Host Defense Peptides in Skin Secretions of Odorrana Tiannanensis: Proof for Other Survival Strategy of the Frog Than Merely Anti-Microbial

Biochimie xxx (2011) 1e7

Contents lists available at SciVerse ScienceDirect

Biochimie

journal homepage: www.elsevier.com/locate/biochi

Research paper Host defense peptides in skin secretions of Odorrana tiannanensis: Proof for other survival strategy of the frog than merely anti-microbial

Weiyu Hea,1, Feifei Fenga,1, Yong Huangd, Huanhuan Guoa, Songyan Zhangb, Zheng Lib, Jingze Liua, Yipeng Wangc,**, Haining Yua,b,* a College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, China b Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116023, China c Biological Resources Laboratory, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, Shandong 264003, China d Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China article info abstract

Article history: Genus Odorrana, among all amphibians studied, is generally reported to have the most abundant and diver- Received 6 August 2011 sified anti-microbial peptides even from a single individual frog. In our previous work, 46 cDNA sequences Accepted 16 September 2011 encoding precursors of 22 different anti-microbial peptides (AMPs) were characterized from the skin of frog, Available online xxx Odorrana tiannanensis. In this work, we reported the purification of three AMPs from skin secretions of O. tiannanensis. Their amino acid sequences matched well with the sequences deduced from cDNAs and they Keywords: were designated as Odorranain-C7HSa, Brevinin-1-OT2 and Odorranain-G-OT, respectively. Furthermore, we Amphibian selected to analyze the four most structurally diversified sequences among the 22 AMPs that are significantly Diversity Anti-microbial peptide (AMP) different from all reported AMPs. By structural characterization, three of them were designated as pleurain-E- fi Antioxidant OT, odorranain-G-OT, odorranain-A-OT, belonging to AMP families already identi ed. The forth one with Odorrana tiannanensis a unique 14-mer sequence of AILTTLANWARKFLa and C-terminal amidation represents the prototypes of a new class of amphibian AMP, and thereby named tiannanensin. Such broad diversity in sequences and structures are consistent with other species in Genus Odorrana. Multi-functions of the synthesized four special AMPs were screened, including anti-microbial, antioxidant, cytotoxic and hemolytic activities. The results suggest that these AMPs may employ sophisticated mechanisms of action in host defense in addition to anti-microbial, although their precise contribution to host defense still seems unclear. Ó 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction to the development of antibiotic resistance, they have attracted considerable attentions as a new generation of antibiotics. Over the past several decades, extensive studies have focused on So far, there have been hundreds of AMPs of different families the bioactive compounds present in amphibian skin secretions, characterized from ranid frogs, including gaegurins, brevinins-1 especially some small peptides [1]. Those peptides are very func- and -2, ranalexin, ranatuerins-1 and -2, esculentins-1 and -2, pal- tionally different, and among these functions, the anti-microbial ustrin, japonicin-1 and -2, nigrocin-2, rugosins and temporin [6e8], activities are commonly considered to be the most important for based on their structural characteristics. Besides, there are usually the amphibian staying safe and defending against invasion of more than one anti-microbial peptide families in a single microorganisms in their habitats. Generally, these anti-microbial amphibian species. Most of the AMPs, 10e50 residues in length, peptides (AMPs) are cationic, amphipathic, and central effector have a common highly conserved N-terminal preproregion, fol- molecules of all forms of lives’ innate immunity [2e4]. AMPs can lowed by a markedly different C-terminal domain that corresponds rapidly kill a broad range of bacteria, yeasts, and fungi by forming to mature AMPs [6,7,9]. Functionally, different amphibian anti- pores in the membranes of target organisms, thus disrupting their microbial peptides have different anti-microbial spectrum. metabolic activities [5]. Because these peptides are less susceptible Odorrana is agenusoftrue frogs(Ranidae) from East Asia and surrounding regions. Many of these frogs inhabit in fast-ﬂowing * Corresponding author. College of Life Sciences, Hebei Normal University, Shi- mountain streams. Odorrana tiannanensis, a characteristic species jiazhuang 050016, China. Tel./fax: þ86 311 86268842. of China, is mainly found distributed in Hainan and Yunnan prov- ** þ Corresponding author. Tel./fax: 86 311 86268842. inces of China. Previous work by Li et al puriﬁed and characterized E-mail addresses: [email protected] (Y. Wang), [email protected] (H. Yu). 1 These authors have the same contribution to this paper. 107 novel AMPs belonging to 30 different families, including

