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A Novel Antimicrobial Peptide from Skin Secretions of the Tree FrogTheloderma kwangsiensisAuthor(s): Hongli Yan , Yingying Liu , Jing Tang , Guoxiang Mo , Yuzhu Song , Xiuwen Yan , LinWei , and Ren LaiSource: Zoological Science, 30(9):704-709. 2013.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.30.704URL: http://www.bioone.org/doi/full/10.2108/zsj.30.704

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2013 Zoological Society of JapanZOOLOGICAL SCIENCE 30: 704–709 (2013)

A Novel Antimicrobial Peptide from Skin Secretions

of the Tree Frog Theloderma kwangsiensis

Hongli Yan1†, Yingying Liu3†, Jing Tang1, Guoxiang Mo1, Yuzhu Song2,

Xiuwen Yan1, Lin Wei1*, and Ren Lai1

1Life Sciences College of Nanjing Agricultural University, Nanjing, Jiangsu 210095, China2Life Science and Technology College, Kunming University of Science and Technology,

Kunming, Yunnan 650093, China3Department of Endocrinology, The No.3 People Hospital of Yunnan Province,

Kunming 650011, China

Most of amphibians belonging to family Rhacophoridae live in arboreal habitats. A large number

of antimicrobial peptides (AMPs) have been identified from amphibian skins. No antimicrobial pep-

tide from Rhacophoridae amphibians has been reported. In this study, we purified and character-

ized a novel antimicrobial peptide, pleurain-a1-thel from skin secretions of the tree frog, Theloderma

kwangsiensis. Its amino acid sequence was determined as RILTMTKRVKMPQLYKQIVCRLFKTC by

Edman degradation, mass spectrometry analysis and cDNA cloning. There are two cysteines, which

form an intra-molecular disulfide bridge, in the sequence of pleurain-a1-thel. Pleurain-a1-thel

exerted potential antimicrobial activities against wide spectrum of microorganisms, including

Gram-negative and -positive bacteria and fungi. It exerted little hemolytic activity in human or rabbit

red cells. To the best of our knowledge, this is the first report of antimicrobial peptide from

Rhacophoridae amphibians.

Key words: Amphibia, antimicrobial peptide, tree frog, skin secretion, Theloderma kwangsiensis

INTRODUCTION

There are many pharmacological compounds in

amphibian skins, some of which have defensive functions.

Amphibian skins lack protective covering, and are this fragile

and easily injured by predators, microorganisms, and para-

sites. Amphibians have adapted by developing effective

chemical defensive systems in their skins. Antimicrobial

peptides are the main components of the chemical defen-

sive system in amphibian skins. Hundreds of antimicrobial

peptides of different lengths, net charges, and isoelectric

points (pI) have been purified and characterized from skin

granular glands of anuran amphibians, particularly those

belonging to the families of Pipidae, Hylidae, Hyperoliidae,

Pseudidae, and Ranidae (Barra and Simmaco, 1995; Bevins

and Zasloff, 1990; Conlon et al., 2004; Chen et al., 2006;

McGillivary et al., 2007; Nicolas and Mor, 1995; Simmaco et

al., 1999; Lu et al., 2006, 2008; Li et al., 2007; Lai et al.,

2002; Xu et al., 2006; Zasloff, 1987, 1992; Zhou et al., 2007;

Zheng et al., 2010). Most amphibian antimicrobial peptides

are 10–50 residues in length, contain no or two cysteines (Li

et al., 2007), and are positively charged. Amphibian

antimicrobial peptides have shown anti-bacterial, anti-fungal,

anti-viral, anti-parasite, and anti-tumor activities. They are

attractive targets for research and development of anti-infec-

tive agents (Conlon et al., 2004; Simmaco et al., 1999).

Theloderma is a genus of frogs belonging to the family

Rhacophoridae, which mainly occurs in tropical regions of

Asia. Most Theloderma species are arboreal, some of which

reproduce in trees (Frost and Darrel, 2011). Although many

antimicrobial peptides have been identified from other

amphibians, no antimicrobial peptide from Rhacophoridae

has been reported. Only a protein with antimicrobial activity

in the skin of Schlegel’s green tree frog Rhacophorus

schlegelii (Rhacophoridae) was identified as histone H2B

(Kawasaki et al., 2003). In the present report, we describe a

novel antimicrobial peptide from skin secretions of the tree

frog, T. kwangsiensis.

