k squares for halocyntin and black omplement like substances [23], ty was assessed toward sheep ery- 600 (A negative control) 600 = A − -helical conformations. α Peptides hemolytic activity. positive control AMP have been isolated from bacteria, viruses, plants, and We report here the screening for antibacterial peptides in five Our results show that all tested species possess antimicrobial 600 structure. With 50% offor TFE, halocyntin 60 and and 74%two papillosin, of spectra respectively. helix remained were At verysignificant observed 70% conformational similar, changes for of indicating a higher percentage that TFE, ofThe TFE. there the 204-nm are isodichroic no pointrandom suggests coil and an equilibrium between (A www.interscience.wiley.com/journal/psc lysozymes [24], or polyhydroxylate Discussion In a context of new bioactive moleculesresearch,marineenvironmentisprobablytheonewiththegreatest with antimicrobial activity potential. Indeed, its biodiversity isbetween unique 50 as and it 80% is of estimatedOn 33 all that known life animal phyla, forms 32 are are presentamong present in which aquatic only 21 environment, in are exclusively oceans. marine. . They are alsoof extraordinary called pharmaceutical ‘natural interest. antibiotics’,already Around been and 700 isolated AMP they have until are marine now invertebrates [1]. [13] and several originate from marine invertebrate species.because We it focused represents the onhave largest cationic shown group that peptides of hemocytes AMP.in are Previous marine a invertebrates works tissue [4,6,7,18–20]. of Ourthen choice screening not for for exhaustive, AMP screening but was voluntaryin oriented hemocytes. on cationic peptides activity in crudethese extracts. activities For wereThis the rapidly indicates three lost thatbe echinoderm after the associated species, first detected to other purificationextracts. antimicrobial This molecules is steps. in activities than accordance with previous could cationic workpresence that peptides of revealed many the different in antimicrobial nonpeptidic the compounds in echinoderms. These molecules[21], are steroidal naphtoquinone glycosides pigments [22], c in various tissue fromOnly the one coelomocytes to work the indicate body the walls [26]. potential presence of an AMP in 190, 208, and 222 nm (50%mean TFE residue spectrum) and ellipticity an at increase of 222 the nm consistent with an Figure 2. control of 100% hemolysis and an acid ac triangles for papillosin. ysis values are represented by blac ative control.throcytes. Hemolytic Percentage activi equation: hemolysis was % hemolysis calculated with the following

52 53 -helix α P. lividus -helical structure sp. and α -helical, (ii) ß-sheet α -helical structure for both peptide, α -helices for both peptides. It also revealed www.interscience.wiley.com/journal/psc α H. tubulosa, Cucumaria 6 10.6 52 41153 ). The spectra were obtained in water, and in presence + + 1 − l -helical structures in presence of TFE. The structure tool.html). α M 4 − Moreover, alignments with the most similar peptide in the In conclusion, this study shows that hemolymphs of the three Cationic host defense peptides can be classified in four groups, residues point hydrophobic ratio(%) APD, find pleurocidinulin (AP00166) (AP00001) for for papillosin, halocyntin two amphiphatic and adenoreg- echinoderm species contain no cationic peptide hostwith defence, which previous is attempts in agreement other to echinoderm isolate species this [26]. kind However, this of study molecules reveals from the structures stabilized with several disulphidestructures, bonds, (iii) and extended (iv)[13]. loop Analyses structures by withGOR the one 4 secondary disulphide [37], structurefrom bond revealed residue prediction 7 an to software 17papillosin. Both for for halocyntin, halocyntin and and papillosin, the from predicted residue 9 to 30 for that such helices mightfor be a amphiphatic as totalsurface at of for least halocyntin, 26 11 and at residues mayamphiphatic least nature of 14 be both from peptides which 34 situated mean the forof segregation on papillosin. hydrophobic The and the nonhydrophobic same residueswheel diagrams is (Figure hydrophobic represented 4). by peptides. This kind of peptideplasmic is assumed membrane, to causing destabilize its thefor cyto- permeabilization. microbial membrane The is thought selectivity to be dueof to anionic their lipids high on content the surface, theent high across this electrical-potential membrane, gradi- and their lack of cholesterol,a supporting good interaction with cationic peptides [39]. based on their structure: (i) amphiphatic was experimentally supported by CDsynthetic experiments peptides performed (Figure on 3). Indeed,which the two are synthetic in peptides randomto coil generate in waterprediction displayed tool a of high the antimicrobial propensity free peptide online database at (APD) http://aps.unmc.edu/AP/main.php, [38] the also possibility confirmed to form ) and papillosin (1.0 10 1 − l M 4 − ences and physico-chemical properties ulated from ADP web site (http://aps.unmc.edu/AP/main.php). Isoelectric points were defined . Moreover, this last column (Figure 1(c) M 8 µ 2009 European Peptide Society and John Wiley & Sons, Ltd. c for halocyntin (0.74 10 , provided two other AMP. The first, . Both molecules are strongly active against -terminal position that is considered to play N :48–55Copyright 15 in vitro Halocyntin and papillosin amino acid sequ 2009; Circular dichroism spectra Halocynthia aurantium -terminally amidated and linked