24 novel families from Odorrana grahami [9]. Totally 372 different protocols. After the cleavage and deprotection of side-chain, the cDNAs encoding these anti-microbial peptides were identified in crude synthetic peptide was purified on a Vydac C18 RP-HPLC that work. In our previous work, 46 cDNA sequences encoding column (25 cm 1 cm), eluting at a flow rate of 1 ml/min by precursors of 22 different AMPs from skin cDNA library of frog, a linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water. O. tiannanensis were successfully cloned. In current work, the Identity of the peptide was confirmed by automated Edman structures and properties of the four most structurally diversified degradation with a protein sequencer and MALDI-TOF-MS analysis. AMPs present in the skin of O. tiannanensis were investigated, and The synthesized peptides containing two cysteins were further furthermore the actual contributions of the general AMPs to the subject to oxidation to form an intrapeptide disulphide bridge. host innate system were discussed to a certain extent. 2.5. Anti-microbial assay 2. Materials and methods Standard and clinical-isolated drug-resistant strains of bacteria 2.1. Collection of frog skin secretions and fungi used in assays were listed in Table 1. The assay was conducted as described in our previous paper [10]. Minimal Adult specimens of O. tiannanensis (n ¼ 20) were captured in inhibitory concentration (MIC) was determined in 96-well micro- Sanya, Hainan Province, China. They were put into a cylinder titer plate by a standard dilution method. Bacteria were incubated container and stimulated gently with an electrical device (10 V, with in MuellereHinton broth (MH) at 37 C to exponential phase of pulse duration of 3 ms). The skin secretions were collected by washing growth and diluted with fresh MH broth to 106 CFU/ml 50 mlof the dorsal region with 0.9% Sodium chloride solution. Totally 200 ml serial dilutions of the peptides in MH were prepared in 96-well solution was collected and centrifuged at 12,000 rpm for 20 min. microtiter plates and mixed with 50 ml of bacteria inoculum. The supernatant was removed, lyophilized and stored at 20 C. Plates were incubated at 37 C for 18 h and the minimal concentration at which no visible growth occurred was recorded. 2.2. Peptide purification and sequencing 2.6. Hemolysis assay Lyophilized sample (0.8 g, total OD280 nm of 200) was dissolved in 10 ml 0.1 M phosphate buffer (PB), pH 6.0, and then applied to Hemolysis assay was conducted as previously reported [11]. The a Sephadex G-50 (Superfine, Amersham Biosciences, 1.6 cm serial dilution of peptides were incubated with washed human 90 cm) column equilibrated with 0.1 M PB, pH 6.0 buffer (Na2H- erythrocytes at 37 C for 30 min and centrifuged at 2000 rpm for PO4$12H2O 4.41 g, NaH2PO4$2H2O 13.69 g, H2O 1000 ml). Elution 5 min. The supernatant was removed and the absorbance at 540 nm was performed using the same buffer with collecting fractions of was measured. 