leptoglycin: a new glycine/leucine-rich antimicrobial peptide isolated from the skin secretion of...

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ARTICLE IN PRESSToxicon xxx (2009) 1–10

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Toxicon

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Leptoglycin: A new Glycine/Leucine-rich antimicrobial peptide isolatedfrom the skin secretion of the South American frog Leptodactyluspentadactylus (Leptodactylidae)

Juliana C. Sousa a,1, Raquel F. Berto a,1, Elicelia A. Gois b, Nauıla C. Fontenele-Cardi b,Jose E.R. Honorio-Junior a, Katsuhiro Konno c, Michael Richardson d, Marcos F.G. Rocha e,Antonio A.C.M. Camargo c, Daniel C. Pimenta f, Bruno A. Cardi a, Krishnamurti M. Carvalho a,g,*

a Laboratorio de Toxinologia e Farmacologia Molecular, Instituto Superior de Ciencias Biomedicas, Universidade Estadual do Ceara, Fortaleza, CE 60.740-000, Brazilb Hospital Sao Jose de Doenças Infecciosas, Fortaleza, CE 60.000-000, Brazilc CAT/CEPID, Instituto Butantan, Sao Paulo, SP 05503-900, Brazild Fundaçao Ezequiel Dias, Belo Horizonte, MG 30510-010, Brazile Faculdade da Veterinaria, Universidade Estadual do Ceara, Fortaleza, CE 60.740-000, Brazilf Laboratorio de Bioquımica e Biofısica, Instituto Butantan, Sao Paulo, SP 05503-900, Brazilg GENPHARMA, Fortaleza, CE 60.000, Brazil

a r t i c l e i n f o

Article history:Received 7 November 2008Received in revised form 26 February 2009Accepted 3 March 2009Available online xxxx

Keywords:Antimicrobial peptidesLeptoglycinLeptodactylus pentadactylusGly/Leu-rich peptidesNatural peptides

Abbreviations: Abs, absorbance; ACN, acetonitrilemedical mycology center; LPG, leptoglycin; MIC, minreversed-phase high performance liquid chromatog

* Corresponding author at: Universidade EstaduFarmacologia Molecular, Av. Paranjana, 1700 - Camp

E-mail address: [email protected] (K.M. C1 These authors contributed equally to the paper.

0041-0101/$ – see front matter � 2009 Elsevier Ltddoi:10.1016/j.toxicon.2009.03.011

Please cite this article in press as: Juliana Cfrom the skin secretion of the South Amerj.toxicon.2009.03.011

a b s t r a c t

Antimicrobial peptides are components of innate immunity that is the first-line defenseagainst invading pathogens for a wide range of organisms. Here, we describe the isolation,biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named lep-toglycin. The amino acid sequence of the peptide purified by RP-HPLC (C18 column) wasdeduced by mass spectrometric de novo sequencing and confirmed by Edman degradation:GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-nega-tive bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimalinhibitory concentrations (MICs) of 8 mM, 50 mM, and 75 mM respectively, but it did notshow antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micro-coccus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) anddermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activitywas observed at the 2–200 mM range concentration. The amino acid sequence of lep-toglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusualcentral proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides.Taken together, these results suggest that this natural antimicrobial peptide could be a toolto develop new antibiotics.

� 2009 Elsevier Ltd. All rights reserved.

; AMPs, antimicrobial peptides; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; CEMM, specializedimal inhibitory concentration; MS, mass spectrometry; MS/MS, tandem mass spectrometry; RP-HPLC,

raphy; TFA, trifluoroacetic acid.al do Ceara, UECE, Instituto Superior de Ciencias Biomedicas, ISCB, Laboratorio de Toxinologia eus do Itaperi, CEP: 60.740-000, Fortaleza, Ceara, Brazil. Tel.: þ55 85 3248 4082; fax: þ55 85 3486 6221.arvalho).

. All rights reserved.

