Published: July 11, 2011

r 2011 American Chemical Society 4230 dx.doi.org/10.1021/pr200393t | J. Proteome Res. 2011, 10, 4230–4240

ARTICLE

pubs.acs.org/jpr

Proteomic Analysis of Skin Defensive Factors of Tree FrogHyla simplexJing Wu,†,‡,§ Han Liu,†,‡,§ Hailong Yang,†,‡,§ Haining Yu,|| Dewen You,† Yufang Ma,†

Huahu Ye,^ and Ren Lai*,†,z

†Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences,Kunming 650223, Yunnan, ChinazLife Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu, China^Institute of Microbiology and Epidemiology, the Academy of Military Medical Sciences, Beijing 100071, China

)Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116023, China‡Graduate School of the Chinese Academy of Sciences, Beijing 100009, China

bS Supporting Information

’ INTRODUCTION

Among vertebrates, amphibian may have the most susceptibleskins because they are directly exposed to various living environ-ments laden with pathogens, predators, and noxious abioticfactors.1 Therefore, their skins are endowed with an excellentchemical defense system composed of pharmacological smallcompounds and gene-encoded peptides/proteins with versatilefunctions including cardiotoxic, neurotoxic, analgesic, antimicro-bial, and antioxidant activities.1�8 All of the properties clearly andadversely affect potential predators, pathogens, or nonbiologicalinjuries.

The number and diversity of compounds in amphibian skinsecretions is surprisingly high, even within a single species.Generally, four categories of compounds including biogenicamines, bufogenines, alkaloids, and peptides/proteins are identi-fied from amphibian skin secretions.1 A large amount of compo-nents in the skin secretions remain to be known, although manycompounds, such as antimicrobial peptides, tachykinins, dermor-phins, cholecystokinin, and bradykinins, have been extensivelystudied.9�15 Trying to reveal the detailed molecular defensivemechanism and high level of biochemical diversity make amphi-bian skins attractive subjects for chemical prospecting.

According to their habitats, there are four types of anuransincluding aquatic (e.g., genus of Xenopus), semiterrestrial (e.g.,genus ofRana), terrestrial (e.g., genus ofBufo), and arboreal (e.g.,genus of Hyla) species. Most Hyla amphibians occur on vegeta-tion near water bodies in forest and in open areas and they aretree-adapted species. In this kind of habitats, they may encountermany biotic and abiotic risk factors. They are green or brown indorsal coloration that facilitate their living in forest canopy. Inaddition, there is an excellent chemical defense system in thesefrogs for maintaining their survival. To investigate skin defensivechemicals of tree frogs, the skin secretions of H. simplex wereanalyzed and studied by proteomics or peptidomics and tran-scriptome analysis coupling with pharmacological tests.

’EXPERIMENTAL SECTION

Collection of Frog Skin SecretionsAdult H. simplex of both sexes (n = 150; weight range 3�5 g)

were collected from vegetation near water bodies in forest ofGuang Xi Province of China. Their skins were subjected to a 6-V

Received: May 1, 2011

ABSTRACT: Tree frogs produce a variety of skin defensive chemicalsagainst many biotic and abiotic risk factors for their everyday survival. Byproteomics or peptidomics and coupling transcriptome analysis withpharmacological testings, 27 peptides or proteins belonging to 9 families,which act mainly as defensive functions, were identified and characterizedfrom skin secretions of the tree frog, Hyla simplex. They are: (1) a novelfamily of peptides with EGF- and VEGF-releasing activities; (2) a novelfamily of analgesic peptides; (3) a family of neurotoxins acting on sodiumchannel; (4) a snake venom-like presynaptically active neurotoxin; (5) asnake venom-like neurotoxin targeting cyclic nucleotide-gated ion chan-nels; (6) a tachykinin-like peptide, which is the first report from tree frogs;(7) two antimicrobial peptides; (8) a alpha-1-antitrypsin-like serpin; and(9) a wasp venom-like toxin with serine protease inhibitors activity.Families of 1, 2, 4, 5, and 8 proteins or peptides are first reported in amphibians. The chemical array in the tree frog skin sharessome similarities with snake venoms. Most of these components in this tree frog help defend against predators, heal wounds, orattenuate suffering.

KEYWORDS: amphibian, Hyla, skin, arboreal, defense

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electronic stimulation for 3�5 s to collect skin secretionsaccording to our previous methods.6 Skin secretions werecollected by washing the dorsal region of each frog with 0.1 MNaCl solution (containing protease inhibitor cocktail, Sigma).The collected solutions (1500 mL of total volume) were quicklycentrifuged, and the supernatants were lyophilized.

