Extremely Abundant Antimicrobial Peptides Existed in the Skins of

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Extremely Abundant Antimicrobial Peptides Existed in the Skins of Nine Kinds of Chinese Odorous Frogs Xinwang Yang,†,‡ Wen-Hui Lee,*,† and Yun Zhang*,† †

Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Kunming, Yunnan 650223, China ‡ Graduate School of the Chinese Academy of Sciences, Beijing 100049, China

bS Supporting Information ABSTRACT:

Peptide agents are regarded as hopeful candidates to solve life-threatening resistance of pathogenic microorganisms to classic antibiotics due to their unique action mechanisms. Peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) from amphibian skin secretions can provide a large amount of structurefunctional information to design peptide antibiotics with therapeutic potential. In the present study, we identified a large number of AMPs from the skins of nine kinds of Chinese odorous frogs. Eighty AMPs were purified from three different odorous frogs and confirmed by peptidomic analysis. Our results indicated that post-translational modification of AMPs rarely happened in odorous frogs. cDNAs encoding precursors of 728 AMPs, including all the precursors of the confirmed 80 native peptides, were cloned from the constructed AMP cDNA libraries of nine Chinese odorous frogs. On the basis of the sequence similarity of deduced mature peptides, these 728 AMPs were grouped into 97 different families in which 71 novel families were identified. Out of these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species. Our results revealed that identical AMPs were widely distributed in odorous frogs; 49 presently identified AMPs could find their identical molecules in different amphibian species. Purified peptides showed strong antimicrobial activities against 4 tested microbe strains. Twenty-three deduced peptides were synthesized and their bioactivities, including antimicrobial, antioxidant, hemolytic, immunomodulatory and insulin-releasing activities, were evaluated. Our findings demonstrate the extreme diversity of AMPs in amphibian skins and provide plenty of templates to develop novel peptide antibiotics. KEYWORDS: AMPs, extreme diversity, odorous frogs

’ INTRODUCTION Bacterial resistance to current antibiotic drugs is a threat to human beings all over the world. There is a considerable interest in the development of antimicrobial agents as a novel therapeutic approach to treat infections.1 Antimicrobial peptides (AMPs) are considered to be excellent templates for the design of novel antibiotics, owing to their unique mechanism that differs from the conventional clinical drugs.2,3 AMPs are distributed widely in nature and have been characterized from many different animal species, including invertebrates, fish, amphibians, birds and mammals.4 Amphibian skins, which are exposed directly to various survival conditions and can secret a remarkable array of AMPs, have aroused great attention in the past decades.5 Up to now, many investigations have been conducted on the AMPs from amphibian skins. However, most of them were focused on one or only a few AMPs from a specific amphibian species. With the genomic and peptidomic methodology achievements, papers dealing with the whole AMPs from specific amphibian species provide much information on the nature of r 2011 American Chemical Society

amphibian AMPs. Two representative studies among them are from Odorrana grahami and Rana nigrovittata. In the skin of O. grahami, 372 cDNA sequences encoding 107 novel AMPs belonging to 30 different families were reported previously.6 Thirtyfour AMPs belonging to nine families were identified from R. nigrovittata.7 We also carried out systemic AMP work on Bombina maxima; 56 different AMP cDNAs were found from two constructed skin cDNA libraries of two individuals. Meanwhile, 7 gene structures of AMPs from B. maxima were characterized from skin or liver genomic DNA, and these AMP genes have been experiencing rapid diversification driven by Darwinian selection.8 More recently, a large number of AMPs existing in the brain of this species was reported.9 A considerable variety of AMPs has been characterized from the skins of amphibians, and these AMPs are grouped into Special Issue: Microbial and Plant Proteomics Received: August 15, 2011 Published: October 26, 2011 306

dx.doi.org/10.1021/pr200782u | J. Proteome Res. 2012, 11, 306–319

Journal of Proteome Research

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Figure 1. Photographs and collecting areas of nine kinds of odorous frogs. Collecting area for each frog species is indicated by the arrow.

O. livida, O. tiannanensis and O. macrotympana, were collected in different areas of China. Animal collection sites and the photographs of these nine odorous frogs were shown in Figure 1. Animal care and handling were conducted in accordance with the requirements of the Ethics Committee of Kunming Institute of Zoology, the Chinese Academy of Sciences.

distinct families by virtue of the structure characteristics, such as Brevinin-1 and -2, Esculentin-1 and -2, Nigrocin, Palustrin, Odorranain-A to -Z and Temporin et al. All of these AMPs share a common and highly conserved precursor structure, which provides the possibility to identify AMPs easily from an established AMP cDNA library, and this strategy was successfully demonstrated in our previous reports and by other works.8,1014 There are about 18 kinds of odorous frogs all over the world, but the properties of AMPs in odorous frog skins were still largely unknown except O. grahami.6 The exclusive resource advantage of 13 kinds of odorous frogs distributed in China facilitated us to carry out systemic investigations on the skin AMPs of nine kinds of Chinese native odorous frogs. Our results reveal the extreme diversity of AMPs in odorous frogs and provide plenty of templates to explore novel peptide antibiotics.

Collection of the Skin Secretions

The frogs of O. andersonii, O. rotodora, O. wuchuanensis, O. margaratae were stimulated by alternative current (6 V) for about 6 s and skin secretions were collected by washing frog bodies with 25 mM Tris-HCl buffer (pH 7.8). The collected solutions were centrifuged at 4000 rpm for 15 min and the supernatants were lyophilized and stored at 80 °C until use. The rest five odorous frogs did not experience this procedure due to limited samples. Peptides Purification

’ EXPERIMENTAL SECTION

The dissolved samples of lyophilized skin secretions were applied on a Sephadex G-75 (superfine, GE Healthcare) gel filtration column pre-equilibrated with 25 mM Tris-HCl buffer (pH 7.8) containing 0.1 M NaCl. Gel filtrations were performed

Animals and Ethics

Nine kinds of odorous frogs, namely O. andersonii, O. rotodora, O. wuchuanensis, O. margaratae, O. hejiangensis, O. schmackeri, 307

dx.doi.org/10.1021/pr200782u |J. Proteome Res. 2012, 11, 306–319


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peptides of frog AMPs in NCBI, in the sense direction, and 30 PCR primer CDS III as mentioned above were used in PCR reactions. The Advantage polymerase was used in the experiments. The PCR was performed as follows: 2 min at 94 °C and then 30 cycles of 10 s at 92 °C, 30 s at 50 °C, 40 s at 72 °C. The PCR products were recovered by DNA Gel Extraction Kit (Bioteke, China) and ligated into pMD19-T Vector (TaKaRa Biotechnology (Dalian) Co., Ltd., China). Finally, the PCR products were cloned into E. coli DH5α competent cells and a specific AMP cDNA library was constructed. Clones with insert >300bp from each specific AMP cDNA library were randomly chosen to carry out DNA sequencing on an Applied Biosystems DNA sequencer (ABI 3730XL, Foster City, CA).

