Acta Biochim Biophys Sin, 2017, 49(6), 550–559 doi: 10.1093/abbs/gmx032 Advance Access Publication Date: 10 April 2017 Short Communication

Short Communication Identification and functional analyses of novel antioxidant peptides and antimicrobial peptides from skin secretions of four East Asian Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021 Xiao Wang1,†, Shuguang Ren1,2,†, Chao Guo1, Weiqi Zhang1, Xiaoli Zhang1, Baowen Zhang1, Sihan Li1, Jian Ren3, Yuhong Hu4,*, and Hui Wang1,*

1Key Laboratory of Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, , 2The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China, 3College of Basic, Tianjin Agricultural University, Tianjin 300384, China, and 4Instrumental Analysis Center, Hebei Normal University, Shijiazhuang 050024, China

†These authors contributed equally to this work. *Correspondence address. Tel/Fax: +86-311-8078-7551; E-mail: [email protected] (H.W.) / Tel/Fax: +86-311-8078-6450; E-mail: [email protected] (Y.H.)

Received 24 January 2017; Editorial Decision 21 February 2017

Abstract In the present study, we identified 50 peptides that are classified into 21 peptide families with anti- oxidant and/or antimicrobial activity from Amolops daiyunensis, hubeiensis, Hylarana maosuoensis and Nanorana pleskei, which belong to four different genera in the Ranidae and Dicroglossidae families. These four frog species were found for the first time to express antioxidant peptides (AOPs) and antimicrobial peptides (AMPs). These peptides include seven newly discovered families daiyunin-1, daiyunin-2, daiyunin-3, maosonensis-1MS1, pleskein-1, pleskein-2, and pleskein- 3. Antioxidant and antimicrobial activity assays showed that some of these peptides have good bio- logical activities. For example, at a concentration of 50 μM, nigroain-B-MS1, and nigroain-C-MS1 both exhibited relatively strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylben- zothiazoline-6-sulfonicacid) (ABTS) free radical scavenging ability, with eradication rates of 99.7% and 68.3% (nigroain-B-MS1), and 99.8% and 58.3% (nigroain-C-MS1), respectively. These peptides are potential candidates for the development of novel antioxidant or AMP preparations.

Key words: amphibia, skin, antioxidant peptide, antimicrobial peptide, free radical

Introduction the world [1]. The skin secretions of contain multiple pep- Amphibia is a kind of vertebrates that inhabit in water during juvenile tides that comprise their defence system in the skin, among which anti- stage and in both water and land during adult stage. The outer layer microbial peptides (AMPs) have been widely studied [2]. Due to the cells of the bare skin of amphibians only have slight cornification and improvement of research methods, more and more peptides with dif- are easy to be invaded by harmful factors in their living environment. ferent structures and novel functions are being discovered [3–7]. In the In order to adapt to the living environment, amphibians successfully mean time, antioxidant peptides (AOPs) begin to attract more and develop their own skin defence system that helps them thrive all over more attention as they are being discovered from amphibians [3–7].

© The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: [email protected] 550 Identification novel AOPs and AMPs from four species of 551