Peptide/Protein PurificationThe lyophilized skin secretion sample of H. simplex (3 g, total

absorbance at 280 nm is 1000) was dissolved in 10 mL of 0.1 Mphosphate buffer solution, pH 6.0 (PBS) and then was applied to

a Sephadex G-50 (Superfine, Amersham Biosciences, 2.6 �100 cm) gel filtration column equilibrated with 0.1 M PBS. Elutionwas performed with the same buffer, collecting fractions of 3.0 mL.The absorbance of the eluatewasmonitored at 280 nm(Figure 1A).Every fraction was subjected to pharmacological tests includingmicrobe-killing, inhibition of serine protease inhibition, analgesia,and effects on isolated guinea pig ileum contraction, humanumbilical vein endothelial cells (HUVECs) growth, and tetrodotox-in sensitive (TTX-S) sodium current as indicated in experimentalprotocols. The protein peaks containing tested pharmacologicalactivities were pooled and purified further as illustrated in Figure 1.

Figure 1. Purification of bioactive proteins or peptides from skin secretions of H. simplex. (A) Lyophilized skin secretion sample of H. simplex wassubjected to Sephadex G-50 gel filtration; the elution was performed by 0.1MPBSwith a flow rate of 0.3mL/min. (B) Fraction 1 from (A) was subjectedto AKTA Mono S cationic exchange column equilibrated with 0.05 M PBS. The elution was performed at a flow rate of 1 mL/min with the indicatedNaCl gradient. (C) Fraction 2 from (A) was subjected to FPLCMono S cationic exchange equilibrated with 0.05 M PBS. (D) Fraction 3 from (A) waspurified further by RP-HPLC (Hypersil BDS C18, 25� 0.46 cm) column. The elution was performed at a flow rate of 0.7 mL/min with the indicatedgradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA) in water. (E�G) Eluted peaks indicated as “3.1”, “1.1”, and “2.1” in D, B, and C,respectively, were subjected to further purification by (E) C18, (F) C4, and (G) C8 RP-HPLC, respectively. The elution was performed at a flow rate of0.7 mL/min with the indicated gradients of acetonitrile in 0.1% (v/v) TFA in water. (Inset in B, F, and G) SDS-PAGE analysis of peak S1, PT, and AT asindicated in (B), (F), and (G) in 15% gel concentration. R, reduced; NR, Nonreduced.

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Structural AnalysisPartial or complete amino acid sequences were determined by

the automated Edman degradation on an Applied Biosystemspulsed liquid-phase sequencer, model 491. Mass fingerprints(MFPs) were obtained using a Voyager-DETM (Voyager DEPro, Applied Biosystems) or AXIMA CFR (Kratos Analytical)Matrix-Assisted Laser Desorption Ionization Time-Of-Flightmass spectrometry (MALDI-TOF-MS) in positive ion and linearmode.16,17 The specific parameters were as follows: the ionacceleration voltage was 20 kV, the accumulating times of singlescanning were 50, and the polypeptide mass standard (AppliedBiosystems or Kratos Analytical) served as the external standard.The accuracy of mass determinations was within 0.1%.

SMART cDNA Synthesis and cDNA CloningTotal RNA was extracted using TRIzol (Life Technologies,

Ltd.) from the skin of a single sample of amphibian. cDNA wassynthesized by using a SMART PCR cDNA synthesis kit(Clontech, Palo Alto, CA) according to the manufacture’sinstruction. Two primers provided by the manufacture (30-SMART CDS Primer IIA, 50-AAGCAGTGGTATCAACGCA-GAGTACT(30)N-1N-30 (N = A, C, G or T; N-1 = A, G or C),and SMART II oligonucleotide, 50-AAGCAGTGGTAT-CAACGCAGAGTACGCGGG-30) were used for the first strandsynthesis. The second strand was amplified using Advantagepolymerase by primer IIA (50-AAGCAGTGGTATCAACGCA-GAGT-30) provided by the manufacture. A directional cDNAlibrary was constructed with a plasmid cloning kit (SuperScriptPlasmid System, GIBCO/BRL) following the instructions ofmanufacturer, producing a library of about 3.5 � 105 indepen-dent colonies.

A PCR-based method for high stringency screening of DNAlibraries was used for screening and isolating the clones. Theoligonucleotide primers (as listed in the Table S1, SupportingInformation) in the antisense direction were designed accordingto the amino acid sequences determined by Edman degradation.Primer II A was used as the sense direction primer. The DNApolymerase was Advantage polymerase from Clontech (PaloAlto, CA). The template was the cDNA from the library. DNAsequencing was performed on an Applied Biosystems DNAsequencer, model ABI PRISM 377.

PLA2 Activity AssayPLA2 activity was measured by a kinetic method according to

the method described by Lobo de Ara�ujo and Radvanyi.18 Thesubstrate was soybean phosphatidylcholine (0.265%) in thesolution of 10 mM CaCl2, 100 mM NaCl, 100 mM PhenolRed, and 7 mM Triton X-100. PLA2 activity (based on the pHchange due to the liberation of fatty acids) was measured aschanges of absorbance at 558 nm per minute, using a UV/visiblespectrophotometer 2100 pro (Amersham Biosciences).