1% v/v Triton X-100 was used to determine the 3.0 ml/10 min, and monitored at 220 nm. The anti-microbial maximal hemolysis. activity of fractions was screened, and interesting peaks were fi further puri ed by C18 reversed phase high performance liquid 2.7. Cytotoxic activity chromatography (RP-HPLC, Hypersil BDS C18, 30 cm 0.46 cm) column. Complete peptide sequencing was determined by Edman In vitro cytotoxic activity of odorranain-G-OT, odorranain-A-OT degradation method on an Applied Biosystems pulsed liquid-phase and tiannanensin were examined using tumor cell lines, SGC7901, fi fi sequencer, model 491. Mass ngerprints (MFPs) of puri ed AMPs Hela and MCF-7. The cells were cultured in Dulbecco’s Modified were obtained using electrospray ionization, quadrupole orthog- Eagle’s Medium (DMEM, 11960-044, Gibco, USA) supplemented fl onal time-of- ight mass spectrometry (ESI-QTOF-MS Applied Bio- with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 U/ml of systems/MDS Sciex Toronto, Canada) instrument. streptomycin in a humidified 5% CO2 atmosphere at 37 C. Cells (2 104 per well) were seeded in 96-well plates and cultured 2.3. Construction of cDNA library and screening of cDNAs overnight until adhered to the plate. Various concentrations of encoding AMPs AMPs dissolved in the corresponding culture medium were added to the wells and the plates were incubated at 37 C for 48 h. The dorsal skin of frog was removed and cut into small pieces, Cytotoxicity of three AMPs were measured by the MTT (3-(4,5- which then were quickly frozen with liquid nitrogen and grinded to dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. powder. Total RNA was extracted using Trizol reagent (Invitrogen, IC50 was defined as the concentration of AMP at which the absor- USA) according to the manufacture instruction. mRNA was isolated bance at 490 nm was reduced by 50%. using a mRNA isolation kit (Dynal Biotec., UK), and cDNA library was Ô Ô constructed using a Creator SMART cDNA Library Construction 2.8. Anti-oxidant activity Kit (Clotech). According to the conserved signal peptide domain of previously characterized AMPs from ranid frogs, a sense oligonu- 0 0 Free radical scavenging activity of the peptides was determined cleotide primer (5 -CCCCATGTTCACCTTGAAG-3 ) was designed and by using a stable DPPH radical (sigma) following previous report 0 0 coupled with 3 antisense primer (5 -ATTCTACAGGCCGAGGCGG [12]. The assay mixture contained 92 mlof6 10 5 M DPPH radical CCGACATG-30) supplied by the Kit to screen the cDNA Library. The PCR procedure was: 5 min of denaturation at 94 C; 30 cycles: Table 1 denaturation at 94 C for 30 s, primer annealing at 56 C for 30 s, Structural parameters of the four AMPs. extension at 72 C for 1 min. The PCR product was purified by gel Peptide GRAVY Number of Net charge Theoretical pI electrophoresis, cloned into pGEM-T vector (Promega Corporation). amino acids Pleurain-E-OT 0.446 26 þ3 11.70 2.4. Peptide synthesis Odorranain-G-OT 1.800 13 þ1 8.06 ODORRANAIN-A-OT 0.350 16 þ4 10.11 The peptides subject to the further analysis were synthesized by Tiannanensin 0.707 14 þ2 11.00 solid phase synthesis on an Applied Biosystems model 433A GRAVY: grand average of hydropathicity. Structural parameters of each peptide peptide synthesizer according to the manufacturer’s standard calculated using the ProtParam tool.