with the same buffer at a flow rate of 0.3 mL/min for O. andersonii or 0.1 mL/min for the other 3 kinds of odorous frogs mentioned above. Fractions were collected every 10 min. The absorbance of each tube was monitored at 280 nm. Fractions with antimicrobial activity were collected and desalted by reverse phase HPLC (RPHPLC) on a C4 column (4.6 250 mm, Elite, China) then lyophilized. The lyophilized samples were dissolved in 20 mM NaAc-HAc buffer (pH 4.0) and applied on a Resource S column (1 mL, GE Healthcare) pre-equilibrated with the same buffer on € KTA explorer FPLC system. The elution was performed with a A linear gradient of the same buffer containing 1 M NaCl at a flow rate of 1 mL/min and monitored at 215 nm. The peaks showing antimicrobial activity were collected separately and then applied to a C18 RP-HPLC column (Hypersil BDS C18, 4.0 300 mm, Elite, China) pre-equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA) in water. The elution was achieved by a linear gradient of 0.1% (v/v) TFA in acetonitrile at a flow rate of 0.7 mL/min and monitored at 215 nm. Peaks with antimicrobial activity were collected and lyophilized for further analysis. A highly sensitive radial diffusion method was adopted to track the AMPs in these purification procedures. Peptides were quantified by UV absorbance at 215 and 220 nm using the following formula: concentration (mg/mL) = (A 215 nm A 225 nm) 0.144.15

Peptides Synthesis

All peptides used for bioactivity evaluation were synthesized by solid phase synthesis on an Applied Biosystems model 433A peptide synthesizer according to the standard protocols as previously reported.16 After cleavage and side-chain deprotection, the crude synthetic peptide was purified on a Vydac 218TP 510 C18 reverse phase-HPLC column (25 1 cm). Elution was performed at a flow rate of 2 mL/min by a linear gradient of acetonitrile in 0.1% TFA in water. Identity of peptides were confirmed by automated Edman degradation with a protein sequencer and mass spectrometry analysis. The purity of the synthetic peptides used for evaluating biological activities was higher than 95%.

Structure Determination of Peptides

The average molecular weight and the purity of each fraction with antimicrobial activity from the HPLC purification procedure was determined on an AXIMA-CFRTM plus MALDI-TOF mass spectrometer (Shimadzu/Kratos, Manchester, U.K.) in a linear mode with α-cyano-4-hydrorycinnamic acid as the matrix. All procedures were carried out according to manufacturer’s standard protocols, and the data were analyzed by the software package provided by the manufacturer. The complete primary sequences of peptides were determined by Edman degradation method on a PPSQ-31A protein sequencer (Shimadzu, Japan) according to the standard GFD or PVDF protocols instructed by manufacturer.

Bioinformatic Analysis

The complete sequences of each mature peptide were analyzed through the protein blast item of Basic Local Alignment Search Tool in NCBI (http://www.ncbi.nlm.nih.gov/). If the sequences showed high structure similarity with known AMP families, the molecules were therefore designed into the specific AMP families and named according to traditional method. If not, each peptide was classified as novel family and named by the genus name of the amphibian species. All the obtained cDNA sequences were aligned using the CLUSTAL W program software package (www.ebi.ac.uk/clustalw).

Construction of Skin AMP cDNA Libraries

Antimicrobial Activity Assay

Each skin AMP cDNA library was constructed from individual skins of the nine odorous frog species. Briefly, total RNA was extracted by TRIzol Reagent (Invitrogen, Carlsbad, CA). Treatment of the skin sample was as follows: one frog was picked out and washed with deionized water and sacrificed. The skin was immediately stripped, cut into pieces and ground in liquid nitrogen. mRNAs were purified by an Absolutely mRNA Purification Kit according to the standard protocols provided (Stratagene, Canada). cDNA was synthesized by SMART techniques using a SMART cDNA Library Construction Kit (Clontech, Canada). The first strand of cDNA was synthesized by using 30 SMART CDS III/30 PCR primer 50 -ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N1N-30 (N = A, G, C, or T; N1 = A, G, or C), SMART IV oligonucleotide 50 -AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-30 and SMART MMLV Reverse Transcriptase (Clontech, Canada). The second strand was amplified by using Advantage polymerase, with 50 PCR primer (50 -AAGCAGTGGTATCAACGCAGAGT-30 ) and 30 PCR primer, CDS III (50 -ATTCTAGAGGCCGAGGCGGCCGACATG-30 ) according to the protocols of the kit. The cDNAs synthesized by SMART techniques were used as templates for high stringent PCR to screen the cDNAs encoding AMPs precursors. Two primers, (50 -CCAAA(G/C)ATGTTCACC(T/A)TGAAGAAA-30 ) designed according to the signal

A high sensitive radial diffusion method was performed according to our previously reported method.17 Briefly, Grampositive bacterial strains Staphylococcus aureus (ATCC 25923), Gram-negative bacterial strains Escherichia coli (ATCC 25922), Bacillus pyocyaneus (CMCCB 10104), and fungal strains Candida albicans (ATCC 2002), were obtained from Kunming Medical College. Microbes were grown in LuriaBertani (LB) broth to an OD600 of 0.8. A 10 μL aliquot of the bacteria was then taken and added to 10 mL of fresh LB broth with 1% Type I agarose (Sigma-Aldrich, St Louis, MO) and poured over on a 90 mm Petri dish. After the agar hardened, small hole was dug and a 7 μL aliquot of the sample was added into the hole of the agar and completely dried before being incubated overnight at 37 °C. If an examined sample contained antimicrobial activity, a clear zone formed on the surface of the agar representing inhibition of bacterial growth. Minimal inhibitory concentration (MIC) was determined in liquid LB medium at pH 7.0 by conventional serial dilution method in 96-well microtiter plates. The MIC value, at which no obvious bacterial growth occurred, was recorded by measuring the absorbance at 600 nm after incubating at 37 °C for 1618 h. Antioxidant activity assay

Free radical scavenging test was adopted to evaluate the antioxidant activity of samples according to the reported method 308



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incubating 2.8 mM potassium persulfate (Sigma-Aldrich, St Louis, MO) with 7 mM ABTS in water, allowing at least 6 h for the reaction in the dark and used immediately. The stock solution was diluted 125 times with ddH2O. Samples dissolved in water were added and solvents of the same volume were used as negative controls, BHT (Sigma-Aldrich, St Louis, MO) dissolved in methanol was used as positive control. The reaction was kept from light and last for 10 min, the absorbance value at 415 nm was read and its decrease implied the antioxidant activity of samples. The antioxidant activity (free radical scavenging rate, %) was evaluated by the formula (Ablank Asample) 100/Ablank. Hemolytic Assay

Hemolytic assay was tested with health human red blood cells in liquid medium as reported.1 Briefly, serial dilutions of the peptides were incubated with washed human red blood cells at 37 °C for 30 min, the cells were centrifuged and the absorbance of the supernatant was measured at 540 nm. Maximum hemolysis was determined by adding 1% Triton X-100 to the cell samples. Immunomodulatory Assay