. Sousa et al., Leptoglycin: A new Glycine/Leucine-rich antimicrobial peptide isolatedican frog Leptodactylus pentadactylus (Leptodactylidae), Toxicon (2009), doi:10.1016/

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1. Introduction

Antimicrobial peptides (AMPs) are relevant compo-nents of innate immunity that is the first-line defense fora wide range of organisms against invading pathogens(Hancock, 2001; Kimbrell and Beutler, 2001; Brown andHancock, 2006). Classic AMPs are mainly cationic andamphipathic peptides with generally 12–50 amino acids inlength, have a net positive charge due an excess of basiclysine and arginine residues over acid residues, and containaround 50% hydrophobic amino acids (Hancock, 2001;Bulet et al., 2004; Brogden, 2005; Brown and Hancock,2006). These peptides, also know as host-defense peptides,have been isolated and characterized from diverse organ-isms (Sitaram and Nagaraj, 2002; Zasloff, 2002; Bulet et al.,2004) such as plants (Condit, 1993; Pelegrini and Franco,2005; Pelegrini et al., 2008), arthropods (Bulet et al., 1999;Lorenzini et al., 2003; Herbiniere et al., 2005; Dubovskiiet al., 2006), amphibians (Zasloff et al., 1988; Bevins andZasloff, 1990; Apponyi et al., 2004; Conceiçao et al., 2006;Pukala et al., 2006) and mammals (Mallow et al., 1996;Johansson et al., 1998; Rieg et al., 2004; Chromek et al.,2006; Zasloff, 2006).

Among amphibians, different antimicrobial peptideshave been isolated from the granular gland skin secretionof anurans, such as Leptodactylus genus. It contains 64species, spread out from South America, mainly in Braziland Antilles (Frost, 2009). Biochemical analyses using theskin secretions of five Leptodactylus species revealeda family of eight structurally homologous AMPs that tend toadopt a-helical conformations in a membrane-mimeticsystem (King et al., 2005; Nielsen et al., 2007; Nascimentoet al., 2007). Thus, the AMPs isolated from Leptodactylusocellatus, ocellatins 1, 2 and 3, displayed growth-inhibitoryactivity against the Gram-negative bacterium Escherichiacoli (Nascimento et al., 2004), and ocellatin 4 isolated fromthe same species presented cytolytic properties andgrowth-inhibitory activity against the Gram-negativeE. coli and Gram-positive Staphylococcus aureus bacteria(Nascimento et al., 2007). The antibacterial peptidefallaxin, isolated from West Indian mountain chicken frogLeptodactylus fallax showed growth-inhibitory activityagainst several Gram-negative bacteria strains, such asE. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae andEnterobacter cloacae, but not toward to Gram-positivebacterium S. aureus or the yeast Candida albicans (Rollins-Smith et al., 2005). Other peptide, termed pentadactylin,purified from Leptodactylus pentadactylus skin secretions,presented a better inhibition against the growth of severalGram-negative bacteria than Gram-positive bacteria andshowed a very weak hemolytic activity (King et al., 2005).Laticeptin (Leptodactylus laticeps) (Conlon et al., 2006) andsyphaxin (Leptodactylus syphax) (Dourado et al., 2007)peptides were tested against both Gram-positive andGram-negative bacteria showing a high growth-inhibitoryactivity. More recently, AMPs with very low antimicrobialpotency, named ocellatins-V1, 2 and 3, were isolated fromLeptodactylus validus (King et al., 2008).

In the present study, we describe the isolation, bio-logical characterization and amino acid sequencing of anovel neutral Glycine/Leucine-rich antimicrobial peptide,

Please cite this article in press as: Juliana C. Sousa et al., Leptoglycifrom the skin secretion of the South American frog Leptodactylus pj.toxicon.2009.03.011

containing a central proline, active against Gram-negativebacteria, named Leptoglycin, from skin secretion of L. pen-tadactylus. This peptide was screened through biologicalassays and presented no hemolytic activity at the MIClevels. Its unique amino acid sequence was deduced bymass spectrometric de novo sequencing and confirmed byEdman degradation.