Please cite this article in press as: W. He, et al., Host defense peptides in skin secretions of Odorrana tiannanensis: Proof for other survival strategy of the frog than merely anti-microbial, Biochimie (2011), doi:10.1016/j.biochi.2011.09.017 W. He et al. / Biochimie xxx (2011) 1e7 3 dissolved in methanol and 8 ml of sample solution (0.2 mg/ml). 3. Results After 30 min incubation at room temperature, the samples reacted with DPPH and converted them into colorless compounds. The 3.1. Purification of AMPs amount of reduced DPPH was quantified by measuring a decrease in absorbance at 520 nm. Inhibition of free radical by DPPH The supernatant of skin secretions was fractionated into three in percentage (I %) was calculated according to the formula: peaks (I, II, III) by Sephadex G-50 (Fig. 1A), and the anti-microbial I%¼ (Ablank Asample) 100/Ablank。Deionized water was used as activity was detected in the peak III. This peak was collected and negative control. subjected further purification by RP-HPLC column as illustrated in

Fig. 1. Fractionation of O. tiannanensis skin secretion. (A) Sephadex G-50 gel filtration. (B) Peak III was purified on RP-HPLC. The purified anti-microbial peptides are indicated by a, b, and c.

Fig. 1B. More than 30 peaks were eluted by the RP-HPLC separation and their anti-microbial activities were screened using the inhibition zone assay. Peaks a, b and c indicated with an arrow in Fig. 1B showed anti-microbial peptides and they were subjected further structural and functional analysis.

3.2. Peptide sequences characterization

Their amino acid sequences of three purified AMPs (peaks a, b and c in Fig. 1B) were determined by automated Edman degradation. The amino acid sequences of a, b and c were SLLGTV KDLLIGAGKSAAQSVLKGLSCKLSKDC, FLPLIASLAANFVPKIFCKITKKC, and FVPAILCSILKTC, respectively. After comparison with sequences deposited in protein database of NIH, sequence a was found iden- tical with previously reported amphibian AMPs, Odorranain- C7HSa. Due to the primary structure similarities with Brevinin-1 and Odorranain-G families, sequence b and c were designated as Brevinin-1-OT2 and Odorranain-G-OT, respectively. As to AMPs deduced from other cDNA sequences cloned, they were either not purified in current experiment, or not obtained enough amount and purity for further structural analysis. All of the three purified peptides have two cysteines at C- terminals, which formed an intramolecular disulfided bridge. However, such C-terminal cyclic heptapeptide domain are suggested having no effect on peptides’ antibacterial properties, but might be important in maintaining their hemolytic activity [13].

3.3. cDNA cloning and mature peptide characterization

Totally 46 cDNAs encoding precursors of 22 different AMPs were cloned from O. tiannanensis skin cDNA library (GenBank Accession: JF329971-JF330012, JN544925-JN544928). The deduced 22 mature AMPs belong to 14 different AMP families already identified in other Ranid species (Fig. 2). Among these 22 AMPs, we choose the four sequences with structures of significantly different from other known AMPs for further chemical synthesis and biochemical analyses. The four mature peptides including one purified and the other three deduced are designated as odorranain-G-OT, pleurain- E-OT, odorranain-A-OT and tiannanensin, respectively. Their cDNAs and corresponding sequences are listed in Fig. 3.

Fig. 3. Nucleic acid sequences of cDNAs encoding the anti-microbial peptides pleurain- E-OT, odorranain-G-OT, odorranain-A-OT and tiannanensin cloned from the O. tiannanensis. Putative mature peptides are boxed, and stop codons are indicated by asterisks (*).

All four precursors share quite similar structural organization, including an N-terminal signal peptide sequence, a spacer peptide region containing several aspartic (D) and glutamic (E) acid residues that was terminated in a dibasic cutting site LyseArg (K-R) for trypsin-like proteases cleavage to release mature C-terminal AMPs, except the N-R for odorranain-A-OT (Fig. 4). The protein physical and chemical parameters of the four AMPs, such as the theoretical pI, net charge and grand average of hydropathicity, were computed by ProtParam and listed in Table 1. The helical wheel diagrams for the four peptides were plotted to estimate their amphipathicity. The sequence of amino acids that make up a-helical region of the peptide’s secondary structure were plotted in a rotating manner where the angle of rotation between consecutive amino acids is 100. The plot revealed that only for odorranain-G-OT and tiannanensin, the hydrophobic amino acids (shown as blue squares) are concentrated on one side of the helix, and the polar or hydrophilic amino acids (shown as red diamonds) Fig. 2. The alignment of mature AMPs characterized from O. tiannanensis. The number of amino acid residues of each sequence is shown in the right. The AMP family of each on the other (Fig. 5). This arrangement is common in alpha helices peptide ascribed is shown ahead. within globular proteins, where one face of the helix is oriented