Acute ToxicityThe acute toxicities of peptides or proteins from the frog skin

secretions against several potential aggressors or predatorsincluding insect, snake, bird, and mouse were tested accordingto our previous methods.6 For all of the tested animals, the samevolume of buffers was used as blank control. Bovine serumalbumin (BSA) with the same volume and concentration wasadministrated as a protein control.

Antimicrobial AssayAntimicrobial activities against Gram-positive bacteria Staphy-

lococcus aureus (ATCC 2592), Bacillus subtilis (ATCC 6633) and

B. cereus, Gram-negative bacteria Escherichia coli (ATCC 25922),B. dysenteriae and Psecdomonas aeruginosa, and fungus Candidaalbicans (ATCC 2002) were assayed according to our previousmethods.9

Trypsin-Inhibitory TestingThe inhibition effects of samples on the hydrolysis of synthetic

chromogenic substrates (S-2238, H-D-Phe-Pip-Arg-pNA, KabiVitrum, Stockholm, Sweden) by trypsin were assayed accordingto methods previously described.6

Effects of Tree Frog Toxins on Voltage-Gated ChannelsRat dorsal root ganglion (DRG) neurons were acutely dis-

sociated and kept in a short-term primary culture according toour previous methods.6 Sodium currents from the neurons wererecorded using the whole-cell patch clamp technique by anAoxn 700B patch clamp amplifier (Axon, American). The P/4protocol was used to subtract linear capacitive and leakagecurrents. Experimental data were obtained and analyzed by usingthe program Clampfit10.0 (Axon, American) and Sigmaplot(Sigma).

Effects on Isolated Guinea Pig Ileum ContractionThe effects of the tree frog skin compounds on isolated guinea

pig ileum contraction were tested according to the methodspreviously described.12

HUVEC Proliferation Assay and the Signaling PathwayDetection by ELISA and Western Blot Analysis

The effects of tested samples on endothelial cell proliferationwere assayed by HUVEC obtained from the Cell Bank ofKunming Institute of Zoology (Kunming, China). ELISA andWestern blot analysis were used to detect the signaling pathwaysinvolving in the HUVEC proliferation.

Radiant Heat Tail-Flick TestAntinociception was assessed using the radiant heat tail-flick

test apparatus (model YLS-12A, ZhengHua, China). UsingKunming male mice (20�25 g), tail-flick latency was measuredat 5, 15, 30, and 60 min after sample administration.

The detail methods are described in the Supporting Informa-tion. All of the animal studies were reviewed and approved by theanimal care and use committee of Kunming Institute of Zoology,Chinese Academy of Sciences.

Synthetic PeptidesAll of the peptides used in this work were synthesized by GL

Biochem (Shanghai) Ltd. (Shanghai, China) and analyzed byHPLC and mass spectrometry to confirmed purity greater than98%. All peptides were dissolved in water.

Statistical AnalysisAll the data were presented as means( SEM and analyzed by

one-way or two-way analysis of variance (ANOVA) followed byDuncan’s test or unpaired t test when appropriate. A value of p <0.05 was taken as the level of significance.

’RESULTS AND DISCUSSION

Purification of Bioactive Proteins or Peptides from the FrogSkin Secretions

The supernatant ofH. simplex skin secretions was divided intothree fractions after Sephadex G-50 gel filtration, as illustrated inFigure 1A. Fractions 1 and 2 showed abilities to inhibit serineproteases and phospholipase A2 (PLA2) activity. Fraction 2 couldblock TTX-S (tetrodotoxin-sensitive) sodium current. Peak 3

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had activities to kill microbes, induce contraction of isolatedguinea pig ileum and HUVEC growth. In addition, the analgesicactivity was also concentrated in peak 3.

Fraction 1 from Sephadex G-50 gel filtration was subjected toAKTA Mono S (1 mL volume, Amersham Biosciences) cationicexchange as illustrated in Figure 1B. The eluted peak at 59 minindicated as “S” in Figure 1B could inhibit trypsin and chymo-trypsin. SDS-PAGE analysis showed that this peak is a homo-geneous protein with a molecular weight (MW) of 44�45 kDa(Inset in Figure 1B). The eluted peak (1.1) containing lethaltoxicity against mice in Figure 1B was purified further by C4 RP-HPLC (Figure 1F). The eluted peak at 53 min indicated as “PT”in Figure 1F was a homogeneous toxin protein with a MW25 kDa determined by SDS-PAGE (Inset in Figure 1F).

Fraction 2 in Figure 1A was subjected to AKTA Mono Scationic exchange as illustrated in Figure 1C. The eluted peak at47 and 60 min indicated as “T1” and “T2”, respectively, inFigure 1C had the ability to inhibit trypsin and block TTX-Ssodium current. The eluted peak indicated “S2” in Figure 1Ccould only inhibit trypsin. Peaks S2, T1, and T2 were proven tobe homogeneous by MALDI-TOF MS analysis (Figure S1,Supporting Information). The eluted peak indicated “2.1”

containing PLA2 activity was purified further by C8 RP-HPLC(Figure 1G). The eluted peak at 53 min indicated as “AT” inFigure 1F was a homogeneous PLA2 protein with a MW 14 kDadetermined by SDS-PAGE (Inset in Figure 1G).