2. Materials and methods

2.1. Collection of skin secretion

Adult specimens, both male and female (n¼ 6), of L.pentadactylus were collected in Paraipaba, Ceara, Brazil. TheL. pentadactylus skin secretions were obtained by mildelectrical stimulation (Conlon et al., 1999) and collected ina cooled beaker by washing the skin surface with coldbi-distilled water. This solution was lyophilized and storedat �25 �C. All the procedures involving animals were inaccordance with the guidelines provided by the BrazilianCollege Animal Experimentation.

2.2. Reagents

All chemicals, salts and standards were purchased fromSigma–Aldrich (St Louis, MO, USA), unless stated otherwise.

2.3. Purification of the antimicrobial peptides

Aliquots of lyophilized skin secretion (40 mg) weredissolved in 2.0 ml of 80% (v/v) ethanol/water andcentrifuged at 5000� g for 30 min. The supernatant waspurified on a C18 semi-preparative RP-HPLC column (Shimpack prep. C18, 2.5� 30 cm, 5.0 mm) equilibrated with 0.1%(v/v) TFA/water (solvent A). Elution was performed ata flow rate of 4.5 ml/min using a two-step gradient:initially, from 0% to 50% acetonitrile (ACN) containing 0.1%TFA (solvent B) over 150 min, followed by 50–80% of samesolvent system over 10 min. The RP-HPLC column eluateswere monitored by their UV absorbance at 214 nm. Frac-tions were manually collected and lyophilized. Purity ofthe peptide was evaluated by analytical RP-HPLC usinga Supelco C18 column (0.46� 25 cm, 5.0 mm) eluted witha 40–55% linear gradient of B over 35 min, undera constant flow rate of 1.0 ml/min and/or by MALDI-TOFmass spectrometry.

2.4. Mass spectrometry

Molecular mass analyses of the fractions and purifiedpeptides were performed on a Q-TOF Ultima API (Micro-mass, Manchester, UK), under positive ionization modeand/or by MALDI-TOF mass spectrometry on an EttanMALDI-TOF/Pro system (Amersham Biosciences, Sweden),using a-cyano-4-hydroxycinnamic acid or sinapinnic acidas matrices.

2.5. Peptide sequencing

Mass spectrometric de novo peptide sequencing wascarried out in positive ionization mode on a Q-TOF Ultima

n: A new Glycine/Leucine-rich antimicrobial peptide isolatedentadactylus (Leptodactylidae), Toxicon (2009), doi:10.1016/

J.C. Sousa et al. / Toxicon xxx (2009) 1–10 3

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API fitted with an electrospray ion source (Micromass,Manchester, UK). The samples were dissolved into a mobilephase of 50:50 aqueous formic acid 0.1% acetonitrile anddirectly injected (10 ml) using a Rheodyne 7010 sample loopcoupled to an LC-10A VP Shimadzu pump at 20 ml/min,constant flow rate. The instrument control and dataacquisition were conducted by MassLynx 4.0 data system(Micromass, Manchester, UK) and experiments were per-formed by scanning from a mass-to-charge ratio (m/z) of50–1800 using a scan time of 2 s applied during the wholeinfusion. The mass spectra corresponding to each signalfrom the total ion current (TIC) chromatogram were aver-aged, allowing an accurate molecular mass determination.External calibration of the mass scale was performed withNaI. For the MS/MS analysis, collision energy ranged from18 to 45 eV and the precursor ions were selected undera 1-m/z window. For the confirmation of the amino acidsequence and Leu/Ile alternate assignments, the peptidewas subjected to Edman degradation using a ShimadzuPPSQ-21 automated protein sequencer, following themanufacturer’s standard instructions. Amino acid analyseswere performed to quantify the different peptide batchesused throughout this work.

2.6. Sequence analysis

Sequence alignment was performed with ClustalW(Thompson et al., 1994) imposing the highest penalty forgap opening.