MATERIALS AND METHODS

Collection of frog skin secretions

Adult specimens of Theloderma kwangsiensis (n = 10; weight

range 15–25 g, sex undetermined) were collected in Guangxi

Province of China. Skin secretions were collected as described in

our previous report (Zhang et al., 2013). Contaminants in frog skin

were removed using water, placed in a cylindrical container and

stimulated by volatilized anhydrous ether immersed in absorbent

cotton. After ~3 min treatment by volatilized anhydrous ether, copi-

ous secretions were found to exude from the frog skin surface. Skin

secretions were collected by washing the frog dorsal region with 0.1

M NaCl containing protease inhibitor cocktail (1%, v/v, Sigma). The

collected skin secretions (about 500 ml) were quickly centrifuged to

remove precipitants. The supernatants was lyophilized and kept at

–20°C. All the experiments were approved by Nanjing Agricultural

University.

* Corresponding author. Tel. : +86-25-84396849;

Fax : +86-25-84396849;

E-mail : weilin2020@126.com† These authors contributed equally to this paper.

doi:10.2108/zsj.30.704

Antimicrobial Peptide from Tree Frog 705

Peptide purification

Dried T. kwangsiensis skin secretions (1.5 g, total absorbance

at 280 nm was 500) was dissolved in 10 ml of 0.1 M phosphate buf-

fer, pH 6.0 (PBS), containing 5 mM EDTA and subjected to filter

through a 10-kDa cut-off Centriprep filter (Millipore, Bedford, CA).

The filtrate was purified using a C8 reversed-phase high perfor-

mance liquid chromatography (RP-HPLC, Hypersil BDS C8, 25 ×0.46 cm) column, as described in our previous report (Zhang et al.,

2013). During the elution, the absorbance of the eluate was moni-

tored at 215 nm. The eluted peaks were subjected to antimicrobial

assay as mentioned below.

Structural analysis

Molecular weight and purity were analyzed using a autoflex™

speed MALDI-TOF (TOF) mass spectrometer (Bruker Daltonik

GmbH, LeiPzig, Germany) following the manufacturer’s instruction.

The complete amino sequence of the purified peptide was deter-

mined by automated Edman degradation on an Applied Biosystems

pulsed liquid-phase sequencer (model ABI 491) following the instru-

ment manual.

Construction and screening of cDNA library

Total RNA from the skin of a single frog was extracted by using

TRIzol reagent (Life Technologies Ltd.) as described in our previous

report (Zhang et al., 2013). mRNAs were prepared from the total

RNA of T. kwangsiensis skin by oligo (dT) cellulose chromatogra-

phy. cDNA was synthesized by using a SMARTTM PCR cDNA

synthesis kit (Clontech, PaloAlto, CA, USA) according to the man-

ufacturer’s instructions. The 3′ SMART CDS Primer II A (5′-AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N-3′, where N = A,

C, G or T and N-1 = A, G or C) and SMART II A oligonucleotide (5′-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3′) were used for

the first strand synthesis. The second strand was synthesized by

using Advantage polymerase (Clonetech, CA, USA) and the 5′ PCR

Primer II A (5′-AAGCAGTGGTATCAACGCAGAGT-3′).A PCR-based method for high stringency screening of DNA

libraries was used for screening and isolating the clones from the

constructed T. kwangsiensis skin cDNA library. Two oligonucleotide

primers, S1 (5′-CG(A/T/C/G)AT(A/T/C)(T/C)T(A/TC/G)AC(A/T/C/

G)ATGAC(A/T/C/G)AA(A/G)-3′ designed according to the sequence

determined by Edman degradation) in the sense direction and

SMART II A oligonucleotide (5′-AAGCAGTGGTATCAACGCAGAG-

TACGCGGG-3′) in the antisense direction were used in PCR reac-

tions. For the DNA polymerase in the PCR reaction, Advantage

polymerase from Clontech (Palo Alto, CA, USA) was used. The

reaction conditions were, 95°C (3 min), and 30 cycles of 95°C (30

s), 56°C (30 s), 72°C (3 min) followed by a 10-min extension period

at 72°C. The PCR products were cloned into pGEM®-T Easy vector

(Promega, Madison, WI, USA). DNA sequencing was performed by

Applied Biosystems DNA sequencer, model ABI PRISM 377.