Two cytokines, TNF-α and IL-8, were used to evaluate the immunomodulatory activity of samples as reported previously.19 For TNF-α assay, the human monocyte-like cell line THP-1 (ATCC TIB-202) was grown in RPMI 1640 medium containing 10% fetal calf serum and 1% L-glutamine (Gibco, Carlsbad, CA). THP-1 cells were seeded in 96-well tissue culture plates at a concentration of 2 105 and differentiated into adherent macrophage-like cells by the addition of phorbol myristate acetate, incubated at 37 °C in 5% CO2 for 3 days and then incubated in triplicate for 6 h either in the presence of medium alone or with Escherichia coli O111:B4 LPS (Sigma-Aldrich, St Louis, MO) or a combination of LPS and peptide in medium. Supernatants were collected to measure the production of TNFα by using commercially prepared ELISA kit under the guidance of the manufacture’s protocol. For IL-8 assay, the human bronchial epithelial cell line BEAS2B (ATCC CRL-9609) was cultured in LHC-9 medium (Invitrogen, Carlsbad, CA). Cells were seeded in 96-well tissue culture plates at a concentration of 5 105 cells/ml and incubated at 37 °C in 5% CO2 for 48 h. Cells were then incubated 8 h to measure IL-8 production either in the presence of medium alone or peptides in medium. Supernatants were collected to measure the production of IL-8 by using commercially prepared ELISA plates under the guidance of the manufacture’s protocol. Figure 2. Purification of AMPs from the skin secretions of O. andersonii. (A) Sephadex G-75 gel filtration. The skin secretions was applied on a Sephadex G-75 column (2.6 100 cm) pre-equilibrated with 25 mM Tris-HCl buffer (pH 7.8), containing 0.1 M NaCl. Peaks with antimicrobial activity were indicated. (B) Cationic exchange on Resource S column. The desalted samples from Sephadex G-75 gel filtration were dissolved in 20 mM NaAc-HAc buffer (pH 4.0) and applied on a Resource S column. The elution was performed with a linear gradient of the same buffer containing 1 M NaCl at a flow rate of 1 mL/min and monitored at 215 nm. Eleven peaks with antimicrobial activity were numbered as indicated. (C1C11) Purification by RP-HPLC. The 11 peaks with antimicrobial activity from cationic exchange were respectively applied to a C18 RP-HPLC column pre-equilibrated with 0.1% (v/v) TFA in water, the elution was achieved by a linear gradient of 0.1% (v/v) TFA in ACN at a flow rate of 0.7 mL/min and monitored at 215 nm.

Insulin-releasing Assay

Insulin-releasing activity of different peptides was carried out on cultured INS-1 cells as reported.20 Briefly, INS-1 cell line was cultured in DMEM medium containing 10% fetal calf serum and 1% L-glutamine (Gibco, Carlsbad, CA). Cells were seeded in 96well tissue culture plates at a concentration of 3 105 cells/mL and incubated at 37 °C in 5% CO2 for 24 h. Cells were then incubated in triplicate for 6 h either in the presence of medium alone or peptides. Supernatants were collected to measure the production of insulin by using commercially prepared ELISA under the guidance of the manufacture’s protocol.

’ RESULTS Purification of AMPs

with some modifications.18 Briefly, a stock solution of ABTS radical (Sigma-Aldrich, St Louis, MO) was prepared by

Six main fractions were obtained from the skin secretions of O. andersonii through Sephadex G-75, the fractions with 309



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antimicrobial activity were mainly existed in tube 100140, suggesting that antimicrobial activity of O. andersonii largely came from low molecular weight peptides (Figure 2A). The collected antimicrobial activity fractions from gel filtration were further purified by Resource S column, at least 27 peaks were found in the ion-exchange purification step. However, only 11 peaks showed antimicrobial activity and numbered accordingly (Figure 2B). Each antimicrobial activity peak from Resource S column was separately purified by RP-HPLC on a C18 column (Figure 2C1C11). Finally, 45 fractions with antimicrobial activity were separated, which suggested that at least 45 AMPs exist in the skin secretions of O. andersonii. Owing to sample limitations, we could not get enough skin secretions from O. rodotoda, O. wuchuanensis and O. margaratae. Thus, a small gel filtration column was used to the purification procedures and the rest purification steps were the same as those of O. andersonii. Finally, 42, 75, and 36 HPLC peaks with antimicrobial activity were purified from O. rodotoda, O. wuchuanensis and O. margaratae, respectively (Figures S1S3, Supporting Information).

Odorranain-F (1 peptide) as illustrated in Figure S93 (Supporting Information). Most of the identified native peptides were grouped into known families, such as Brevinin-1 and -2, Esculentin-1 and -2, Nigrocin, Odorranian-F. Only 3 novel families were recognized in the skin secretions of O. andersonii but none in O. rotodora and O. wuchuanensis through the present purification procedure and peptidomic analysis, which indicated that the AMPs in the skin of odorous frogs by conventional purification methods mainly existed in the above-mentioned known families. Thus, structure determination of the rest fractions from both O. wuchuanensis and O. margaratae was aborted. cDNA Clones

A great deal of AMP precursors were identified from the constructed AMP skin cDNA libraries of these nine kinds of odorous frogs. As listed in Table 2, at least 3800 different cDNA sequences were determined. All the precursors of the peptidomic confirmed native 80 AMPs were cloned. However, the precursors of partial N-terminal sequence determined Rotodorin-G members could not be found in the present study. This might be caused by comparatively lower DNA sequencing coverage on the constructed O. rodotoda AMP cDNA library. The cDNA sequences isolated from individual frog skins are universally translated into peptides. Many peptides that could not be purified by conventional biochemistry isolation methods are deduced from cDNA sequences. In total, 728 different AMPs were identified from nine Chinese odorous frogs. These peptides were classified into 97 families that were composed of 26 known families and 71 novel families. Among these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species (Figures S91S99, Supporting Information). To view the structure properties of different AMP families, only one precursor from each family was selected and aligned. All of these AMP precursors shared a highly conserved motif, which were divided into N-terminal hydrophobic signal peptide, an acidic segment and mature peptides at C-terminal. The most common typical enzyme cleavage sites composed of “Lys-Arg” followed by “Val-Arg” and “Asn-Arg” existed in the deduced precursors except Rotodorin-G (Figure 3). Surprisingly, as many as 49 identical AMPs belonging to 12 different AMP families crossly distributed in the skins of present used odorous frogs. These 49 identical AMPs were divided into 21 different AMP molecules. Thus, 28 repeated AMP molecules existed at least 2 different frog species of present studied nine odorous frogs (Table 3). Moreover, 28 AMPs characterized in the present study were identical to the previously reported AMPs in other amphibian species. Interestingly, Odorranain-U-OA1 was identical to the Nigronaian-E identified from R. nigrovittata. The rest identical AMPs were mainly distributed in odorous frogs, as many as 25 identical AMPs of the present study were also distributed in O. grahami (Table S3, Supporting Information). Taken together, 49 identical AMPs could be found in different amphibian species. To our knowledge, this is the first time to reveal that so many identical AMPs could exist in different frogs.