2.7. Antibacterial assays

During the purification procedure, the antibacterialactivity of the peptides was monitored by in vitro inhibitionzone assay on agarose plates (Hultmark et al., 1983). Assayswere performed over the bacteria strains P. aeruginosa LP01,Citrobacter freundii LP02 (isolated from the animal’s naturalenvironment) (Supplemental Data) and S. aureus ATCC25.923. Briefly, bacteria first grew in BHI broth at 37 �C.Then, approximately 2�108 cells/ml were added toagarose medium. Peptides were dissolved in 10% DMSO andaliquots of 10 ml were spotted on the plate. The inhibitionzones were evaluated after overnight incubation at 37 �C.

Minimal inhibitory concentration (MIC) was deter-mined for the microorganisms P. aeruginosa ATCC 9027, E.coli ATCC 28922, C. freundii ATCC 8090, S. aureus ATCC25.923, Micrococcus luteus ATCC 9341 and Enterococcusfaecalis ATCC 29912 by the broth microdilution method, inaccordance with the Clinical and Laboratory StandardsInstitute – CLSI (formerly NCCLS) (NCCLS, 2003). Pre-inocula of strain bacteria isolates were prepared in BHIbroth and incubated for 3–4 h at 37 �C. Lyophilized aliquotsof the purified peptide were dissolved in 10% DMSO (50 ml)and incubated with an inoculum (50 ml of 1�108 colonyforming units/ml with reference to a 0.5 McFarland stan-dard turbidity) from a logarithmic phase growth culture ofstrain bacteria in 96-well microtiter cell-culture plates for20 h at 37 �C, under humidified air atmosphere. Afterincubation, the absorbance at 492 nm of each well wasdetermined using a microtiter plate reader. Assays wererun in duplicate for strain and MIC values were recorded as


the lowest concentration of peptide where no growth couldbe determined.

2.8. Antifungal assays

The inhibitory effect of leptoglycin was determined ongrowth of yeast (C. albicans CEMM 01-3-075, Candida tro-picalis CEMM 01-2-078) and dermatophytes fungi (Micro-sporum canis CEMM 01-2-133, Trichophyton rubrum CEMM01-1-100). MIC for Candida spp. was determined by thebroth microdilution method, in accordance with the Clinicaland Laboratory Standards Institute – CLSI (formerly NCCLS;M27-A2), (NCCLS M27A 2002). The broth microdilutionassay for M. canis and T. rubrum was performed as previouslydescribed (Jessup et al., 2000; Fernandez-Torres et al., 2002;Brilhante et al., 2005; Fontenelle et al., 2008) based on theM38-A document (CLSI; formerly NCCLS M38A 2002).Standardized inocula (2.5–5.0�103 CFU/mL for Candidaspp. and 5�104 CFU/ml for M. canis and T. rubrum) were alsoprepared by turbidimetry. Stock inocula were prepared onday 2 for Candida spp. and day 10 for M. canis and T. rubrumcultures, grown on potato dextrose agar at 28 �C. M. canis,T. rubrum (Fernandez-Torres et al., 2002) conidia withhyphal fragments and suspension of Candida spp. (Britoet al., 2007) blastoconidia suspensions were transferred tosterile tubes, followed by volume adjustment (4 ml withsterile saline solution). Suspensions were allowed to settlefor 5 min at 28 �C, and their density was read (530 nm) andthen adjusted to 95% transmittance. After this, suspensionswere diluted to 1:2000 for Candida spp. and 1:500 for M.canis and T. rubrum, with buffered RPMI 1640 medium (withL-glutamine, without sodium bicarbonate; 0.165 mol/Lmorpholinepropanesulfonic acid, pH 7.0; Sigma ChemicalCo), to obtain the inoculum size of c. 2.5–5�103 CFU/mL forCandida spp. and 5�104 CFU/mL for M. canis.

Leptoglycin (dissolved in 10% DMSO in distilled water)was incubated at 37 �C with previous suspensions in96-well microdilution plates and antifungal activity wasanalyzed visually after 2 days for Candida spp. and 5 daysfor M. canis and T. rubrum as recommended by CLSI. TheMIC was defined as the lowest leptoglycin concentrationthat caused 100% inhibition of visible fungal growth.