Antimicrobial assay

Several microorganism strains including bacteria and fungus

were used for antimicrobial assays. They are Gram-positive bacte-

rium Staphylococcus aureus (ATCC2592), Bacillus subtilis (ATCC

6633), Gram-negative bacteria Escherichia coli (ATCC25922), B.

dysenteriae, and fungi Candida albicans (ATCC2002), and several

clinically isolated Candida albicans strains obtained from Kunming

Medical University. Bacteria were cultured in LB (Luria-Bertani)

broth to an absorbance of 0.8 at 600 nm. A 10 μl aliquot of the bac-

teria culture was then mixed with to 8 ml of fresh LB broth contain-

ing 0.7% agar and applied to a 90 mm Petri dish containing 25 ml

of 1.5% agar in LB broth. After the top agar hardened, a 20 μl ali-

quot of test samples with certain concentration filtered on a 0.22 μm

Millipore filter was plated onto the surface of the top agar and com-

pletely dried before being incubated overnight at 37°C as our pre-

vious method (Lai et al., 2002). For C. albicans culture, yeast

extract-peptone-dextrose broth was used. Minimal inhibitory concen-

tration (MIC) was determined in liquid LB medium by incubating the

bacteria in LB broth with the tested sample with different concentra-

tion. The MIC at which no visible growth occurred was recorded.

Hemolytic testing

Hemolytic testing was performed by using human and rabbit

red cells in Alsever’s solution (1L containing 4.2 g NaCl, 8.0 g citric

Acid·3Na·2H2O, 0.55 g citric Acid·H2O, 20.5 g D-glucose) following

the method described by Bignami (Bignami, 1993). The antimicro-

bial peptide sample was serially diluted by Alsever’s solution and

incubated with red cells at 37°C for 30 min. Red cells were centri-

fuged and the absorbance of the supernatant was measured at 595

nm. 100% hemolysis was determined by adding 1% Triton X-100 to

a sample of cells.

Synthetic peptide

The peptide was synthesized by GL Biochem (Shanghai) Ltd.

(Shanghai, China) and analyzed by HPLC and mass spectrometry

to a confirmed purity higher than 98%.

RESULTS

Purification of antimicrobial peptide

Skin secretions of T. kwangsiensis were divided into

more than 30 peaks by C8 RP-HPLC as reported in our pre-

vious work. The eluted peptide peak at the position of elution

time of ~50 min and acetonitrile concentration of 45% showed

antimicrobial activity, as indicated by the arrow in Fig. 1A.

This peak was collected and further subjected to mass spec-

trometry analysis to determine the purity. As illustrated in

Fig. 1B, it is a homogenous peak with a single molecular

weight.

Structural characterization

The purified antimicrobial peptide was named pleurain-

a1-thel. Automated Edman degradation gave an amino acid

sequence of RILTMTKRVKMPQLYKQIVCRLFKTC (Fig. 2).

The peptide was composed of 26 amino acid residues

including two cysteines, which possibly form an intramolec-

ular disulfide bridge. This peptide, putatively containing a

single disulfide bridge, has a theoretical molecular weight of

3194.79 Da. MALDI-TOF-MS yielded an observed mass of

3194.796 Da as illustrated in Fig. 1B. It accorded well with

the theoretical molecular weight (3194.79), suggesting that

the two cysteines in this peptide form an intramolecular dis-

ulfide bridge. Synthesized peptide showed the same RP-

HPLC elution manner and mass spectrometry analysis

result with the native peptide. BLAST search revealed that

sequence of pleurain-a1-thel showed similarity to the antimi-

crobial family of pleurain-a1 found in the frog skin secretions

of Rana pleuraden (Ranidae) (Fig. 2B) (Wang et al., 2007).

cDNA cloning

Upon screening of a skin cDNA library, several clones

containing inserts of around 316-base pairs were identified

and isolated. Both strands of these clones were sequenced

(Fig. 2A). The nucleotide sequence (GenBank accession

number KC572126) encoding pleurain-a1-thel and the

deduced amino acid sequence are shown in Fig. 2A. The

nucleotide sequence was found to contain a coding region

of 207 nucleotides. The encoded amino acid sequence cor-

responds to a polypeptide of 67 amino acids (aa), which is

the precursor of pleurain-a1-thel. The precursor is com-

H. Yan et al.706

posed of predicted signal peptide, acidic spacer peptide

containing multiple acidic amino acid residues (aspartate

and glutamate) and mature peptide of pleurain-a1-thel. Two

possible enzymatic processing sites (-K22-R23- and -K41-K42-

K43-R44-) are found in the sequence of the precursor. It

shows significant sequence similarity to the precursor of

pleurain-a1 found in the frog skin of R. pleuraden (Fig. 2B).