Fig. 4. The amino acid sequences deduced from the cDNA clones encoding anti-microbial peptides. Gaps () have been introduced to optimize the sequence homology. The sequences of mature peptides are underlined. toward the hydrophobic core and one face is oriented toward the inherit the direct anti-microbial activity, such as pleurain-E-OT solvent-exposed surface. Usually such amphipathic alpha-helix versus Pleurain-E [12]. It is demonstrated that homology alone is structural feature is believed to be important for the anti-micro- insufficient to ascribe a function to these peptides. bial function. Thus, it’s rational for us to speculate that odorranain- Such results seem inconsistent with the hypothesized activity G-OT and tiannanensin might possess potent anti-microbial from peptides’ secondary structures. Although in nearly all cases, activities. the strategy employed in enhancing activity involved increasing the amphipathic alpha-helical character of the peptide, recent study by 3.4. Anti-microbial and hemolytic assays Houston et al suggested that the preformation of alpha-helix in solution was not necessarily beneficial to anti-microbial activity The anti-microbial activities of the four AMPs were examined [14]. To date, no one has attempted a comprehensive study of the firstly using the inhibition zone assay. Totally about 50 microor- structureefunction relationships of a family of closely related linear ganisms were tested, including bacteria and fungi, even some peptides with simple sequences designed to adopt different clinically isolated (IS) drug-resistant strains. However, in dose of secondary structures. Further experiments are needed to reveal the 2 mg/ml, only odorranain-G-OT and tiannanensin showed slight importance of different secondary structures with varying levels of anti-microbial activity toward Candida tropicalis 092422(IS) and amphipathic character in determining anti-microbial activity and Staphylococcus aureus ATCC2592 & Candida albicans ATCC2002, selectivity between bacterial and mammalian membranes. respectively (Table 2). In the further MIC determination assay, only Since among the four peptides synthesized only odorranain-G- MIC of tiannanensin against S. aureus ATCC2592 was determined as OT and tiannanensin inherited the traditional bactericidal char- high as 75 mg/ml, while odorranain-G-OT and tiannanensin did not acter of AMP though in the slight way, we only tested the hemolytic totally inhibit cell growth in liquid medium in a dose up to 100 mg/ activity of these two peptides using the fresh-prepared human ml (Table 2). The sensitive strain could not continue to grow on agar erythrocytes. Odorranain-G-OT and tiannanensin at a concentra- plates after a 6 h treatment with concentration above the corre- tion of 100 mg/ml had hemolytic activities on blood cells of 9.22% sponding MIC. Whilst the peptides are homologous to previously and 10.5%, respectively. reported anti-microbial peptides, the current peptides do not 3.5. Cytotoxic activity

Some amphibian AMPs have been demonstrated to exert cytotoxic and antiproliferative efﬁcacy to certain tumor cells, such as magainin II, which kills bladder cancer cells by pore formation but has no effect on normal murine or human ﬁbroblasts [15]. However, in current study, all the four peptides did not show cytotoxicity in vitro to tumor cell lines of SGC7901, Hela and MCF-7 in the MTT assay. Judging by the same strategy exploited in breaking down the cell membrane, current result appears accordant with previous anti-microbial activity.

3.6. Free radical scavenging activity

The production of free radicals signiﬁcantly increases in critical situations (oxidative stress, contamination, UV exposure, etc.) [10].

Table 2 Anti-microbial activity of peptides.

MIC(mg/ml)

Microorganism Odorranain-G-OT Tiannanensin Amp Gram-positive S. aureus ATCC2592 ND 75 4.69 Fungi C. albicans ATCC2002 ND >100 4.69 Candida tropicalis 092422(IS) >100 ND >100

MIC: minimal inhibitory concentration. These concentrations represent mean Fig. 5. Helical wheel diagrams for four peptides. Aliphatic residues are shown as blue values of three independent experiments performed in duplicates. Amp: Ampicillin. squares, polar or negatively charged residues as red diamonds, and positively charged ND: no detectable activity in inhibition zone assay in dose of 2 mg/ml; >100: have residues as black octagons. (For interpretation of the references to colour in this ﬁgure detectable anti-microbial activity in inhibition zone assay, but not totally inhibit cell legend, the reader is referred to the web version of this article.) growth in liquid medium in a dose up to 100 mg/ml; IS: clinically isolated strain.

Fig. 6. DPPH radical scavenging activity of the four peptides at a concentration of 80 mg/ml. The peptides were incubated with DPPH solution at room temperature for 30 min, and then the amount of reduced DPPH was quantiﬁed by measuring a decrease in absorbance at 520 nm. Values are mean of triplicate determinations (n ¼ 3) standard deviation.