Fraction 3 from Figure 1A was subjected to C18 RP-HPLC asillustrated in Figure 1D. Several eluted peaks indicated as G1, F1,G2, Ta, G3, and A1 were proven to be homogeneous byMALDI-TOF MS analysis (Figure S1, Supporting Information). Theeluted peak indicated by an arrow in Figure 1D was subjected tofurther purification by C18 RP-HPLC as illustrated in Figure 1E.Four eluted peaks (G4, A1, G5, G5) containing single observedmolecular mass were collected from this RP-HPLC purification(Figure S1, Supporting Information). In total, 16 pure proteinsor peptides were purified from skin secretions of the frog.

Purification, cDNA Cloning, Structure, and Function ofSerine Protease Inhibitor

As illustrated in Figure 1B and C, two serine proteaseinhibitors named hylaserpin S1 and S2 (Indicated as S1 andS2) were purified from the frog skin secretions, respectively.SDS-PAGE and mass spectrometry analysis indicated thatthey had molecular weight of 44 kDa (Figure 1B) and 6.1 kDa

Figure 2. Amino acid sequences of two purified serine proteinase inhibitors and bacteriostatic activity of hylaserpin S2. (A) Amino acid sequence ofhylaserpin S1. Amino acid sequences determined by Edman degradation are underlined. (B) Amino acid sequence of hylaserpin S2 and the sequencecomparison with other serine proteinase inhibitors. Sequence of trypsin inhibitor wasp venom peptide, ASPI2, Rana trypsin inhibitor, Bombina trypsininhibitor is from reference,17�20 respectively. Gaps (�) have been introduced to optimize the sequence homology. Stars (*) indicate identical amino acidresidues. (C) Bacteriostatic activity of hylaserpin S2 with different concentration (2.5, 5, and 10 μM). The experiment was performed in triplicate on B.subtilis (ATCC 6633) by measuring the absorbance of the bacterial suspension at 600 nm at different intervals (0�7 h). Bacterial growth in the absenceof hylaserpin S2 (0 μM) was served as positive control.

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(Figure S1A, Supporting Information), respectively. For hyla-serpin S1, its amino-terminal and partial interior amino acidfragments recovered from trypsin hydrolysis were subjected toEdman degradation sequencing. Complete amino sequence ofhylaserpin S2 was determined by Edman degradation. On thebasis of the amino acid sequences of theN-terminus, degeneratedprimers were designed to screen the cDNA sequences. The fulllength the cDNA sequence (GenBank Accession HM747289)encoding hylaserpin S1 precursors were cloned from the skincDNA library ofH. simplex. Hylaserpin S1 precursor is composedof 415 amino acid residues (aa) including a signal peptide of 23 aaand mature hylaserpin S1 of 392 aa. Hylaserpin S1 showssimilarity with the alpha-1-antitrypsin-like serpin superfamily. Itis the first report of purification and characterization of serpinprotein from amphibian skin.

The cDNA encoding hylaserpin S2 precursor was also clonedfrom the frog skin library (Figure 2B) (GenBank AccessionHM747290). Hylaserpin S2 precursor is composed of 83 aaincluding a predicted signal peptide (23 aa) and a maturehylaserpin S2 (60 aa). The mass spectrometry analysis gave anobserved mass of 6132.3 that matched well with the theoreticalmolecular weight of 6133.02 (Figure S1A, Supporting In-formation). Hylaserpin S2 showed similarity with wasp venompeptide (Cysteine-rich venom protein 6),19 amphibian serineprotease inhibitors containing 10 half-cysteines from genus ofRana20 and Bombina,21 and ASPI-2 from the nematode, Anisakissimplex.22 They had identical half-cysteine motifs as illustrated inFigure 2B.

Hylaserpin S1 and S2 inhibited the hydrolytic activitiesof trypsin and/or chymotrypsin on chromogenic substrates.Hylaserpin S1 had an inhibitory constant (Ki) of 5.5 � 10

�8

Mand 3.1 � 10�7 M for trypsin and chymotrypsin, respectively.Hylaserpin S2 only inhibited trypsin with aKi of 7.2� 10

�8

M. Inaddition, hylaserpin S2 exhibited bacteriostatic effect againstGram-positive bacteria B. subtilis (ATCC 6633). Hylaserpin S1had direct microorganism-killing abilities. It had a minimalinhibitory concentration of 15, 35, and 3.5 μg/mL againstB. subtilis (ATCC 6633), Escherichia coli (ATCC 25922), andCandida albicans (ATCC 2002), respectively.

Many pathogens are found to secret extracellular proteinases,and recent findings suggest that proteinases play an importantrole in the development of diseases.23 Much evidence suggeststhat a major function of proteinase inhibitors in hosts is tocombat the proteinases of pests and pathogens.23 Combinedwith their antimicrobial activities, the skin protease inhibitors ofthe frog likely play important roles in resistance to invasion ofpathogens and pests.