Structural characterization comparison

A comparison of pleurain-a1-thel precursor structure

with that of other AMP precursors belonging to the pleurain

family (He et al., 2012; Wang et al., 2007; Yang et al., 2009)

are shown in Fig. 3A. Although all of AMP precursors shared

highly homologous preproregions, which are composed of

signal peptide, acidic spacer peptide. The signal peptide

shared a similarity of 80–100%. The mature peptides

adopted divergent primary structures containing two

cysteines to form an intramolecular disulfide bridge, or one

or even no cysteine, variable in length (15–31 amino acids)

and broad range in theoretical isoelectric points (6.07–

10.86). Most, however, are positively charged. Pleurain-a1-

thel exploits a pI of 10.57 and net charge of +7, respectively,

which facilitates it to bind with the negatively charged bac-

terial membranes (Li et al., 2007). The sequence compari-

son of pleurain-a1-thel precursor with precursors of other

amphibian bioactive peptides including protease inhibitors

(OGSI and Odorranain-B-RN1) (Li et al., 2008a; Yan et al.,

2012), lectin (Odorranalectin) (Li et al., 2008b), tachykinin

(Tachykinin OG1) (Li et al., 2006), bradykinin (Ranakinin-N)

(Liu et al., 2008) and dermorphin (Montecucchi et al., 1981),

which exert defensive functions are presented in Fig. 3B.

These precursors share a common N-terminal preproregion,

which is highly conserved, followed by a variable C-terminal

domain that corresponds to the mature peptides. In particu-

lar, their signal peptides (composed of 22–23 amino acid

residues) share 70–100% identity (Fig. 3B).

Antimicrobial activities assay

As listed in Table 1, pleurain-a1-thel showed antimicro-

bial activity against most microorganism strains tested, other

than B. subtilis ATCC 6633 and clinically isolated C.

albicans 08022710. It exerted the strongest antimicrobial

ability against C. albicans ATCC2002. The MIC is 10 μg/ml.

Its effects on several clinically isolated C. albicans strains

were tested. Pleurain-a1-thel exerted antimicrobial activity

against them with MIC of 25–200 μg/ml. The antimicrobial

activities of AMPs in the pleurain family are shown in Table

2. Most exhibited a similar antimicrobial spectrum, although

three (pleurain-a1-thel, e1, n1) were not susceptible to B.

subtilis. Pleurain-a1-thel showed moderate antimicrobial

activity among the AMPs of pleurain family.

Fig. 1. Purification of antimicrobial peptide from the skin secre-

tions of T. kwangsiensis. (A) The filtrate of the skin secretions of T.

kwangsiensis by 10 kDa cut-off was divided by a Hypersil BDS C8

RP-HPLC column (25 × 0.46 cm) equilibrated with 0.1% (v/v) trifluo-

roacetic acid/water. The elution was performed with the indicated

gradient of acetonitrile at a flow rate of 0.7 ml/min. The purified anti-

microbial peptide is indicated by an arrow. (B) MALDI-TOF mass

spectrometry analysis of the purified antimicrobial peptide.

Fig. 2. cDNA sequence of pleurain-a1-thel and sequence compar-

ison. (A) The nucleotide sequence encoding pleurain-a1-thel pre-

cursor and the deduced amino acid sequence. The sequence of

mature pleurain-a1-thel is boxed. The bar (–) indicates the stop

codon. (B) The precursor sequence comparison of pleurain-a1-thel

with pleurain-a1 found in R. pleuraden. Mature peptides are boxed.

The asterisk (*) indicates the identical amino acid residue.