2.9. Hemolysis assay

The hemolysis assay protocol was modified fromOnuma et al. (1999). The cells were separated from theplasma of healthy donors by sedimentation and a 1%suspension of human red blood cells (RBC) (4.99�108/ml),washed three times by centrifugation with 0.15 M PBS, pH7.4, was prepared. In order to determine the hemolyticactivity, 150 ml PBS/peptide solution (2–200 mM) containing10% DMSO were added to 50 ml 1% RBC suspension andmixed. After 60 min incubation at 37 �C, the samples werecentrifuged at 3000� g for 5 min and a 100 ml aliquot wastransferred to microtiter plates and the absorbance wasmeasured at 405 nm. Reference samples for 100% hemo-lysis were 1% of RBC incubated with 0.1% Triton X-100, andfor no hemolysis, 1% of blood suspension, subject to thesame manipulation.



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3. Results

3.1. Purification and characterization of leptoglycin

The supernatant fraction of the centrifuged L. penta-dactylus crude skin secretion was fractionated by semi-preparative RP-HPLC, as shown in Fig. 1, panel A. Initially,peaks were tested for antibacterial activity against P. aeru-ginosa (LP01), C. freundii (LP02) and S. aureus LP03. Signifi-cant biological activity against Gram-negative bacteriacould be found for the peak containing leptoglycin (LPG,indicated by the arrow, Fig. 1, panel A). The peptide wasfurther purified as observed by MALDI/TOF-MS (Fig.1, panelB), which shows the purified peptide and sodium and aceticacid adducts. Once the peak containing the purified peptideretained its antibiotic activity, it was selected for de novomass spectrometric sequencing.

3.2. De novo peptide sequencing

The purified active selected peak containing leptoglycinwas submitted to de novo sequencing. Accurate molecularmass measurement was performed (Fig. 2, panel A, for thedoubly charged ion) which was dissociated by CIF withargon, generating the daughter ion spectrum depicted inFig. 2, panel B. Peptide processing according to a modifiedprotocol of Westermeier and Raven (2002) showed nocysteine or disulfide bridges in the molecule, for nomolecular mass shift could be observed after the sampleprocessing (data not shown). The MS/MS spectra wereanalyzed by the BioLynx software module of MassLynx 4.0(Fig. 3) and manually verified for accuracy in the amino acidsequence interpretation. The peptide was fully sequencedby mass spectrometry and identified as a novel bioactivepeptide and termed Leptoglycin (LPG, for it comes fromLeptodactylus and has high glycine content). It was possible

Fig. 1. (A) RP-HPLC profile on a preparative C18 column of pooled skin secretion of Larrow indicates the fraction containing leptoglycin (LPG). (B) MALDI-TOF/MS profileone acetic acid adduct. Inset: Analytical RP-HPLC profile of purified LPG (C18 colum


to fully sequence the complete peptide without enzymaticdigestion; nevertheless, additional Edman degradation wasperformed to validate the sequence and to check for Leu/Ilealternates (data not shown). Thus, chemical sequencingconfirmed the MS/MS deduced amino acid sequence: Gly-Leu-Leu-Gly-Gly-Leu-Leu-Gly-Pro-Leu-Leu-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Leu-Leu.

3.3. Sequence analysis

Leptoglycin was aligned with other Leptodactylus AMPs,Xenopus AMPs and the two isoforms of acanthoscurrin,a spider hemolymph antimicrobial protein together withthe homolog core of protein 4 (a transcription protein fromXenopus spp., which homology to leptoglycin was foundthrough BLAST searches) as depicted in Fig. 4. These resultsshow that leptoglycin presented important homology withXenopus spp. protein 4 (89%) and acanthoscurrin isoforms(59%), but lower than 50% homology with other amphibianAMPs aligned.