covalently with a single C Analysis of halocyntin and papillosin sequence properties Papillosin GFWKKVGSAAWGGVKAAAKGAAVGGLNALAKHIQ 34 Peptidethanks to the Compute pI/Mw tool of the Expasy website (http://www.expasy.org/tools/pi Amino acid sequence Total Charge Isoelectric Total Net charges and total hydrophobic ratio were calc Halocyntin FWGHIWNAVKRVGANALHGAVTGALS 26 Table 2. -helical conformation. It possesses a broad activity spectrum Marine invertebrate antibacterial peptide J. Pept. Sci. Figure 3. (Table 2) clearlyand shows chemical that characteristicshalocyntin of both and has AMP. also peptides a Papillosin higherdata net present is suggest charge longer (Table that physical 2). halocyntin than Theoretical papillosin. is This slightly was confirmed more by hydrophobiclater HPLC data: than (28% halocyntin acetonitrile) is than eluted the papillosin last reverse-phase (25% purification step acetonitrile) on a during C immune system ofspecies, mammals [35]. Finally, a fourth tunicate against Gram-negative and Gram-positivehalocidin, bacteria. is The second, composedamino of acid residues two that are linked subunits by ahas containing single been disulfide demonstrated bond 18 [3]. that It the and 18active 15 residues than monomer the was heterodimer more ordifferent bacterial the strains. 15 residue monomer against dicynthaurin, is composed of twoany 30-residue sequence monomers without homologyare to other tunicatecysteine AMP disulfide bond. [5]. In a Molecules membrane mimeticα system, it adopts peptide presents no hemolytical activitycells toward (in sheep contrast red to blood halocyntin). Inbe conclusion, a papillosin promising seems candidate to forputative future use studies as aimed therapeutics. to evaluate its of 20, 30, and 50% of TFE. and (d)). Moreover,(phenylalanine) halocyntin in possesses ana aromatic role in residue was antimicrobial assessed activity [36]. Their bacteriolytic activity Gram-positive bacteria. However, papillosinuntil presents 8 an times activity higher thanPapillosin halocyntin, also possess depending a on high the antibacterial activityof bacteria. Gram-negative against bacteria, a while panel halocyntin only presentactivity a limited against these bacterialpapillosin strains. are more often The around or MBC below 1 determined for : : . 32 265 et al 2000; 1991; :48–55 1997; 15 1999; FEBS Lett. Comp. Biochem. 2009; R. Galinier J. Biochem. (Tokyo) J. Biol. Chem. : 1723–1729. : 121–126. 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Mitta G, Hubert F, Noel T, Roch P. Myticin, a novel cysteine-rich 20. Miyata T, Tokunaga F, Yoneya T, Yoshikawa K, Iwanaga S, Niwa M, 21. Service M, Wardlaw AC. Echinochr 22. Andersson L, Bohlin L, Iorizzi M, Riccio R, Minale L, Moreno-Lopez W. and FEBS 2009 European Peptide Society and John Wiley & Sons, Ltd. : 3645–3654. c Comp. Biochem. 48 M. sabatieri : 141–148. 2004; : 81–86. Copyright 521 1527 : 515–521. cytes of the solitary tunicate, 118 olar residues imparts amphipathicity. 2002; 2001; -helical structure was predicted tides from tunicate hemocytes. α 1997; Residues 1–18 of halocyntin (a) and residues 2–19 of papillo .An : 149–168. FEBS Lett. 198 : 158–162. Antimicrob. Agents Chemother. 2004; H. papillosa Biochim. Biophys. Acta 400 . We characterized two peptides, halocyntin and 1997; Helical wheel diagrams. 27 for papillosin. These two low values indicate that . 0 Halocynthia aurantium. antimicrobial peptide from hemo Romestand B. Insights intoinvertebrates: the penaeid the shrimps and anti-microbial the oysterImmunol. defense Crassostrea Rev. gigas. of marine invertebrates. alpha-helical antimicrobial pep Sherman MA, Waring A,peptide Lehrer RI. from Dicynthaurin:aurantium. hemocytes an of antimicrobial the solitary tunicate, Halocynthia peptides from thePhysiol., solitary B Biochem. tunicate, Mol. Biol. Styela clava. Lett. − 3. Jang WS, Kim KN, Lee YS, Nam MH, Lee IH. Halocidin: a new 2. Bachere E, Gueguen Y, Gonzalez M, de Lorgeril J, Garnier J, 1. Tincu JA, Taylor SW. Antimicrobial peptides from marine 6. Lee IH, Zhao C, Cho Y, Harwig SS, Cooper EL, Lehrer RI. Clavanins, 5. Lee IH, Lee YS, Kim CH, Kim CR, Hong T, Menzel L, Boo LM, Pohl J, 4. Lee IH, Cho Y, Lehrer RI. Styelins, broad-spectrum antimicrobial ´ ezians Dupont for their participation in some HPLC purification Acknowledgements Collection of theducted tunicate by and Mikel echinoderm Becerro.in samples Thanks recording were CD to data. con- Daniel TheV Auguin authors are for grateful his tosteps help Jean and Bobo antimicrobial activity and tests. both peptides selectively interact withmembrane, the and lipids of not the with microbial represent proteins. a good It antibacterial means drug candidate thateffects. without In both combination many with side peptides the lack of hemolyticalsheep activity red toward blood cells for papillosinconcentration, these and data also indicate that for the halocyntin two peptides atpotential represent low candidates for pharmaceutical assays.are These protected molecules by two international patentsand WO (WO 2004/081 2004/081 214 024 A1). A2 www.interscience.wiley.com/journal/psc References for each peptide, and weeither found against that both a molecules panelwith were active of a Gram-positive higher and antimicrobialbinding -negative activity potential bacteria, for or papillosin.and Boman The index protein- [40] is 0.06 for halocyntin presence of AMP in both tunicate species, Figure 4. H. papillosa papillosin from clearly evident clustering of polar and ap

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