To evaluate the antioxidant capacities in a convenient way, the four attenuated the potency so that symbiotic cutaneous microbes are peptides reacted with 2,20-diphenyl-l-picrylhydrazyl (DPPH), due able to survive in the antibacterial skin secretions [22,23]. to the relative stability, easy measurement, and good reproduc- The other explanation by us is that these AMPs defend host ibility [12]. The assay is based on the decolorization by monitoring innate system by means other than bactericidal. The current finding absorbance decreases at 520 nm. At a concentration of 80 mg/ml, that AMPs of low anti-microbial activities usually have rather high apparently odorranain-A-OT has the strongest antioxidant activity antioxidant activities might offer firm evidence to support our by scavenging 89.24% DPPH radicals, followed by odorranain-G-OT claim. Our previous work about some multi-functional (anti- and pleurain-E-OT with 70.41% and 49.16%, respectively. While microbial and antioxidant) peptides from Xizang plateau frog also tiannanensin only scavenge little part about 4.24% of free radicals suggest the frog skin AMPs may participate in the frog’s innate generated (Fig. 6). Such scavenging activity of the peptides would defense from different perspectives [10]. be related primarily to the disulfide bridge, and secondarily to the Although the importance of these AMPs in the survival strategy hydroxyl groups present in the synthesized peptide sequence, thus of the amphibians is not clearly understood, their presence as contributing to their hydrogen donating ability [16]. The antioxi- antibacterial agents in skin secretions seems not essential for the dant activity of the current four peptides is correlated directly with hosts to survival. Among species that do produce dermal anti- the number of cysteine residues and disulfide bridges, confirming microbial peptides, their precise contribution to host defense in the the theory above. environment seems offer evolutionary advantage to other roles, such as antioxidant agent. 4. Discussion Acknowledgments Amphibians, being the first group of organisms forming a con- necting link between land and water, are forced to adopt and This work was supported by the grants from Chinese National survive in a variety of conditions laden with pathogens and pred- Natural Science Foundation (30900240, 41076098). ators [17]. Thus, it is not surprising that amphibians among verte- brates could have the most susceptible skins because they are References directly exposed to the harsh conditions of the habitat. Therefore, they are endowed with an excellent chemical defense system [1] C.L. Bevins, M. Zasloff, Peptides from frog skin, Annual Review of Biochemistry 59 (1990) 395e414. composed of pharmacological gene-encoded peptides [4,17,18]. [2] K. Radek, R. Gallo, Antimicrobial Peptides: Natural Effectors of the Innate Among these peptides, anti-microbial peptides (AMPs), families of Immune System. Springer, 2007, pp. 27e43. cationic peptides that adopt an amphipathic a-helical conformation [3] H. Jenssen, P. Hamill, R.E.W. Hancock, Peptide antimicrobial agents, Clinical Microbiology Reviews 19 (2006) 491. in a membrane-mimetic environment have been studied the most [4] M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature 415 comprehensively. It is generally presumed that AMPs play a key (2002) 389e395. role in amphibian host innate immunity and defend the animal [5] P. Nicolas, Multifunctional host defense peptides: intracellularÓ\targeting e against invasion by pathogenic microorganisms [19]. antimicrobial peptides, FEBS Journal 276 (2009) 6483 6496. [6] J.M. Conlon, The therapeutic potential of antimicrobial peptides from frog Although people hope that the frog skin AMPs could provide skin, Reviews in Medical Microbiology 15 (2004) 17. more leading templates for designing novel anti-infection agents, [7] T.F. Duda, D. Vanhoye, P. Nicolas, Roles of diversifying selection and coordi- these peptides are usually found having a low bactericidal potency nated evolution in the evolution of amphibian antimicrobial peptides, Molecular Biology and Evolution 19 (2002) 858. toward readily available microorganisms that have relevance to [8] B. Matutte, K.B. Storey, F.C. Knoop, J.M. Conlon, Induction of synthesis of an human disease. One explanation for such low potency is the antimicrobial peptide in the skin of the freeze-tolerant frog, Rana sylvatica, in hypothesis that cutaneous symbiotic bacteria may provide the response to environmental stimuli, FEBS Letters 483 (2000) 135e138. [9] J. Li, X. Xu, C. Xu, W. Zhou, K. Zhang, H. Yu, Y. Zhang, Y. Zheng, H.H. Rees, R. Lai, major defense system against pathogens in the habitats of certain Anti-infection peptidomics of amphibian skin, Molecular & Cellular Proteo- frog species [20,21]. In these species, natural selection may have mics 6 (2007) 882.