Structure Determination of Purified AMPs

The mass spectrometry graphs of 45 fractions with antimicrobial activity from the skin secretions of O. andersonii were shown in Figures S4S48 (Supporting Information). Among these 45 samples, the amino acid sequences of 35 peaks with good purity were separately determined and their complete amino acid sequences were shown in Figure S91 (Supporting Information). The rest 10 peaks, including peak 5, 6 in Figure 2C2, Peak 1 in Figure 2C3, Peak 4 in Figure 2C5, Peak 3, 4, 6, 11 in Figure 2C10, Peak 3, 6 in Figure 2C11, were not pure and not suitable for primary structure determination. The determined molecular weights by mass spectrometry of these 35 AMPs matched well with their theoretical molecular weights (Table 1). By BLASTp search in NCBI and by virtue of the structure similarity, these AMPs were grouped into 11 antimicrobial families, including 8 previously reported families, Brevinin-1 (3 peptides), Brevinin-2 (8 peptides), Esculentin-1 (6 peptides), Esculentin-2 (3 peptides), Nigrocin (3 peptides), Odorranain-A (1 peptides), Odorranain-F (4 peptides) and Odorranain-J (2 peptides). Meanwhile, 3 families were found to be novel, which were named as Andersonin-W (2 peptides), Andersonin-X (1 peptide) and Andersonin-Y (2 peptides). The 42 mass spectrometry graphs in Figures S49S90 (Supporting Information) revealed the observed molecular weights and the purity of fractions isolated from the skin secretion of O. rodotoda. Four fractions, including peak 1 in Figure S1C8, peak 1, 2 in Figure S1C11, peak 3 in Figure S1C13, were not of ideal purity and the amino acid sequences of the rest 38 fractions were determined. However, peak 1 and 2 in Figure S1C12 only gave partial N-terminal sequences of 19 amino acid residues. These two samples shared identical partial N-terminal sequences and showed no obvious structure similarity with known AMPs and named as Rotodorin-G. The rest full sequence confirmed 36 peptides were classified into 6 families, including Brevinin-1 (11 peptides), Brevinin-2 (10 peptides), Esculentin-1 (5 peptides), Esculentin-2 (6 peptides), Nigrocin (3 peptides) and Odorranain-F (1 peptide) (Figure S92, Supporting Information). The observed molecular weights of the peptides were consistent with the theoretical molecular weight (Table S1, Supporting Information). Furthermore, nine fractions from the skin secretions of O. wuchuanensis were confirmed by primary structure and mass spectrometry determinations (Table S2). These nine peptides were identified as Brevinin-2 (3 peptides), Nigrocin (5 peptides) and

Primary Structure Diversity of AMPs

Linear Peptides. Linear peptides were very rich in the skins of these nine kinds of odorous frogs. These linear peptides were divided into true linear peptides with no cysteine residue and linear peptides with one free cysteine. The length of linear peptides with no cysteine differed remarkably, AMPs composed of 11 to 56 amino acid residues were characterized 310



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Table 1. Peptidomic Analysis of AMPs Purified from the Skin Secretions of O. andersonii TMWa Da AMPs name

HPLC position in Figure 2

reduced

oxidized

OMWb Da

difference Da

figure

Brevinin-2-OA1

Peak 1 in C1

3370.00

3368.00

3368.50

0.50

S4

Brevinin-2-OA2

Peak 1 in C2

3370.00

3368.00

3368.20

0.20

S5

Brevinin-2-OA3

Peak 2 in C2

3381.07

3379.07

3379.20

0.13

S6

Brevinin-2-OA6

Peak 3 in C2

3290.84

3288.84

3292.10

3.26

S7

Brevinin-2-OA7

Peak 4 in C2

3292.85

3290.85

3291.10

0.25

Peak 5 in C2

S8 S9

Peak 6 in C2

S10

Nigrocin-OA1

Peak 7 in C2 Peak 1 in C3

1973.6

1971.46

1972.50

0.96

S11 S12

Brevinin-2-OA8

Peak 2 in C3

3291.78

3289.78

3290.00

0.22

S13

Brevinin-2-OA4

Peak 1 in C4

3368.91

3366.91

3367.10

0.19

S14

Esculentin-1-OA1

Peak 2 in C4

4859.96

4857.96

4858.70

0.74

S15

Odorranain-F-OA1

Peak 1 in C5

3113.72

3111.72

3111.70

0.02

S16

Nigrocin-OA2

Peak 2 in C5

2154.67

2152.67

2152.90

0.23

S17

Brevinin-2-OA5

Peak 3 in C5

3295.82

3293.82

3293.10

0.72

S18

Odorranain-F-OA2


3142.80

3140.80

3141.10

0.3

S19 S20

Odorranain-F-OA3

Peak 2 in C6

3111.64

3109.64

3110.10

0.46

S21

Brevinin-1-OA1

Peak 3 in C6

2586.14

2584.14

2583.50

0.64

S22

Odorranain-F-OA4

Peak 1 in C7

3142.80

3140.80

3140.80

0.00

S23

Brevinin-1-OA2

Peak 2 in C7

2584.12

2582.12

2581.80

0.32

S24

Andersonin-W1

Peak 1 in C8

1953.35

2951.35

1951.60

0.25

S25

Andersonin-W2

Peak 1 in C9

1951.38

1949.38

1949.30

0.08

S26

Esculentin-2-OA1 Andersonin-X1


3821.65

3819.65 2121.50

3819.10 2121.30

0.55 0.20

S27 S28

Andersonin-Y1

Peak 2 in C10

2156.71

2156.70

0.01

Peak 3 in C10 Peak 4 in C10 Brevinin-1-OA19

Peak 5 in C10

S29 S30 S31

4544.55

4542.55

4542.80

0.25

Peak 6 in C10

S32 S33

Esculentin-1-OA2

Peak 7 in C10

4879.86

4877.86

4878.30

0.44

S34

Esculentin-1-OA3 Esculentin-1-OA4


4864.85 4878.87

4862.85 4876.87

4863.10 4875.30

0.25 1.57

S35 S36

Peak 10 in C10

3838.64

3836.64

3836.40

0.24

Esculentin-2-OA2

Peak 11 in C10 Odorranain-J-OA1

Peak 12 in C10

Esculentin-2-OA3

Peak 13 in C10

S37 S38

3821.65

2095.48

2096.00

0.52

S39

3819.65

3820.40

0.75

S40

Esculentin-1-OA6

Peak 1 in C11

2718.24

2718.40

0.26

S41

Andersonin-Y2

Peak 2 in C11

1594.96

1596.10

1.14

S42

Odorranain-A-OA1


1755.98

1753.98

1753.60

0.38

S43 S44

Peak 5 in C11

4863.86

4861.86

4861.40

0.46

Esculentin-1-OA5

Peak 6 in C11 Nigrocin-OA3

Peak 7 in C11

Odorranain-J-OA2

Peak 8 in C11

S45 S46

2090.66

2088.66

2088.80

0.14

S47

2096.43

2095.70

0.63

S48

a

TMW, theoretical average molecular weight calculated at PeptideMass.30 b OMW, observed average molecular weight achieved by mass-spectrometry analysis.

“X1-C-Xn”. Moreover, many other motifs that described as “X3-C-Xn”, “X4-C-Xn”, “X6-C-Xn”, “X7-C-Xn”, “X8-C-Xn”, “X9-C-Xn”, “X10C-Xn”, “X11-C-Xn”, “X12-C-Xn”, “X14-C-Xn”, “X19-C-Xn”, “X20C-Xn”, “X21-C-Xn”, “X22-C-Xn”, “X23-C-Xn”, “X29-C-Xn” and “X31-C-Xn” were represented by families identified in this research.

(Figure 4A). Twenty-three families were found to be true linear peptides with one free cysteine. The cysteine position in these peptides varied considerably (Figure 4B). In the case of AndersoninT1, the free cysteine was located in the second position from N-terminus to C-terminus, which represented the motif of 311



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Table 2. Summary of Mature Peptides Identified in the Skins of Nine Odorous Frog Species mature peptides

families figure

cDNA sequences

total

confirmeda

total

reported

O. andersonii

700

197

35

47

22

25

S91

O. rotodora

650

97

34

20

11

7 + 2b

S92

O. wuchuanensis

350

60

9

20

12

6 + 2c

S93

O. margaratae

350

72

21

14

4 + 3d

S94

O. schmackeri

350

57

19

10

6 + 3e

S95

O. hejiangensis

350

77

29

16

9 + 4f

S96

O. tiannanensis

350

40

17

11

3 + 3g

S97

O. livida O. macrotympana

350 350

64 64

20 17

12 9

5 + 3h 6 + 2i

S98 S99

species

new

a Confirmed by Peptidomics analysis. b 7 unique families termed as Rotodorin-A to -G + 2 families, Andersonin-O and -W. c 6 unique families termed as Wuchuanin-A to -F + 2 families, Andersonin-W and Rotodorin-F. d 4 unique families termed as Margaratain-A to -D + 3 families, Andersonin-W, Rotodorin-C and -F. e 6 unique families termed as Schmacherin-A to -F + 3 families, Andersonin-W and Rotodorin-C and -F. f 9 unique families termed as Hejiangin-A to -I + 4 families, Andersonin-O, and -W, Rotodorin-C and -F. g 3 unique families termed as Tiannanin-A to -C + 3 families, Andersonin-I, -O, -W. h 5 unique families termed as Lividin-A to -F + 3 families, Andersonin-K, -O, and Rotodorin-F. i 6 unique families termed as Macrotympanain-A to -F + 2 families, Andersonin-W and Tiannanin -A.