Antimicrobial Peptide from Tree Frog 707

Hemolytic activity

Red blood cells were used to evaluate the hemolytic

capability of pleurain-a1-thel. This peptide exerted little

hemolytic activity on both human and rabbit red cells. At

concentrations of 100 and 200 μg/ml, hemolysis of human

red cells induced by pleurain-a1-thel was 2.5 and 3.7%,

respectively. For rabbit blood red cells, hemolysis was 3.2

and 3.5%, respectively.

DISCUSSION

Rhacophoridae is a family of frog species distributed in

regions of Asia and Africa. There are almost 300 species

belonging to the family of Rhacophoridae, which covers two

subfamilies and 12 genera. Rhacophoridae frogs inhabit

unique arboreal environments, mostly living in damp broad-

leaf forest or vegetation near water bodies in forests (Frost

and Darrel, 2011). Although many antimicrobial peptides

have been identified from amphibians belonging to the

Ranidae and Hylidae, there are no reports of antimicrobial

peptides from the family Rhacophoridae.

Fig. 3. Structural characteristics comparison. (A) Comparison of pleurain-a1-thel with other antimicrobial peptides belonged to pleurain family

from Ranidae amphibians. (B) Comparison of pleurain-a1-thel precursor with other precursors of bioactive peptides from amphibians. The

identical amino acid residue is indicated by the asterisk (*). The region of signal peptide is italic. The predicted enzymatic cleavage site is bold

and italic. Mature peptides are underlined. The cysteines of mature peptide to form an intramolecular disulfide bridge are bold. The bar (–) was

introduced for optimal comparison. AA: Number of mature peptides’ amino acids. NC: Net charge. PI: Theoretical isoelectric point. MW: Molec-

ular weight.

Table 1. Antimicrobial activity of pleurain-a1-thel.

MIC, minimal inhibitory concentration. These concen-

trations represent mean values of three independent

experiments performed in duplicates. NA, no activity at

the concentration of 200 μg/ml. CI, clinically isolated

strain.

Microorganisms MIC (μg/ml)

Gram-negative bacteria

E. coli ATCC25922 100

B. dysenteriae 50

Gram-positive bacteria

S. aureus ATCC2592 50

B. subtilis ATCC 6633 NA

Fungi

C. albicans ATCC2002 10

C. albicans 08030401 (CI) 50

C. albicans 08022821 (CI) 25

C. albicans 08032815 (CI) 200

C. albicans 08030809 (CI) 25

C. albicans 08030102 (CI) 100

C. albicans 08022710 (CI) NA

Table 2. Antimicrobial activity comparison of pleurain-a1-thel with

other AMPs belonging to the pleurain family. MIC, minimal inhibitory

concentration. These concentrations represent mean values of

three independent experiments performed in duplicate. NA, no activ-

ity at the concentration of 200 μg/ml.

AMPs MIC (μg/ml)

E. coli S. aureus B. subtilis C. albicans

pleurain-a1-thel 100 50 NA 10

pleurain-a1 60 15 120 30

pleurain-b1 25 3.1 3.1 1.6

pleurain-c1 50 6.3 3.1 25

pleurain-d1 25 12.5 50 25

pleurain-e1 25 6.3 NA 25

pleurain-g1 50 50 100 12.5

pleurain-j1 12.5 12.5 50 6.3

pleurain-m1 6.3 6.3 25 12.5

pleurain-n1 50 12.5 NA 12.5

pleurain-r1 100 25 100 50

H. Yan et al.708

We purified and characterized a novel antimicrobial pep-

tide, pleurain-a1-thel from skin secretions of the frog, T.

kwangsiensis (Fig. 1). To our knowledge, this is the first

published report of an antimicrobial peptide from a

Rhacophoridae amphibian. Sequence comparison indicated

that pleurain-a1-thel belongs to the antimicrobial peptide

family of pleurain-a1, which was originally identified in the

frog of R. pleuraden (Wang et al., 2007). Both sequences of

mature peptides and precursors of these pleurain-a1 antimi-

crobial peptides are highly conserved (Fig. 2). R. pleuraden

and O. tiannanensis belonging to the amphibian family Rani-

dae (He et al., 2012; Wang et al., 2007; Yang et al., 2009).

Furthermore, pleurain-a1-thel shares a highly homologous

propreregion with other AMP precursor members of the

pleurain AMP family. These results indicate that both

Ranidae and Rhacophoridae share the same antimicrobial

peptide family, and may provide the evolution of amphibian

antimicrobial peptides.