Purification, cDNA Cloning, Structure, and Function ofAnalgesic Peptides

Two novel peptides named analgesin a1 and a2 were purifiedfrom the skin secretions as illustrated in Figure 1D and 1E(Indicated as A1 and A2). By Edman degradation, their aminoacid sequences were determined as FLPFL (analgesin a1) andFLPIL (analgesin a2), respectively. Treating with carboxypepti-dase Y did not lead to the release of free amino acids underconditions that free amino acids were released from a peptidewith free C-terminal �COOH group. This result indicates thatC-terminal ends of analgesin a1 and a2 are amidated, which isfurther confirmed by mass spectrometry analysis. MALDI-TOF-MS analysis gave observed molecular masses of 635.4 and 600.5(Figure S1B and S1C, Supporting Information), respectively,

which matched well with the calculated molecular masses (635.8and 600.8). Therefore, the amino acid sequence of analgesin a1and a2 is FLPFL-NH2 and FLPIL-NH2, respectively.

By random screening, two cDNA clones (T6 and T28,GenBank Accession HM747287 and HM747288) encodinganalgesin a1 and a2 precursors were obtained as illustrated inFigure 3A. Clone T6 encodes a precursor with 147 aa including 5copies of analgesin a1, 2 copies of analgesin a2, and one copy ofanalgesin homologue (FWPFL). Clone T6 encodes a precursorwith 186 aa including 12 copies of analgesin a1. Bibasic enzymaticprocessing sites (-KR- and �RR-) are positioned at N- and C-terminus of these mature analgesins, respectively.

Radiant heat Tail-Flick tests indicated that both analgesin a1and a2 have antinociceptive ability as illustrated in Figure 3B.After 5min intraperitoneal administration, the tail-flick latency ofanalgesin a1 and a2was 4.3 and 6.9 s, respectively, while their baselinelatency was 3.9 s. After 15 min intraperitoneal administration, they

Figure 3. Amino acid sequence and function of analgesic peptides fromskin secretions of H. simplex. (A) Precursors of analgesins. Matureanalgesins are boxed. (B) Antinociceptive assay of analgesins by radiantheat Tail-Flick tests. Mice were randomly assigned to each group (eitherexperimental or control, n = 9). Sample dissolved in saline wasintraperitoneally administered at a dose of 2.5 mg/kg body weight.Tail-flick latency was measured at 5, 15, 30, and 60 min after sampleadministration. Control groups received an injection of the same volumeof saline. (C) Inhibition of analgesin a1 on isolated guinea pig ileumcontraction induced by bradykinin.

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showed the strongest antinociceptive ability, and the tail-flicklatencywas 6.7 and 7.5 s, respectively. Their antinociceptive effectswere lasted for at least 30 min (Figure 3B). In order to investigatethe possible antinociceptive mechanism, the effect of analgesin a1on isolated guinea pig ileum contraction induced by bradykininwas tested. As illustrated in Figure 3C, analgesin a1 significantlyinhibited ileum contraction induced by bradykinin. Bradykinin is akind of nociception inducer. This result suggests that analgesina1 acts on signaling pathway involved in bradykinin to exert

antinociception. Further work is necessary to identify the actualmechanism.

Tree frogs may encounter more injuries such as predatorattacks, insect biting or stinging, and pathogen invasion thanother amphibians because of their arboreal life. Nociceptivestresses from injuries will normally lead to an inflammatoryresponse with a great deal of leukocyte activation.24 Antinoci-ceptive peptides (analgesins) are able to block pain and inflam-mation for self-protection, and lighten suffering.

Figure 4. Amino acid sequence comparison of (A) tachykinin-H precursor with tachykinin-O precursor and (B) the contraction of isolated guinea pigileum induced by tachykinin-H. The sequence of tachykinin-O is from ref 12. Mature tachykinins are boxed. Gaps (�) have been introduced to optimizethe sequence homology. Stars (*) indicate identical amino acid residues.

Figure 5. (A) Diversity of neurotoxins acting on sodium channels from the frog skin of H. simplex and (B) effects of anntoxin-S1 and�S2 on sodiumcurrent. The predicted signal peptides are in italic. Gaps (�) have been introduced to optimize the sequence homology. Stars (*) indicate identical aminoacid residues.

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Purification, cDNA Cloning, Structure, and Function ofTachykinin-Like Peptide

A peptide exerting contractile activity on isolated guinea pigileum was purified from the skin secretions of the frog asillustrated in Figure 1D (indicated as Ta). The purified peptidewas subjected to the amino acid sequence analysis by automatedEdman degradation. The amino acid sequence is PRPDQ-FYGLM-NH2 (named tachykinin-H1). MALDI-TOF-MS anal-ysis gave an observed molecular mass of 1222.7 (Figure S1D,Supporting Information), which matched well with the calcu-lated molecular mass (1223.4). It shares the similar C-terminalsequence of F(Y/I)GLM-NH2 consensus motif as found in othertachykinin-like peptides, but with variable N-terminus. Com-bined with mass spectrometry analysis, it was speculated that theC-terminal end of this peptide was amidated. Tachykinin-H1 isthe first report on tachykinin-like peptide from tree frogs. ThecDNA encoding tachykinin-H1 precursor was cloned from thecDNA library of the frog (GenBank AccessionHM747308). Thisprecursor is composed of 91 aa containing a copy of maturetachykinin-H1.