Various novel AOPs are found in the secretions of skin. Dalian, China) as previously described [3]. Six specific sense primers These AOPs can be considered as the third antioxidant system except and CDS III/3′ polymerase chain reaction (PCR) antisense primer for several antioxidant enzymes and low-molecular-weight antioxi- described before [3], were used in PCR reaction to clone the cDNAs of dants [4]. Some members among these AOPs have extremely strong AOPs and AMPs. PCR procedure was set as previously described [3]. antioxidant activities and high free radical scavenging rate [3–5]. In addition, these peptides also have low cytotoxicity, and are promising Protein digestion to become novel antioxidants [3,6,7]. Lyophilized secretions were prepared in four different ways as The Nanorana belongs to Dicroglossidae, and currently described previously, and ready for peptidomic analysis [14]. includes 28 species [8]. To date, little is known about the AMPs and Secretions were resolubilized in Guanidine–HCl (pH = 8.0) and AOPs in Dicroglossidae family [6,7,9–12]. Plateau frog Nanorana pleskei equally divided. Then, samples were incubated with 20 mM dithio- is one of the species in the genus Nanorana, which distributes in swamps threitol at 37°C for 30 min to break the disulfide bonds to reduce cyst- on plateau with 3300–4500 m elevation [13]. In such bad living environ- eine side-chain sulfhydryl groups. Then the samples were incubated ment, N. pleskei still survive, which attracts a lot of concerns from with 50 mM iodoacetamide at 25°C in dark with constant shaking for researchers. In addition, Amolops daiyunensis (Daiyun Torrent Frog), Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021 45 min in order to stabilize sulfhydryl groups by alkylation. Samples Pelophylax hubeiensis (Hubei Gold-striped Pond Frog), and Hylarana without the necessity for tryptic digestion were desalinated with SPE maosuoensis (Maoson Frog) all belong to the family Ranidae [13]. (C18; DiKMA, Beijing, China) before liquid chromatography–mass To our knowledge, there has been no report on the functional pep- spectrometry (LC–MS) analysis. Equal amounts of materials with and tides secreted from the skin of the four East Asian species. Here, we without reductive alkylation were subject to tryptic digestion. Sample report the identification and functional analyses of AMPs and AOPs precipitation was processed in the acetic acid:acetone:ethanol mixture from these four frog species, including seven new peptide families. The (0.1:50:50; v/v) at −20°C for 6 h, followed by rinsing with 70% etha- peptides, nigroain-B-MS1 and nigroain-C-MS1, are found to have nol (1 ml) and centrifugation (12,000 g,4°C). The precipitate was potentials to be developed into new antioxidant drugs due to their evenly suspended in 0.1 M NH HCO buffer (pH = 8.0) and then split antioxidant activity with no hemolytic activity against erythrocytes. 4 3 into three equal portions and mixed with trypsin (substrate to enzyme, 100:1; w/w). All portions were digested at 37°C while three reactions Materials and Methods Table 1. MICs (μM) against microorganisms of peptides from the Sample collection skin of A. daiyunensis, P. hubeiensis, H. maosuoensis, and N. Amolops daiyunensis (20 males and 9 females) were collected from pleskei Dehua, Fujian Province, China. P. hubeiensis (21 males and 18 females) were obtained from Wuhan, Hubei Province, China. H. mao- Microorganism Gram-positive Gram-negative Fungi suoensis (three males and two females) were captured from Shangsi, bacteria bacteria Guangxi Zhuang Autonomous Region, China. N. pleskei (11 males ABCDEFGH and 23 females) were caught in Zoigê plateau in Sichuan Province, China, which is in the east part of Qinghai-Tibet Plateau of China. Temporin-DY1 150 NA 150 NA NA NA NA NA The frogs were fed for several days in the lab before being set free alive Brevinin-1DY1 37.5 37.5 37.5 NA NA 37.5 NA NA Palustrin-2DY1 NA NA 2.3 NA NA 150 NA NA at the location of collection. Skin secretions were acquired by electric Daiyunin-1 NA NA NA NA NA NA NA NA stimulations as previously described [3]. All protocols were permitted Daiyunin-2 NA NA NA NA NA NA NA NA by the Animal Ethics Committee of Hebei Normal University. Daiyunin-3 NA NA NA NA NA NA NA NA Temporin-HB1 18.8 18.8 2.3 NA NA 150 NA NA Peptides purification and sequencing Temporin-HB2 150 18.8 75 NA NA NA NA NA Brevinin-1HB1 4.7 9.4 1.2 75 75 37.5 NA 37.5 Phosphate buffer (0.1 M) with a final concentration of 5 mM EDTA Pelophylaxin-HB1 NA NA 9.4 NA NA 75 NA 150 was adjusted to pH 6.0. Lyophilized skin secretions of A. daiyunen- Ranacyclin-HB1 18.8 NA 150 NA NA NA NA NA sis, P. hubeiensis, H. maosuoensis,orN. pleskei were resolubilized Palustrin-2HB1 37.5 NA 9.4 NA 150 75 NA NA fi in the buffer. The puri cation of AOPs and AMPs was done as pre- Temporin-MS1 150 18.8 37.5 150 150 37.5 NA 150 viously described [3]. After being purified by gel filtration (Sephadex Temporin-MS4 9.4 18.8 4.7 NA NA 150 NA NA G-50) and reversed phase-high performance liquid chromatography Maosonensis-1MS1 NA NA NA NA NA NA NA NA (RP-HPLC), the elution fractions were collected and subject to anti- Odorranaopin-MS1 NA NA NA NA NA NA NA NA oxidative or antimicrobial activity assays. Fractions representing Odorranaopin-MS2 NA NA NA NA NA NA NA NA either activity were collected for sequencing with Model 491 sequen- Brevinin-2MS1 150 NA 9.4 150 75 18.8 150 75 cer (Thermo Fisher Scientific, Waltham, USA) using Edman degrad- Nigroain-B-MS1 4.7 NA 18.8 NA NA NA NA NA Nigroain-C-MS1 NA NA 150 NA NA NA NA NA ation method. Mass measurements were carried out on an LTQ-XL Nigroain-D-SN1 NA NA NA NA NA NA NA NA mass spectrometer (Thermo Fisher Scientific). Nigroain-K-SN1 75 NA 4.7 NA NA 75 NA 150 Pleskein-1 NA NA 37.5 NA NA NA NA NA Polymerase chain reaction Pleskein-2 NA NA 150 NA NA NA NA NA mRNA was isolated from a single frog skin using mRNA separation kit Pleskein-3 NA NA NA NA NA NA NA NA (Thermo Fisher Scientific) according to the manufacturer’s manual. The NA: not active. A: Staphylococcus aureus (ATCC 25923), B: Enterococcus quantity and quality of mRNA were analyzed using the NanoDrop faecalis 981 (IS), C: Nocardia asteroides 201118 (IS), D: Pseudomonas fi 2000 UV spectrophotometer (Thermo Fisher Scienti c). The cDNA aeruginosa (CGMCC 1.50), E: Klebsiella Pneumoniae 08040724 (IS), F: library was created using Switching Mechanism At 5′ end of the RNA Escherichia coli (ATCC 25922), G: Candida albicans (ATCC 10231), H: Transcript (SMART™) cDNA library construction kit (TaKaRa, Candida glabrata 090902 (IS). IS: isolated strain. 552 Identification novel AOPs and AMPs from four species of frogs lasted respectively for 1 h (incomplete digestion), 2 h (incomplete diges- The nucleotide and peptide sequences of A. daiyunensis, P. hubeien- tion), and 8 h (complete digestion) to allow graded digestion. sis, H. maosuoensis, and N. pleskei identified in this study were added to this Nr database. The proteomics calculation parameters LC–MS analysis were set as previously described [3]. Theoretical physicochemical properties and signal peptides of each peptide were predicted by the Tryptic peptides were loaded on a C18 RP column (0.75 mm × Innovagen’s peptide calculator and SignalP 4.1 software. 150 mm; Thermo Fisher Scientific), and then eluted using linear gra- dient (0%–80%) solvent B [solvent A: pure H2O + 0.1% formic acid (FA); solvent B: pure acetonitrile (ACN) + 0.1% FA] over Peptide synthesis 240 min at a flow rate of 400 nl/min. Eluted peptides were directly Syntheses of all peptides tested in bioactivity assays were performed electrosprayed at 2.0 kV by a nano-ESI emitter and delivered into on a Model 433 A peptide synthesizer (Thermo Fisher, Scientific). The LTQ-XL (Thermo Fisher Scientific) via an uncoated spraying needle disulfide bridge in the peptide was formed by N, N-dimethylforma- (inner diameter: 15 μm) at MS capillary transfer temperature of mide (DMF) and C-terminal amidation was conducted by the a- 200°C. LC–MS data were acquired using Xcalibur 2.0 software amidating enzyme. The synthetic peptides were purified by RP-HPLC Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021 (Thermo Fisher Scientific). The analysis process was optimized by (C18; 3 cm × 25 cm; Kromasil, Bohus, Sweden). After loading, the employing gas-phase fractionation that can improve the perform- concentration of 0.1% (v/v) trifluoroacetic acid (TFA)/ACN in the ance of peptide identification [15]. Each sample was analyzed eluting solvent was raised from 10% to 100% (v/v) over 25 min at a according to five scans varied in m/z ranges, including four full MS flow rate of 22 ml/min using a linear gradient. The effluent absorbance scans with different narrow m/z range (400–600 m/z, 600–800 m/z, was monitored at 214 nm. Purity was >95%, and the identity was 800–1000 m/z, 1000–2000 m/z) and one scan of normal range confirmed by an ESI-LTQ (Thermo Fisher Scientific) instrument with (400–2000 m/z). Other LC–MS calculation parameters were set as a 3.0 kV electrospray voltage at mass spectrometer capillary transfer previously described [3]. temperature of 350°C.