3.4. Antimicrobial activity and hemolytic assay

Leptoglycin was assessed for antimicrobial activity aspresented in Table 1. MICs of leptoglycin obtained againstGram-negative bacteria P. aeruginosa ATCC 9027, E. coliATCC 28922, C. freundii ATCC 8090 were 8.0 mM, 50.0 mMand 75.0 mM, respectively. On the other hand, no activitywas observed toward Gram-positive bacteria S. aureusATCC 25923, E. faecalis ATCC 29912 and M. luteus ATCC9341. Leptoglycin was also tested against human patho-genic fungi: C. albicans, C. tropicalis, M. canis and T. rubrum.None of these fungi showed growth inhibition in thepresence of leptoglycin. No hemolytic activity wasobserved at 2–200 mM concentration.

. pentadactylus. Right axis: Acetonitrile concentration along the gradient. Theof purified LPG. This profile also contains one and two sodium adducts and

n Shim pack 0.46� 25 cm).


XV

m/z877 878 879 880 881 882 883 884 885 886 887 888 889

%

0

100261005AF 31 (1.166) Sm (Mn, 2x3.00); Cm (30:32) TOF MS ES+

92.0882.031

881.534

882.528

883.037

883.522886.011886.509884.032

XV MSMS 881.5

m/z150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0

100261005AG 44 (1.660) Sm (Mn, 2x3.00); Cm (35:54) TOF MSMS 881.50ES+

102882.044

881.535

758.409624.444

511.336

284.201

171.121228.141

183.127

398.252285.165341.231

296.206370.258

483.345409.290439.192

609.301

596.435

587.331

625.433

681.460627.313 740.401

815.992

760.445 872.520

816.994871.507

882.541

883.038

1138.666883.5351004.686

891.608 1005.6831081.645

1139.657

A

B

Fig. 2. (A) ESI-Q-TOF/MS accurate molecular mass determination of the doubly charged LPG ion. (B) Collision induced fragmentation daughter ion spectrumgenerated through dissociation.


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Please cite this article in press as: Juliana C. Sousa et al., Leptoglycin: A new Glycine/Leucine-rich antimicrobial peptide isolatedfrom the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae), Toxicon (2009), doi:10.1016/j.toxicon.2009.03.011

M/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

%

0

100

XV MSMS 881.5261005AG MaxEnt 3 44 [Ev-50123,It50,En1] (0.050,200.00,0.200,1400.00,2,Cmp) 1: TOF MSMS 881.50ES+

bMax yMax

1762.07(M+H) +

1630.98b21

1517.89b20758.41

y11624.44b7

511.34b6

284.20b3171.12

b2143.12

a2

398.26b5341.23

b4

483.34a6 609.30

596.44a7

681.46b8

1138.67y151004.76

b11871.50y12778.51

b9 891.68b10

1081.64y14

1460.86b191251.74

y161175.72b14

1403.83b18

1602.98a21

1744.06

1726.041763.19

1769.12

GL L G G L L G P L L L G G G G G G G G G L

L G GG G G G G G G L L LP G L LGG LLG

Fig. 3. Representative deconvoluted de novo sequencing profile of purified leptoglycin. The doubly charged ion was selected for dissociation and the daughter ionspectra were processed by ByoLynx (Micromass). The spectrum is annotated for b and y ions. The deduced peptide sequence is shown on the top of the graph, forb and y series.


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4. Discussion

In the present study, we report the isolation, amino acidsequence and antimicrobial effects of Leptoglycin, a novelantimicrobial peptide purified from the skin secretion ofL. pentadactylus (Anura, Leptodactylidae).

In spite of the deduced (and chemically confirmed)amino acid sequence (Gly-Leu-Leu-Gly-Gly-Leu-Leu-Gly-Pro-Leu-Leu-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Leu-Leu),leptoglycin would not be an ideal candidate for MS/MSsequencing due to the hydrophobicity and repetitive-ness of the sequence. However, the peptide yieldeda rather complete dissociation spectrum, as represented inFig. 2(B). Spectra were then deconvoluted and processedaccordingly, as shown in Fig. 3 being the deducedMS/MS sequence thoroughly confirmed by the Edmandegradation.