Pleurain is a super family that contains many antimicro-

bial peptides (He et al., 2012; Wang et al., 2007; Yang et al.,

2009). The discovery of pleurain-a1-thel from the tree frog

T. kwangsiensis contributes a novel member to the pleurain

family. To date, the already discovered AMPs of pleurain

family were from R. pleuraden and O. tiannanensis (He et

al., 2012; Wang et al., 2007; Yang et al., 2009), which live

in a slightly damper environment and face much higher

microbiological diversity than those of the tree frog T.

kwangsiensis. This may support the notion that the antimi-

crobial activity of some AMPs discovered in R. pleuraden

and O. tiannanensis are stronger than that of pleurain-a1-

thel.

Interestingly, pleurain-a1-thel precursor shares a com-

mon N-terminal preproregion, which is highly conserved,

with precursors of amphibian lectin, protease inhibitor, bra-

dykinin, tachykinin, and dermorphin (Fig. 3B). All the peptide

groups found in these amphibian skins have different biolog-

ical activities, but these activities are related to defensive

functions and self-protection. Antimicrobial peptides, lectins

and protease inhibitors act in antimicrobial defense; pro-

tease inhibitors also act as anti-parasitic agents; algesic

peptides including tachykinin and bradykinin, and antinoci-

ceptive peptide (dermorphin) could help hosts avoid injury or

alleviate noxious stimuli. It appears that all these defensive

peptides in amphibian skin originate from a common ances-

tor, however, much more work is needed to address this

hypothesis.

ACKNOWLEDGMENTS

This work was supported by the Chinese National Natural Sci-

ence Foundation (31025025, 31000960, 31025025, U1132601 and

31201717), the Ministry of Science and Technology (2010CB529800),

Jiangsu Province (BK2012365, BE2012748, CXZZ11_0649), Yunnan

Province (2011CI139 and 2012BC009), and Nanjing Agricultural

University (KJ2012023).

REFERENCES

Barra D, Simmaco M (1995) Amphibian skin: a promising resource

for antimicrobial peptides. Trends Biotechnol 13: 205–209

Bevins CL, Zasloff M (1990) Peptides from frog skin. Annu Rev

Biochem 59: 395–414

Bignami GS (1993) A rapid and sensitive hemolysis neutralization

assay for palytoxin. Toxicon 31: 817–820

Boman HG (1991) Antibacterial peptides: key components needed

in immunity. Cell 65: 205–207

Chen T, Zhou M, Rao P, Walker B, Shaw C (2006) The Chinese

bamboo leaf odorous frog (Rana (Odorrana) versabilis) and

North American Rana frogs share the same families of skin

antimicrobial peptides. Peptides 27: 1738–1744

Conlon JM, Kolodziejek J, Nowotny N (2004) Antimicrobial peptides

from ranid frogs: taxonomic and phylogenetic markers and a

potential source of new therapeutic agents. Biochim Biophys

Acta 1696: 1–14

Dubois A (1992) Notes sur la classification des Ranidae (Amphibiens;

Anoures). Bull Mens Soc Linn Lyon 61: 305–352

Frost DR (2011) Amphibian Species of the World: an Online Refer-

ence. Version 5.5 (31 January, 2011). Electronic Database

accessible at http://research.amnh.org/vz/herpetology/amphibia/

American Museum of Natural History, New York, USA

He W, Feng F, Huang Y, Guo H, Zhang S, Li Z, Liu J, et al. (2012)

Host defense peptides in skin secretions of Odorrana tiannan-

ensis: Proof for other survival strategy of the frog than merely

anti-microbial. Biochimie 94: 649–655

Kawasaki H, Isaacson T, Iwamuro S, Conlon JM (2003) A protein

with antimicrobial activity in the skin of Schlegel’s green tree

frog Rhacophorus schlegelii (Rhacophoridae) identified as his-

tone H2B. Biochem Biophys Res Commun 312: 1082–1086

Lai R, Zheng YT, Shen JH, Liu GJ, Liu H, Lee WH, et al. (2002) Anti-

microbial peptides from skin secretions of Chinese red belly

toad Bombina maxima. Peptides 23: 427–435

Li J, Liu T, Xu X, Wang X, Wu M, Yang H, Lai R (2006) Amphibian

tachykinin precursor. Biochem Biophys Res Commun 350:

983–986

Li J, Xu X, Xu C, Zhou W, Zhang K, Yu H, et al. (2007) Anti-infection

peptidomics of amphibian skin. Mol Cell Proteomics 6: 882–894

Li J, Wu J, Wang Y, Xu X, Liu T, Lai R, Zhu H (2008a) A small

trypsin inhibitor from the frog of Odorrana grahami. Biochemie

90: 1356–1361

Li J, Wu H, Hong J, Xu X, Liu T, Lai R (2008b) Odorranalectin is a

small peptide lectin with potential for drug delivery and target-

ing. PLoS ONE 3: e2381

Liu X, You D, Chen L, Wang X, Zhang K, Lai R (2008) A novel bra-

dykinin-like peptide from skin secretions of the frog, Rana

nigrovittata. J Pept Sci 14: 626–630

Lu Y, Li J, Yu H, Xu X, Liang J, Tian Y, et al. (2006) Two families of

antimicrobial peptides with multiple functions from skin of

rufous-spotted torrent frog, Amolops loloensis. Peptides 27:

3085–3091

Lu Y, Ma Y, Wang X, Liang J, Zhang C, Zhang K, et al. (2008) The

first antimicrobial peptide from sea amphibian. Mol Immunol 45:

678–681

McGillivary G, Ray WC, Bevins CL, Munson RS, Jr, Bakaletz LO

(2007) A member of the cathelicidin family of antimicrobial pep-

tides is produced in the upper airway of the chinchilla and its

mRNA expression is altered by common viral and bacterial co-

pathogens of otitis media. Mol Immunol 44: 2446–2458

Montecucchi PC, de Castiglione R, Piani S, Gozzini L, Erspamer V

(1981) Amino acid composition and sequence of dermorphin, a

novel opiate-like peptide from the skin of Phyllomedusa

sauvagei. Int J Pept Protein Res 17: 275–283

Nicolas P, Mor A (1995) Peptides as weapons against microorgan-

isms in the chemical defense system of vertebrates. Annu Rev

Microbiol 49: 277–304

Simmaco M, Mignogna G, Barra D (1999) Antimicrobial peptides

from amphibian skin: what do they tell us? Biopolymers 47:

435–450

Wang X, Song Y, Li J, Liu H, Xu X, Lai R, Zhang K (2007) A new

family of antimicrobial peptides from skin secretions of Rana

pleuraden. Peptides 28: 2069–2074

Xu X, Li J, Han Y, Yang H, Liang J, Lu Q, Lai R (2006) Two antimi-

Antimicrobial Peptide from Tree Frog 709

crobial peptides from skin secretions of Rana grahami. Toxicon

47: 459–464

Yan X, Liu H, Yang X, Che Q, Liu R, Yang H, et al. (2012) Bi-func-

tional peptides with both trypsin-inhibitory and antimicrobial

activities are frequent defensive molecules in Ranidae amphib-

ian skins. Amino Acids 43: 309–316

Yang H, Wang X, Liu X, Wu J, Liu C, Gong W, et al. (2009) Antioxi-

dant peptidomics reveals novel skin antioxidant system. Mol

Cell Proteomics 8: 571–583

Zasloff M (1987) Magainins, a class of antimicrobial peptides from

Xenopus skin: isolation, characterization of two active forms,

and partial cDNA sequence of a precursor. Proc Natl Acad Sci

USA 84: 5449–5453

Zasloff M (1992) Antibiotic peptides as mediators of innate immu-

nity. Curr Opin Immunol 4: 3–7

Zhang H, Wei L, Zou C, Bai JJ, Song Y, Liu H (2013) Purification

and characterization of a tachykinin-like peptide from skin

secretions of the tree frog Theloderma kwangsiensis. Zool Sci

7: 529–533

Zheng R, Yao B, Yu H, Wang H, Bian J, Feng F (2010) Novel family

of antimicrobial peptides from the skin of Rana shuchinae.

Peptides 3: 1674–1677

Zhou M, Wang L, Owens DE, Chen T, Walker B, Shaw C (2007)

Rapid identification of precursor cDNAs encoding five structural

classes of antimicrobial peptides from pickerel frog (Rana

palustris) skin secretion by single step “shotgun” cloning.

Peptides 28: 1605–1610

(Received February 3, 2013 / Accepted April 3, 2013)