Tachykinin-H1 induced contraction of isolated guinea pigileum in a dose-dependent manner as illustrated in Figure 4B.The tachykinin peptide family is one of the most prevalentpeptide families described in animals. They perform multiplephysiological functions including smooth muscle contraction,

vasodilation, inflammation, nerve signal processing, neuropro-tection, and neurodegeneration.25 Tachykinins are involved inpain transmission at the spinal level.25 The skin tachykinin maymake frogs avoid injury.

Purification, cDNA Cloning, Structure, and Function ofNeurotoxins Acting on Sodium Channels

Two peptides (anntoxin-S1 and �S2) with observed mass of6528.9 and 6431.5 were purified from the skin secretions,respectively (Indicated as T1 and T2 in Figure 1C and FigureS1, Supporting Information). Their amino acid sequences aredetermined as ASDYRCNLSRSYGKGSGSFTNYYYDKATN-SCKTFTYRGTGGNGNRFKTLEECETTCVTG and AAADH-RCGLIRNLGKGSGSFTNYYYDKATNSCKTFTYRGTG GNGN-RFKTLEECGTTCVTG, respectively. The cDNAs encoding pre-cursors of anntoxin-S1 and -S2 were cloned from the skin cDNAlibrary of the frog. The other four homologues of anntoxin-S1 and -S2 were also identified by cDNA screening as indicated in Figure 5(GenBank Accession HM747291�6). All the six precursors werecomposed of 77�80 aa. They showed sequence similarity with thefirst amphibian gene-encoded neurotoxin (anntoxin) identifiedfromH. annectans, which contained four half-cysteines to form twointramolecular disulfide bridges.6 Five of them contain the samehalf-cysteinemotifs with anntoxin.One of them (450�8) containsonly one-half-cysteine (Figure 5A).

Figure 6. Diversity of EGF or VEGF-releasing peptides from the skin ofH. simplex and their effects on EGF or VEGF releasing. (A) Diversity of EGF orVEGF-releasing peptides. Mature peptides are boxed. Gaps (�) have been introduced to optimize the sequence homology. Stars (*) indicate identicalamino acid residues. (B) Effects of different concentration of hylareleasin 3 and 4 on HUVEC proliferation. (C) Effects of different concentration ofhylareleasin 3 and 4 on EGF releasing. (D) Effects of different concentration of hylareleasin 3 and 4 on VEGF releasing. These results represent meanvalues of five independent experiments performed in duplicates. The values for groups treated by samples were significant different from the values forcontrols (*p < 0.05 and **p < 0.01).

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Considering that anntoxin acts as a tetrodotoxin-sensitive(TTX-S) voltage-gated sodium channel (VGSC) blocker,6 the

effects of anntoxin-S1 and -S2 on TTX-S VGSC were assayed.They inhibited TTX-S sodium current in a dose-dependentmanner as anntoxin did (Figure 5B). Anntoxin showed lethaltoxicities against several kinds of tested animals like insect,reptile, bird, and mammalian.6 Administered by i.p., the toxicdoses of anntoxin-S1 (LD50) against L. exigua Hubner, E.plumbea, C. coturnix, and Kunming mice are 0.1, 0.63, 4.7, and10 mg/kg body weight, respectively.

Anntoxin-S1/-S2 is the second report of gene-encoded neu-rotoxins from amphibians after the first gene-encoded neurotox-in from H. annectans. Anntoxin has been proven to be animportant component of skin chemical defense system of thetree frog, H. annectans. Its strong and fast-acting toxicity againstpotential predators and skin distribution with high concentrationmake it an excellent chemical component.6 The current workproved that another tree frog skin also had the same family ofgene-encoded neurotoxins. These results suggest that this familyof neurotoxins act as the first-line array of defense.

cDNA Cloning, Structure, and Function of EGF or VEGF-Releasing Peptides

Six peptides with molecular weights ranging from 1500 to1800Da (Figure S1G�L, Supporting Information) were purifiedfrom the frog skin secretions (Indicated as G1-G6 in Figure 1D

Figure 7. Effects of hylareleasin 3 and 4 on protein kinases (ERK, JNK, and AKT) phosopholyation. (A)Western blot shows that effects of hylareleasin3 (H3) and 4 (H4) (1, 5, 25, 125 μg/mL) on protein kinases phosopholyation. Con: control. (B and C) Relative activation of protein kinases byhylareleasin 3 and 4 in a dose-dependent manner, respectively. (D)Western blot shows the time-course of the phosphorylation induced by hylareleasin3. Con: control. (E) Relative activation of protein kinases by hylareleasin 3 in time-dependent manner.