FASTA search using leptoglycin sequence, when per-formed over sequence clusters and not individual proteins,gives match with 89% homology with a cDNA-deducedsequence named Xenopus protein 4 (UniProt entry #UPI000069F3E7) (Fig. 4). These sequences share homologywith bactericidal/permeability-increasing (BPI)/lipopoly-saccharide-binding protein (LBP) protein family. Theseproteins serve as primary defense mechanism by recog-nizing and removing potentially harmful odorants or path-ogenic microorganisms from the mucosa (Beamer et al.,1998). Also, leptoglycin presents 59% homology with thelong palate, lung and nasal epithelium carcinoma-associated


protein 4 (Ligand-binding protein RY2G5) (UniProt entryP59827) of human origin (Fig. 4).

It is interesting to note that BPI/LBP proteins and BPI/LBP-originated peptides, share high-affinity binding tolipopolysaccharide (Beamer et al., 1998), a glycolipid foundin the outer membrane of Gram-negative bacteria. Thesepeptides have been considered as cryptides, a new conceptof bioactive peptides generated through the action ofproteolytic enzymes on precursor proteins (Pimenta andLebrun, 2007; Someya et al., 2007). Then, although theexistence of protein 4 in L. pentadactylus was not demon-strated yet, the strong homology of leptoglycin with Xen-opus protein 4 and its action against Gram-negativebacteria suggest that leptoglycin could be a cryptide of thisprotein.

On the other side, leptoglycin’s unique amino acidsequence aligns better with antimicrobial peptide from thehemocytes of tarantula spider Acanthoscurria gomesiana(acanthoscurrins 1 and 2) (Lorenzini et al., 2003) thanamphibian AMPs known at present date. Both possessa higher hydrophobic content that may be associated tohigher Gly and Leu prevalence in these sequences (Fig. 4).

Furthermore, the XTs peptides, isolated from Siluranatropicalis (formerly Xenopus tropicalis) (Ali et al., 2001),particularly XT6 and XT7 lie closer to LPG (45% homology)in the alignment tree than other Leptodactylus AMPs(Fig. 4). Besides both peptides possess a higher hydro-phobic content, XT7 is the only AMP that contains anidentical sequence of seven amino acids (GLLGPLL) with


Fig. 4. Multiple sequence alignment as performed by ClustalW. Leptoglycin overlapping regions with the other peptides are depicted. The arrow indicates thatthe sole proline residue of leptoglycin aligns with XT7 and XT1 prolines, indicating a possible similar mechanism of action. The number in parenthesis corre-sponds to the UniProt (http://www.uniprot.org) entry, when available. Leptoglycin is currently in process in UniProt. (*) Gly-Leu-rich peptides from differentspecies of hylid frogs: AA¼ Agalycnis annae, AC¼ Agalycnis callidryas, PB¼ Phyllomedusa bicolor, PD¼ Phyllomedusa dacnicolor.


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Please cite this article in press as: Juliana C. Sousa et al., Leptoglycin: A new Glycine/Leucine-rich antimicrobial peptide isolatedfrom the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae), Toxicon (2009), doi:10.1016/j.toxicon.2009.03.011

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Table 1Antimicrobial activity assays of leptoglycin.

Microorganisms MIC (mM)

Gram-negative bacteriaPseudomonas aeruginosa ATCC 9027 8Escherichia coli ATCC 28922 50Citrobacter freundii ATCC 8090 75

Gram-negative bacteriaStaphylococcus aureus ATCC 25.923 N.A.Enterococcus faecalis ATCC 29912 N.A.Micrococcus luteus ATCC 9341 N.A.

YeastCandida albicans CEMM 01-3-075 N.A.Candida tropicalis CEMM 01-2-078 N.A.

Filamentous fungiMicrosporum canis CEMM 01-2-133 N.A.Trichophyton rubrum CEMM0 1-1-100 N.A.Hemolysis % N.A.