Cyclic Peptides. Most of AMPs from rained frogs had a disulfide bridge at their C-terminus. In the skin of these nine odorous frog species, 36 families were found to possess disulfide bond located in the C-terminal end and the intramolecularlly disulfide-bridged AMPs accounted up to 84.15% of all the peptides identified in this research. It was interesting that the number of amino acid residues contributed to the cyclic motif showed extreme diversity as shown Figure 4C. The motif of cyclic heptapeptides “C-X5-C”, represented by Brevinin-1,-2, Esculentin-1,-2, Nigrocin, Odorranain-F, -G, Pulustrin, Schmackerin-C, Andersonin-F, was termed as “Rana box” and considered to play very import role in the innate immune system of frogs. In this report, two families, including Schmackerin-C, Andersonin-F increased the diversities of this cyclic motif. Moreover, other “Rana box” domains were also observed. All of these cyclic motifs, including “C-X2-C”, “C-X3-C”, “C-X4-C”, “C-X6-C”, “CX7-C”, “C-X8-C”, “C-X9-C”, “C-X10-C”, “C-X11-C”, “C-X12C”, “C-X13-C”, “C-X14-C”, “C-X15-C”, which differed in size from the conserved disulfide-bridged hepapeptides segment, were rarely found in the skin AMPs of rained frogs. Among these cyclic motifs, the motif of 1113 residues was first reported in O. grahami. The rest motifs were first described in the skins of odorous frogs. Peptides with Three Cysteine Residues. In this research, several AMPs with three cysteine residues were characterized as displayed in Figure 4D. It was very difficult to judge whether the three cysteines formed disulfide-bridge or not. Moreover, if they really did, the motif of disulfide bond was still not easy to identify. Anyhow, the AMPs with three cysteine residues were rarely reported previously and it might be interesting to characterize their structure and biological activities. Neutral and Anionic Peptides. In this research, nine peptides with this special feature were found, including Andersonin-H3 (0 net charge), Andersonin-I 1,2 (0 net charge), Andersonin-N (2 net charge), Andersonin-R (6 net charge), Andersonin-S (6 net charge), Wuchuanin-D (1 net charge), Schmackerin-D (1 net charge), Hejiangin-C (1 net charge), MacrotympanainA (1 net charge) (Figure 4E). The existence of anionic peptides might reflect a compensation to basic AMPs against microbe invasion by amphibians.21,22

Antimicrobial activity

Purified peptides exhibited strong antimicrobial activities against 4 tested microbes including Gram-positive, Gram-negative and fungal strains. The determined MIC values of most of the purified peptides were below 10 μg/mL. Notably, Esculentin-1OA1 and OA2 showed the strongest antimicrobial activities against 4 tested microbes, MIC values of less than 2.5 μg/mL were determined for all the tested strains by both molecules. These 2 molecules might be served as good templates to design novel antimicrobial drugs. On the contrary, the synthesized 23 peptides showed weak antimicrobial activities. Only AndersoninC1, Hejiangin-A1 and Schmackerin-C1 showed killing effects against the tested strains (Table 4). Hemolytic Activity

Only Andersonin-C1 showed obvious hemolytic activity against human red blood cells at the concentration of 50 μg/mL. All of the rest synthesized peptides showed no hemolytic activity under the test conditions (Table 4). Antioxidant Activity

Most of the 23 synthesized peptides showed obvious antioxidant activity at the concentration of 50 μg/mL. However, Andersonin-D1, -Q1 and -S1 showed no detectable antioxidant activity. Odorranain-A-OA11, Hejiangin-A1, MacrotympanainA1 and Wuchuanin-A1, which could scavenge almost all the ABTS+, represented the most potential antioxidant activity among the tested samples (Figure 5A). Immunomodulatory Activity

At the concentration of 50 μg/mL, all the synthesized peptides showed no obvious cytotoxic activity against the tested cell lines (data not shown). Most of the synthesized peptides did not have obvious effects on the TNF-α production induced by LPS. Only two samples, Macrotympanain-A1 and Margaratain-B1 showed potential inhibitory effects on the TNF-α production induced by LPS. Meanwhile, Andersonin-D1 could weakly increase the release of TNF-α induced by LPS (Figure 5B). The effects of synthesized peptides on the IL-8 production were quite different. Odorranain-A-OA11, Andersonin-G1, Margaratain-A1 and B1 could obviously increase the release of IL-8 at the concentration of 50 μg/mL. However, under the same

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Figure 3. Alignment of representative AMP precursors from each family. One precursor of each family was picked out and aligned. Rotodorin-G family with only partial N-terminal amino acid sequence was also listed.

conditions, Odorranian-A-OA12, Andersonin-H3, -N1, -Q1, -R1, -S1, Lividin-D1, Schmackerin-C1, Tiannanin-A1 and Wuchuanin-C1, -D1, F1 all showed obvious inhibitory effect on the production

of IL-8. Among them, Andersonin-Q1 showed the highest inhibitory effect and production of IL-8 was almost undetectable. The rest peptides, including Andersonin-C1, -D1, Hejiangin-A1, 313



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Table 3. Novel AMPs Distributed in Different Species of Nine Odorous Frogs name of families

name of the AMPs

1) Andersonin-O

1) Andersonin-O-OMar1=Andersonin-O-OR1

2) Andersonin-W

2) Andersonin-W-OS1=Andersonin-W-2

3) Brevinin-1

3) Brevinin-1-OMar5=Brevinin-1-OW1=Brevinin-1-OT11=Brevinin-1-OMac21 4) Brevinin-1-OL2=Brevinin-1-OR2

4) Brevinin-2

5) Brevinin-2-OMar2=Brevinin-2-OA15 6) Brevinin-2-OL8=Brevinin-2-OR7

5) Esculentin-1

7) Esculentin-1-OW2=Esculentin-1-OA13

6) Esculentin-2 7) Nigrocin

8) Esculentin-2-OW2=Esculentin-2-OA1 9) Nigrocin-OMar1=Nigrocin-OA10 10) Nigrocin-OMar21=Nigrocin-OA17 11) Nigrocin-OMar3=Nigrocin-OA1 12) NigrocinOH1=Nigrocin-OS5 13) NigrocinOH3=Nigrocin-OMar10=Nigrocin-OL6 14) Nigrocin-OL1=Nigrocin-OR10 15) Nigrocin-OL3=Nigrocin-OR1