Table 1. Antimicrobial Activity of Hylains

MICa (μg/mL)

microoganism Hylain 1 Hylain 2

Gram-positive bacteria

S. aureus 5.50 5.50

B. subtilis 5.50 11.0

B. cereus 11.0 22.0

Gram-negative bacteria

E. coli 44.0 22.0

B. dysenteriae 2.75 5.50

P. aeruginosa 5.50 11.0

Fungus

C. albicans 66.0 44.0aMIC, minimal peptide concentration required for total inhibition ofcell growth in liquid medium. These concentrations represent meanvalues of three independent experiments performed in duplicates.

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and Figure 1E). They are named hylareleasin 1�3 and 9�11,respectively. Their amino acid sequences were determined byEdman degradation as illustrated in Figure 6A. According to theiramino acid sequences, the cDNA sequences encoding theirprecursors were cloned from the frog skin library (Figure 6A).Other five precursors encoding hylareleasins were also obtainedby the cDNA screening. Total 11 hylareleasins were identifiedfrom this frog (GenBank Accession HM747297�307). Most ofthem are composed of 16 aa. Only the predicted hylareleasin 8 iscomposed of 18 aa. They share the same N-terminal amino acidsequence (GLLD/NP). By BLAST search, these peptides do notshow any sequence similarity with known sequences, indicatingthat they are a novel family of peptides.

Synthesized hylareleasin 3 and 4 were used to screen possiblebioactive functions including microorganism-killing, proteaseinhibition, smooth muscle contraction, and induction of HU-VEC growth. They had no ability to kill micoorganisms, inhibitproteases and affect smooth muscle contraction but they in-creased HUVEC growth in a dose-dependent manner as illu-strated in Figure 6B. At the concentration of 125 μg/mL,hylareleasin 3 and 4 induced proliferation rate by 60% and140%, respectively. They also were found to significantly induceEGF and VEGF secretion (Figure 6C and D). Especially, at theconcentration of 125 μg/mL, the concentration of EGF inducedby hylareleasin 3 and 4 is 7 and 15 times of baseline concentration(control), respectively. To investigate the possible signalingpathway, protein kinases (ERK, JNK, and AKT) phosopholya-tion induced by hylareleasin 3 and 4 was tested. As illustrated inFigure 7A�C, they significantly increased protein kinases phos-phorylation. For some protein kinases, such as JNK and AKT,their effects on phosphorylation appeared not to be a dose-dependent manner. The time-course of the phosphorylationinduced by hylareleasin 3 was also assayed (Figure 7D and E).After 15 min of treatment by hylareleasin 3, the phosophoryla-tion of ERK, JNK, and AKT was significantly increased. After 4 hof treatment, the phosphorylation level was back to control.

Skins are the first line of amphibians to defend noxiousenvironmental factors. Tree frogs spend much time living ontrees, so that their fragile skins may encounter more biotic andabiotic injuries than other amphibians. Therefore, there is more

possibility to form wounds in their skins. More than 11 peptidescontaining functions to induce EGF and VEGF releasing andendothelial cell growth have been found in the skin of the treefrog, H. simplex. They possibly take part in the skin woundhealing. The rich diversity of this family of peptides in the skinsuggests that the corresponding genes form a multigene familyoriginating from a common ancestor.

Structure and function of antimicrobial peptidesTwo peptides named hylain 1 and 2 with antimicrobial activities

were purified from the skin secretions. Mass spectrometry gave anobservedmass of 1372.3 and 1370.6, respectively (Figure S1MandN, Supporting Information). Their amino acid sequences weredetermined as GILDAIKAFANALG-NH2 (Hylain 1) and GILD-PIKAFAKAAG-NH2 (Hylain 2), respectively, by Edman degrada-tion. According to their amino acid sequences, they should havetheoretical molecular weights of 1372.6 and 1370.6, respectively,which matched well with the observed masses (1372.3 and1370.6). They showed strong antimicrobial activities againstGram-positive and negative bacteria and fungi as listed in Table 1.They showed the strongest antimicrobial abilities against S. aureusand B. dysenteriae. The MICs are less than 5.5 μg/mL.

Antimicrobial peptides are important components of innatedefense system. They form the first line of host defense againstnoxiousmicroorganisms. Amphibian skins synthesize and secrete aremarkably diverse array of antimicrobial peptides. Most ofamphibian antimicrobial peptides are less than 50 residues inlength.3 They are released onto the outer layer of the skin toprovide an effective and fast acting defense against harmfulmicroorganisms. Similar to other amphibians, tree frog skins havealso antimicrobial peptides to defense microorganism infection.