N.A.: not active up to 200 mM.


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a proline residue aligned with LPG sequence (Fig. 4, arrow).This Pro residue in a-helical can create a kink in the peptidebackbone because the lack of an amide proton, whichnormally provides a hydrogen bond donor, and it iscommonly found within the amphipathic a-helicalpeptides. In particular, this residue often appears in thecentral region of membrane-active peptides, and it maycontrol the folding process and affects the membranetranslocation or penetration (Yang et al., 2006). In addition,a central proline in a-helical peptides is important for fastelectrostatic interaction with negatively charged lipidmembranes, being crucial for effective translocation of theAMPs (Yang et al., 2006). The same feature could beobserved in buforin II, discovered in the stomach of theAsian toad Bufo bufo gargarizans (Park et al., 1996). Thispeptide efficiently crosses lipid bilayers (Kobayashi et al.,2004) and strongly binds to DNA and RNA (Park et al.,1998). There is an internal proline hinge in buforin II, whichis a key structural factor for the cell-penetrating propertyand critical factor for determining the antimicrobialpotency of buforin II (Kobayashi et al., 2000; Park et al.,2000). Thus, it is likely that the strong potency of lep-toglycin over Gram-negative bacteria (Table 1) may beconsequence of the presence of such proline in the centralregion of backbone of the peptide.

Finally, leptoglycin contains the motif GLL repeated 3times and GPL one time along its sequence, similar to themotifs of Gly/Leu-rich dermaseptin family peptides (iso-lated from the skin secretion of South American hylid frogsof the Phyllomedusinae subfamily) that are arranged inregular 3-mer motifs GXL (where X represents any aminoacid) and repeated 2–5 times along the sequence (Vanhoyeet al., 2004). However, in spite of the similarity of the motifsabove, leptoglycin presents only 36–50% homology withthose of dermaseptin family peptides (Fig. 4).

Classic antimicrobial peptides screening are generallyperformed over human pathogenic strains of isolatedbacteria in a search for novel antibiotics. Indeed, very fewstudies have been performed over strains of naturallyoccurring environmental bacteria, such as those that arepresent in amphibian’s skin (Ashcroft et al., 2007). In fact,


the synthesis and release of antimicrobial peptides in frogskin depend on environmental and species-specific factors(Magoni et al., 2001; Matute et al., 2000). Furthermore,species with antimicrobial peptides effective against thepathogen or opportunistic bacteria do have a naturaldefensive capability that may hold the infection in check(Rollins-Smith et al., 2002). Interesting, leptoglycin dis-played antibacterial activity against the L. pentadactylusskin-isolated Gram-negative bacteria P. aeruginosa, andC. freundii (Table 1, Supplementary data), acting as possiblehost-defense peptide. Furthermore, the ability of thispeptide to inhibit the growth of the strains of referencebacteria P. aeruginosa, C. freundii and E. coli (Table 1),suggests that it may be used as a tool to develop newantibiotic drugs.

In summary, leptoglycin is a linear peptide witha particular amino acid composition, characterized bya high level of glycine (59.1%) and leucine (36.4%) anda central proline. The presence of 22 amino acids with nocharged residues confers to this peptide a net charge ofzero and theoretical pI value (5.52). Further studies will benecessary to determine the secondary structure and themechanism of action of this new and uncommon antimi-crobial peptide of Leptodactylus genus.

Acknowledgments

Supported by funds provided by FUNCAP, CAPES,FAPESP, Lab. Cristalia, FAPEMIG (Edital 24000/01[MR]) andCNPq (grants 303516/2005-4 [DCP] & 473614/2006-5[DCP]). Parts of this work were developed at the CAT/CEPID,a FAPESP grant. We are grateful to Rosa G.S. Oliveira forHPLC assistance.

Conflict of interest

The authors of the article declare that there are nopotential conflicts of interest.

Appendix. Supplementary data

Supplementary data associated with this article can befound in the online version, at doi:10.1016/j.toxicon.2009.03.011

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