8) Odorranain-G

16) Nigrocin-OL16=Nigrocin-OR12 17) Odorranain-GOH1=Odorranain-G-OW1

9) Odorranain-O

18) Odorranain-O-OS1=Odorranain-O-OMar1=Odorranain-OOH1=Odorranain-O-OL1

10) Odorranain-T

19) Odorranain-T-OL1=Odorranain-T-OR2

11) Odorranain-U

20) Odorranain-UOH1=Odorranain-U-OMar1=Odorranain-U-OMac2

12) Rotodorin-F

21) Rotodorin-FOH1=Rotodorin-F1=Rotodorin-F-OL1

-E1, -F1, Macrotympanain-A1 and Wuchuanin-A1, showed no obvious effects on the production of IL-8 (Figure 5C).

determinations and mass spectrometry assays from the skin secretions of O. andersonii, O. rotodora and O. wuchuanensis. Peptidomic analysis of purified 80 AMPs revealed that the posttranslational modifications rarely happened in odorous frogs and this result greatly helped us to characterize AMPs by cDNA cloning techniques. By molecular cloning methods, 728 cDNAs coding for different AMPs (including purified and confirmed 80 AMPs) were identified from the skins of nine kinds of odorous frogs after sequencing 3800 AMP DNA sequences (Table 2). According to the structure similarity of the deduced mature AMPs, 97 AMP families were characterized. Most of the peptides were classified into 26 known families, including Brevini-1 and -2, Esculentin-1 and -2, Nigrocin, Palustrin, Temporin, Odorranian-A to -W et al. Seventy-one families were novel and named according to the genus names of these odorous frog species (Figures S91S99, Supporting Information). Among these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species (Table S3, Supporting Information). Although extensive studies have been conducted on the AMPs from amphibian skins, only about 300 AMPs were identified from more than 20 rained frogs.9 This indicated that only 15 AMPs were identified from 1 frog species on an average. In this research, 728 AMPs were identified from only nine odorous frogs suggested that approximately 80 AMPs could be obtained from one odorous frog. Moreover, if the identification of peptides purified from O. wuchuanensis and O. margaratae were conducted deeply, coupled with the peptidomic analysis of the rest 5 frog skin secretions, more AMPs would be certainly identified. Peptidomic analysis combined with genomic approaches proved to be a powerful tool to identify the AMPs in amphibians.8,24 Our research, together with the AMP work on O. grahami,6 implied odorous frogs might represent the most extreme AMP diversity in nature. The primary structures of deduced mature AMPs also showed great variations. Based on the structure characteristics, these

Insulin-releasing Activity

Nine synthesized peptides, Andersonin-C1, -D1, -G1, -N1, -Q1, Hejiangin-A1, Tiannanin-A1, Wuchuanin-C1 and -E1, showed obvious insulin-stimulating activity at the concentration of 50 μg/mL. However, other 14 peptides showed no obvious effects on the release of insulin (Figure 5D).

’ DISCUSSION Odorous frogs were characterized by the skin secretions with unpleasant smells when they were stimulated. Few systematic researches on the AMPs in their skin secretions were conducted. One remarkably laborious work was conducted on the skin of O. grahami. One-hundred seven AMPs belonging to 30 divergent families were reported recently.6 The work on O. grahami inspires us to explore the diversity of AMPs in odorous frogs. Out of the nine odorous frogs used in the present study, there were some investigations concerning AMPs from three odorous frogs reported previously. Three members of Nigrocin, three members of Brevin-1 and three of Brevini-2 AMPs were previously identified in the skin secretions of O. schmacheri.10,13,14 One Nigrocin member was found in the skin of O. hejiangensis.10 Two Nigrocin members, one Brevinin-1 member, two Brevinin-2 members and one Esculentin-2 member were identified from the skin of O. livida.10,23 The AMPs from O. andersonii, O. rotodora, O. wuchuanensis, O. margaratae, O. tiannanensis and O. macrotympana were never reported before. In total, 198 HPLC peaks with antimicrobial activity were purified from the skin secretions of O. andersonii, O. rotodora, O. wuchuanensis and O. margaratae. So many AMP fractions isolated from 4 specific amphibian species implied that the extreme AMP diversity existed in odorous frogs. Out of these 198 HPLC peaks, 80 AMPs were confirmed by a combination of amino acid 314



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Figure 4. Structure diversity of the peptides identified in the skin of nine odorous frog species. (A) Linear peptides without cysteine. (B) Linear peptides with one free cysteine. (C) Peptides with disulfide-bridged motifs. (D) Peptides with three cysteine residues. (E) Neutral and anionic peptides.

AMPs could be classified as: (1) linear peptides, which composed of linear peptides with no cysteine residue and linear peptides with one free cysteine; (2) cyclic peptides; (3) peptides with three cysteine residues and (4) neutral and anionic peptides (Figure 4). Up to now, it is generally accepted that the diversity of amphibian AMPs made it rarely possible to find identical AMPs in two different species, even in those closely related.25 However, our results demonstrated that 21 identical AMP molecules represented by 28 repeated molecules could distribute in at least 2 different frog species of present studied nine odorous frogs (Table 4). Meanwhile, 28 present identical AMPs represented by 38 repeated molecules were found to be existed in other amphibian species (Table S3, Supporting Information). It was not likely to ascribe the cross-pollution occurred in the experimental procedure to this phenomenon. The chances of cross-contamination in our experiments are very little. First, the appearances of these frogs are obviously distinct from each other as shown in Figure 1 and the frogs were carefully chosen. Second, the construction of skin cDNA library for each frog was performed at different time and places, the cDNA clones were stored and sequenced at

different times. Identical biological active peptides found in skins of different amphibian species have already reported.13 Widely distributing of identical AMPs in different odorous species suggested that these molecules might play an important role in odorous frogs and their biological activities need to be further investigated. All the native 80 peptides showed strong direct killing effects on Gram-positive, Gram-negative and fungal strains. Esculentin1-OA1 and -OA2 showed the strongest antimicrobial activities and might be served as good templates to design novel antimicrobial drugs. On the contrary, the 23 synthesized peptides showed weak killing effects against the tested strains. This might be caused by the insensitivity of the synthesized peptides to the tested strains used or the synthesized peptides having other unknown biological activities, since the deduced peptides synthesized were mainly according to their structure features. AMPs having antioxidant, hemolytic, immunomodulatory and insulinreleasing activities have already recognized. Thus, these biological activities were chosen to evaluate the possible functions of the synthesized peptides. 315



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Table 4. Antimicrobial and Hemolytic Activity of Purified and Synthesized Peptides antimicrobial activity (MIC μg/mL) samples