[10] Z. Lu, L. Zhai, H. Wang, Q. Che, D. Wang, F. Feng, Z. Zhao, H. Yu, Novel families [16] W. Brand-Williams, M. Cuvelier, C. Berset, Use of a free radical method to eval- of antimicrobial peptides with multiple functions from skin of Xizang plateau uate antioxidant activity, LWT-Food Science and Technology 28 (1995) 25e30. frog, Nanorana parkeri, Biochimie 92 (2010) 475e481. [17] B.T. Clarke, The natural history of amphibian skin secretions, their normal [11] Y. Wang, Z. Lu, F. Feng, W. Zhu, H. Guang, J. Liu, W. He, L. Chi, H. Yu, Molecular functioning and potential medical applications, Biological Reviews 72 (1997) cloning and characterization of novel cathelicidin-derived myeloid antimi- 365e379. crobial peptide from Phasianus colchicus, Developmental & Comparative [18] K.A. Brogden, Antimicrobial peptides: pore formers or metabolic inhibitors in Immunology (2010). bacteria? Nature Reviews Microbiology 3 (2005) 238e250. [12] H. Yang, X. Wang, X. Liu, J. Wu, C. Liu, W. Gong, Z. Zhao, J. Hong, D. Lin, [19] G. Diamond, N. Beckloff, A. Weinberg, K.O. Kisich, The roles of antimicrobial Y. Wang, R. Lai, Antioxidant peptidomics reveals novel skin antioxidant peptides in innate host defense, Current Pharmaceutical Design 15 (2009) 2377. system, Molecular & Cellular Proteomics 8 (2009) 571e583. [20] D.C. Woodhams, V.T. Vredenburg, M.A. Simon, D. Billheimer, B. Shakhtour, [13] V. Kumari, R. Nagaraj, Structure- function studies on the amphibian peptide Y. Shyr, C.J. Briggs, L.A. Rollins-Smith, R.N. Harris, Symbiotic bacteria Ó brevinin 1E: translocating the cationic segment from the C \terminal end to contribute to innate immune defenses of the threatened mountain yellow- a central position favors selective antibacterial activity, The Journal of Peptide legged frog, Rana muscosa, Biological Conservation 138 (2007) 390e398. Research 58 (2001) 433e441. [21] R.N. Harris, R.M. Brucker, J.B. Walke, M.H. Becker, C.R. Schwantes, [14] M. Houston Jr., L. Kondejewski, D. Karunaratne, M. Gough, S. Fidai, R. Hodges, D.C. Flaherty, B.A. Lam, D.C. Woodhams, C.J. Briggs, V.T. Vredenburg, Skin R. Hancock, Inﬂuence of preformed alpha-helix and alpha-helix induction on microbes on frogs prevent morbidity and mortality caused by a lethal skin the activity of cationic antimicrobial peptides, The Journal of Peptide Research fungus, The ISME Journal 3 (2009) 818e824. 52 (1998) 81e88. [22] J.M. Conlon, Structural diversity and species distribution of host-defense [15] J. Lehmann, M. Retz, S.S. Sidhu, H. Suttmann, M. Sell, F. Paulsen, J. Harder, peptides in frog skin secretions, Cellular and Molecular Life Sciences (2011) G. Unteregger, M. Stockle, Antitumor activity of the antimicrobial peptide 1e13. magainin II against bladder cancer cell lines, European Urology 50 (2006) [23] H.G. Boman, Innate immunity and the normal microﬂora, Immunological 141e147. Reviews 173 (2000) 5e16.