Purification, cDNA Cloning, Structure, and Function of SnakeVenom-like Neurotoxin Targeting Cyclic Nucleotide-GatedIon Channels

As illustrated in Figure 1F, a protein named pseudechetoxin-HS with a MW of 25 kDa was purified. Its amino acid sequencewas determined by Edman degradation and cDNA cloning.Mature pseudechetoxin-HS contains 217 aa, which shares sig-nificant similarity with the cyclic nucleotide-gated ion channelblocker, pseudechetoxin, identified from snake venoms of the

Figure 8. Amino acid sequences of pseudechetoxin-HS and ammodytoxin-HS. (A) Amino acid sequence of pseudechetoxin-HS and its comparisonwith snake venom pseudechetoxin (24). (B) Amino acid sequence of ammodytoxin-HS and its comparison with snake venom ammodytoxin (26, 27).Amino acid sequences determined by Edman degradation are underlined. The predicted signal peptides are in italic. Gaps (�) have been introduced tooptimize the sequence homology. Stars (*) indicate identical amino acid residues.

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coastal taipan (Oxyuranus scutellatus).26 Previous works haveindicated that snake pseudechetoxin has a strong ability to blockthe cGMP-dependent current. Block was independent of voltageand required only a single molecule of toxin.27 Pseudechetoxin-HS from the tree frog skin shares 97 (46%) identical aa residueswith the snake pseudechetoxin. Especially, they have identicalcysteine motifs (Figure 8A). Pseudechetoxin-HS’s effect oncGMP-dependent current is not tested in this study, but itslethal toxicity on mice was tested. Administered by i.p., the LD50

against Kunming mice is 1.5 mg/kg body weight. The skinpseudechetoxin-HS in the tree frog probably acts as a defensivemolecule against predators.

Purification, cDNA Cloning, Structure, and Function ofSnake Venom-like Presynaptically Neurotoxin

A PLA2 neurotxoin named ammodytoxin-HS was purifiedfrom the tree frog skin secretions as illustrated in Figure 1G(indicated as AT). SDS-PAGE analysis indicated that it had aMW of 14 kDa (Inset in Figure 1G). Its amino-terminal andpartial interior amino acid fragments recovered from trypsinhydrolysis were subjected to Edman degradation sequencing.Using specific primers designed from amino acid sequencesdetermined by Edman degradation, the full length cDNA en-coding ammodytoxin-HS was cloned (Figure 8B). Mature ammo-dytoxin-HS contains 148 aa, which shares significant similaritywith the presynaptically neurotoxin, ammodytoxin B identifiedfrom snake venoms of Vipera ammodytes ammodytes.28,29 Previousworks have indicated that the snake ammodytoxin B has the abilityto block acetylcholine release in neuromuscular junction and is aneuro-toxic PLA2.

28�30 Ammodytoxin-HS’s PLA2 activity wastested. The isolated ammodytoxin-HS presented specific PLA2activity (3.2 UA/mg/min). Ammodytoxin-HS also showed lethaltoxicity against mice as pseudechetoxin-HS did. Administered by i.p., the LD50 against Kunming mice is 2.5 mg/kg body weight.Three neurotxins including anntoxins, ammodytoxin-HS, andpseudechetoxin-HS has been found in the tree frog skins. Inter-estingly, all of these neurotoxins are snake venom-like toxins,indicating that some tree frog skin secretions contain multipletoxic components as snake venoms do.

’CONCLUSION

Amphibians can adapt to a wide range of habitats andecological conditions because of their versatile skins. Their skinsare morphologically, biochemically, and physiologically complexorgans, which make amphibians adapt to variable habitats,ranging from arid deserts to deep freshwater lakes, or from a lifeunderground to one spent entirely in the tropical forest canopy.1

Tree frogs are a group of special amphibian species because oftheir unique arboreal environments. Especially, most Hyla frogshave a preference to stay on the top of leaves. In this kind ofenvironment, they are easily attacked by their predators andinjured. In addition, their skin colors can mimic the forestenvironment. Nine families of bioactive peptides or proteinsincluding analgesic peptides, neurotoxins, EGF- or VEGF-releas-ing peptides, tachykinin-like peptide, antimicrobial peptides, andprotease inhibitors are found in the skin secretions of the treefrog, H. simplex. The EGF- or VEGF-releasing peptides, theanalgesic peptides, the snake venom-like presynaptically neuro-toxin, the snake venom-like neurotoxin targeting cyclic nucleo-tide-gated ion channels, and alpha-1-antitrypsin-like serpin arereported for the first time in amphibians.Most of these peptide orprotein families have functions to defend against predators, heal

wounds, or attenuate suffering. In addition, the discovery ofmultiple snake venom-like toxins in the tree frog may shed lighton toxin evolution from amphibian to reptile.

’ASSOCIATED CONTENT

bS Supporting InformationDetailed materials and methods. Figure S1 and Table S1. This

material is available free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Dr. Ren Lai. Tel: +86-871-5196202. Fax: +86-871-5199086.E-mail: rlai@mail.kiz.ac.cn.

Author Contributions§These authors contributed equally to this paper

’ACKNOWLEDGMENT

This work was supported by Chinese National NaturalScience Foundation (30830021, 30800185, 31025025), theMinistry of Science and Technology (2010CB529800,2009ZX09103-1/091), and the Ministry of Agriculture(2009ZX08009-159B).

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