E. coli

C. albicans

S. aureus

B. pyocyaneus

Brevinin-1-OA1

6.2

24.8

12.4

Brevinin-1-OA2

10.4

10.4

20.8

5.2

Brevinin-1-OA12

6.6

3.3

6.6

13.2

Brevinin-1-OR1

5.3

10.6

10.6

42.4

Brevinin-1-OR2

5.8

5.8

11.6

11.6

Brevinin-1-OR3

9.1

18.2

18.2

18.2

Brevinin-1-OR4

8.6

8.6

8.6

8.6

Brevinin-1-OR5 Brevinin-1-OR6

15.2 11.1

7.6 11.1

15.2 22.2

30.4 22.2

12.4

Brevinin-1-OR7

5.7

11.4

11.4

5.7

Brevinin-1-OR8

13.1

13.1

26.2

52.4

Brevinin-1-OR9

9.8

9.8

4.9

9.8

Brevinin-1-OR10

3.4

6.8

6.8

27.2

Brevinin-1-OR11

6.2

12.4

12.4

12.4

Brevinin-2-OA1

12.1

6.0

12.1

12.1

Brevinin-2-OA2 Brevinin-2-OA3

13.2 6.3

6.6 3.2

6.6 6.3

6.6 12.6

Brevinin-2-OA4

20.0

10.0

10.0

20.0

Brevinin-2-OA5

30.6

30.6

30.6

30.6

Brevinin-2-OA6

11.2

11.2

22.4

44.8

Brevinin-2-OA7

5.8

5.8

11.6

23.2

Brevinin-2-OA8

7.9

4.0

4.0

15.8

Brevinin-2-OR1

5.5

11.0

11.0

22.0

Brevinin-2-OR2 Brevinin-2-OR3

12.1 20.6

12.1 10.3

12.1 20.6

6.0 41.2

Brevinin-2-OR4

11.8

11.8

5.9

11.8

Brevinin-2-OR5

13.2

6.6

6.6

6.6

Brevinin-2-OR6

5.8

11.6

5.8

11.6

Brevinin-2-OR7

8.6

17.2

8.6

8.6

Brevinin-2-OR8

13.1

13.1

13.1

13.1

Brevinin-2-OR9

20.4

5.1

10.2

20.4

Brevinin-2-OR10 Brevinin-2-OW1

15.8 4.7

15.8 4.7

7.9 9.4

7.9 4.7

Brevinin-2-OW2

6.6

3.3

3.3

6.6

Brevinin-2-OW3

10.5

10.5

10.5

21.0

Esculentin-1-OA1

1.2

2.4

1.2

1.2

Esculentin-1-OA2

2.5

1.3

1.3

2.5

Esculentin-1-OA3

5.4

2.7

5.4

2.7

Esculentin-1-OA4

5.6

5.6

2.8

2.8


3.4 4.2

1.7 2.1

1.7 4.2

3.4 2.1

Esculentin-1-OR1

8.8

8.8

4.4

17.6

Esculentin-1-OR2

3.3

3.3

6.6

6.6

Esculentin-1-OR3

5.5

11.0

11.0

11.0

Esculentin-1-OR4

8.6

8.6

8.6

8.6

Esculentin-1-OR5

4.1

2.1

4.1

8.2

Esculentin-2-OA1

8.5

8.5

4.3

4.3


4.4 10.2

4.4 5.1

2.2 5.1

2.2 10.2

Esculentin-2-OR1

10.8

10.8

21.6

21.6

Esculentin-2-OR2

8.1

8.1

4.1

16.2

Esculentin-2-OR3

7.9

15.8

7.9

7.9

316

hemolytic activitya (%)



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Table 4. Continued antimicrobial activity (MIC μg/mL) samples

E. coli

C. albicans

S. aureus

B. pyocyaneus

Esculentin-2-OR4

3.6

3.6

3.6

3.6

Esculentin-2-OR5

15.8

7.9

15.8

15.8

Esculentin-2-OR6

4.0

8.0

8.0

16.0

Nigrocin-1-OA1 Nigrocin-1-OA2

5.2 6.3

5.2 3.2

10.4 6.3

10.4 6.3

Nigrocin-1-OA3

10.8

10.8

5.4

21.6

Nigrocin-1-OR1

11.5

11.5

5.8

23.0

Nigrocin-1-OR2

8.6

8.6

8.6

17.2

Nigrocin-1-OR3

10.2

10.2

20.4

20.4

Nigrocin-1-OW1

4.4

4.4

8.8

8.8

Nigrocin-1-OW2

6.2

3.1

6.2

12.4

Nigrocin-1-OW3 Nigrocin-1-OW4

11.3 7.2

6.7 7.2

11.3 3.6

22.6 14.2

Nigrocin-1-OW5

5.1

5.1

5.1

10.2

Odorranain-A-OA1

35.2

17.6

35.2

17.6

Odorranain-F-OA1

5.0

2.5

2.5

10.0

Odorranain-F-OA2

7.5

3.8

3.8

7.5

Odorranain-F-OA3

12.6

6.3

6.3

12.6

hemolytic activitya (%)

Odorranain-F-OA4

8.2

4.1

4.1

4.1

Odorranain-F-OR1 Odorranain-F-OW1

14.5 8.9

14.5 8.9

7.3 17.8

14.5 17.8

Odorranain-J-OA1

40.2

20.1

20.1

40.2

Odorranain-J-OA2

24.3

24.3

12.2

48.6

Andersonin-W1

23.5

47.0

>47.0

>47.0

Andersonin-W2

20.2

20.2

10.1

40.4

Andersonin-X1

30.5

30.5

15.3

15.3

Andersonin-Y1

3.2

1.6

1.6

3.2

Andersonin-Y2 Odorranain-A-OA11

10.5 >200

5.3 >200

21.0 >200

10.5 >200

Odorranain-A-OA12

>200

>200

>200

>200

Andersonin-C1

30

30

>120

30

15.2 ( 2.1

Andersonin-D1

>126

16

16

16

ND

Andersonin-G1

>200

>200

>200

>200

ND

Andersonin-H3

>200

>200

>200

>200

ND

Andersonin-N1

>200

>200

>200

>200

ND

Andersonin-Q1 Andersonin-R1

>200 >200

>200 >200

>200 >200

>200 >200

ND ND

Andersonin-S1

>200

>200

>200

>200

ND

Hejiangin-A1

100

50

25

25

ND

Hejiangin-E1

>200

>200

>200

>200

ND

Hejiangin-F1

>100

100

100

100

ND

ND ND

Lividin-D1

>100

>100

>100

>100

ND

Macrotympanain-A1

>200

>200

>200

>200

ND

Margaratain-A1 Margaratain-B1

>200 >100

>200 >100

>200 >100

>200 >100

ND ND

Schmackerin-C1

100

100

50

50

ND

Tiannanin-A1

>200

>200

>200

>200

ND

Wuchuanin-A1

>200

>200

>200

>200

ND

Wuchuanin-C1

>200

>200

>200

>200

ND

Wuchuannin-D1

>200

>200

>200

>200

ND

Wuchuanin-E1

>100

>100

>100

>100

ND

a

The hemolytic activity of purified peptides was not tested. ND, not detectable. These data represent mean values of three independent experiments performed in triplicates. 317



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Figure 5. Biological activities of 23 synthesized peptides. (A) Antioxidant activity. (B) Effects of synthesized peptides on TNF-α production induced by LPS (1 μg/mL). (C) Effects of synthesized peptides on IL-8 production. (D) Effects of synthesized peptides on insulin production. A fixed concentration of 50 μg/mL was used for the synthesized peptides for all the experimests. * P < 0.05 and ** P < 0.01.

Several evidence showed that a novel antioxidant system composed by gene-coded peptides existed in the amphibian skins and it seemed these antioxidant peptides are structurally related to AMPs.6 One peptide might own both antimicrobial and antioxidant activities. Because of the insensitivity to the tested 4 microbes, we tested the antioxidant activity of the synthesized peptides. Most of the synthesized peptides showed potential scavenging activity against ABTS+. In the cases of Odorranain-A-OA11, Macrotympanain-A1 and Wuchuanin-A1, our results supported the proposal that peptides with potential antioxidant activity always contained a free cysteine residue which was responsible for the radical scavenging activity.26 However, Hejiangin-A1 (without cysteine residue) also showed strong antioxidant activity challenge this proposal. The antioxidant mechanism of linear antioxidant peptides without free cysteine residue needed to be further studied. Furthermore, peptides with a cyclic motif also showed antioxidant activity, such as Andersonin-C1, -G1, -H3, Hejiangin-B1, Schmackerin-C1 et al. Comparatively, the antioxidant activity of cyclic peptides was lower than that of linear peptides. This might imply that linear peptides, especially those with free cysteine residue, play a more important role in the skin antioxidant defense system. In addition to the direct microbicidal activity, AMPs may have diverse and commentary abilities to modulate the innate immune response and the primary role of these agents may not always be the direct killing of microbes.19,27 Peptides with selective

modulation of innate immunity have been considered to be new hope to current anti-infective therapy.28,29 In this research, several peptides were found to have immunomodulatory activity as illustrated in Figure 5B and 5C. Only 3 peptides showed influence on the TNF-α production induced by LPS. Meanwhile, most of these 23 peptides could affect the release of IL-8. Experimental results demonstrated that 4 peptides strongly increased but 12 peptides largely decreased the release of IL-8. Insulin-releasing activity was found in different amphibian species.29 Nine out of 23 synthesized peptides showed obvious insulin-stimulating activities (Figure 5D). Taking the results of biological function assay results together, AMPs having no or weak microbicidal activity might have other important roles in the survival of amphibians.

’ CONCLUSIONS Characterization of AMPs from the skins of nine Chinese odorous frog species was carried out by a combination of both peptidomic and genomic methods. One-hundred ninety-eight AMPs could be isolated from skin secretions of 4 odorous frog species. The identities of 80 native AMPs were confirmed by peptidomic analysis out of these isolated 198 AMPs. Precursors of 728 AMPs were obtained by genomics approach. These 728 AMPs, which almost accounted for 30% of all AMPs yet found in nature, were grouped into 97 different families, including 71 318



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novel families. Our results revealed that different AMPs might have various biological activities. Present findings strongly suggested that odorous frogs might represent the most extreme AMP diversity in nature.

(14) Chen, T.; et al. Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs. Peptides 2006, 27, 42–48. (15) Park, C. H.; et al. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J. Biol. Chem. 2001, 276, 7806–7810. (16) Lee, W. H.; et al. Maximin 9, a novel free thiol containing antimicrobial peptide with antimycoplasma activity from frog Bombina maxima. FEBS Lett. 2005, 579, 4443–4448. (17) Zhao, H.; et al. Identification of novel semenogelin I-derived antimicrobial peptide from liquefied human seminal plasma. Peptides 2008, 29, 505–511. (18) Akerstrom, B.; et al. The lipocalin alpha1-microglobulin has radical scavenging activity. J. Biol. Chem. 2007, 282, 31493–31503. (19) Bowdish, D. M.; et al. Immunomodulatory activities of small host defense peptides. Antimicrob. Agents Chemother. 2005, 49, 1727–1732. (20) Whim, M. D. Pancreatic beta cells synthesize neuropeptide Y and can rapidly release peptide co-transmitters. PLoS One 2011, 6, e19478. (21) Barra, D.; Simmaco, M. Amphibian skin: a promising resource for antimicrobial peptides. Trends Biotechnol 1995, 13, 205–209. (22) Lai, R.; et al. An anionic antimicrobial peptide from toad Bombina maxima. Biochem. Biophys. Res. Commun. 2002, 295, 796–799. (23) Zhou, M.; et al. Lividins: novel antimicrobial peptide homologs from the skin secretion of the Chinese Large Odorous frog, Rana (Odorrana) livida. Identification by 00 shotgun00 cDNA cloning and sequence analysis. Peptides 2006, 27, 2118–2123. (24) Lai, R. Combined peptidomics and genomics approach to the isolation of amphibian antimicrobial peptides. Methods Mol. Biol. 2010, 615, 177–190. (25) Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415, 389–395. (26) Liu, C.; et al. Frog skins keep redox homeostasis by antioxidant peptides with rapid radical scavenging ability. Free Radic. Biol. Med. 2010, 48, 1173–1181. (27) Bowdish, D. M.; et al. A re-evaluation of the role of host defence peptides in mammalian immunity. Curr. Protein Pept. Sci. 2005, 6, 35–51. (28) Scott, M. G.; et al. An anti-infective peptide that selectively modulates the innate immune response. Nat. Biotechnol. 2007, 25, 465–472. (29) Easton, D. M.; et al. Potential of immunomodulatory host defense peptides as novel anti-infectives. Trends Biotechnol. 2009, 27, 582–590. (30) Wilkins, M. R.; et al. Protein identification and analysis tools in the ExPASy server. Methods Mol. Biol. 1999, 112, 531–552.

’ ASSOCIATED CONTENT

bS

Supporting Information Supplemental tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Dr. Yun Zhang, Kunming Institute of Zoology, the Chinese Academy of Sciences, 32, East Jiaochang Road, Kunming, Yunnan 650223, China. Tel and fax: +86-871-5198515. E-mail: [email protected]. Dr. Wen-Hui Lee, Kunming Institute of Zoology, the Chinese Academy of Sciences, 32, East Jiaochang Road, Kunming, Yunnan 650223, China. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by grants from National Basic Research Program of China (973 Program, 2010CB529800) and The National Natural Science Foundation of China (NSFCYunnan-Union-Funding U1132601, and 31071926, 30870304). ’ REFERENCES (1) Zhang, Y.; et al. Structure-function relationship of king cobra cathelicidin. Peptides 2010, 31, 1488–1493. (2) Uccelletti, D.; et al. Anti-Pseudomonas activity of frog skin antimicrobial peptides in a Caenorhabditis elegans infection model: a plausible mode of action in vitro and in vivo. Antimicrob. Agents Chemother. 2010, 54, 3853–3860. (3) Vaara, M. New approaches in peptide antibiotics. Curr. Opin. Pharmacol. 2009, 9, 571–576. (4) Jenssen, H.; et al. Peptide antimicrobial agents. Clin. Microbiol. Rev. 2006, 19, 491–511. (5) Davidson, C.; et al. Effects of chytrid and carbaryl exposure on survival, growth and skin peptide defenses in foothill yellow-legged frogs. Environ. Sci. Technol. 2007, 41, 1771–1776. (6) Li, J.; et al. Anti-infection peptidomics of amphibian skin. Mol. Cell. Proteomics 2007, 6, 882–894. (7) Ma, Y.; et al. Peptidomics and genomics analysis of novel antimicrobial peptides from the frog, Rana nigrovittata. Genomics 2010, 95, 66–71. (8) Lee, W. H.; et al. Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification. Eur. J. Immunol. 2005, 35, 1220–1229. (9) Liu, R.; et al. There are abundant antimicrobial peptides in brains of two kinds of Bombina toads. J. Proteome Res. 10, 1806–1815. (10) Wang, L.; et al. Nigrocin-2 peptides from Chinese Odorrana frogs--integration of UPLC/MS/MS with molecular cloning in amphibian skin peptidome analysis. Febs J. 2010, 277, 1519–1531. (11) Iwakoshi-Ukena, E.; et al. Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae. Peptides 2011, 32, 670–676. (12) Conlon, J. M.; et al. Characterization of antimicrobial peptides from the skin secretions of the Malaysian frogs, Odorrana hosii and Hylarana picturata (Anura:Ranidae). Toxicon 2008, 52, 465–473. (13) Quan, Z.; et al. Novel brevinins from Chinese piebald odorous frog (Huia schmackeri) skin deduced from cloned biosynthetic precursors. Peptides 2008, 29, 1456–1460. 319


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