Mass spectra data and physicochemical properties Minimal inhibitory concentration and free radical analysis scavenging Database analysis was performed using BioWorks 3.3.1 software Table 1 shows the microorganism strains involved in minimal (Thermo Fisher Scientific). Entrez family Ranidae protein sequence inhibitory concentration (MIC) assays conducted using microdilu- non-redundant (Nr) database (15. 11. 2013) from NCBI was used. tion broth method on 96-well plates [16,17]. Comparable amount

Figure 1. Purification of peptides from skin secretions of A. daiyunensis (A), P. hubeiensis (C), and N. pleskei (E) The peaks that possess antimicrobial or anti- oxidative activity are indicated by arrows: palustrin-2DY1: GIMDTIKNAAKDVVQSLLNKASCKLAKTC (Peak D1, B), temporin-DY1: FFPMLADLVSKIF-NH2 (Peak D2, B), brevinin-1DY1: FLPLLAGLAANFLPTIICKIARKC (Peak D3, B), palustrin-2HB1: GLWTTIKEGLKKFSLGVLDKIRCKIAGGC (Peak H1, D), temporin-HB1:

FLPLLAGLAAKWF-NH2 (Peak H2, D), brevinin-1HB1: FLPAIIGMAAKVLPAFLCKITKKC (Peak H3, D), pelophylaxin-HB1: GILMDTFKGAAKNVAGFLLDKLKCKISGGC

(Peak H4, ), temporin-HB2: FLPFLAGLFGKIF-NH2 (Peak H5, D), pleskein-1: FFPLIPGVRCKILRTC (Peak P1, F), pleskein-2: FFLLPIPNDVKCKVLGICKS (Peak P2, F). The line shows the percentage of acetonitrile in a linear gradient. Identification novel AOPs and AMPs from four species of frogs 553 of corresponding strains in log-phase growth were mixed with The mixtures were heated at 100°C for 10 min to determine the proper media to a final volume of 100 μleachwell(1× 106 cfu/ml), value for maximal inhibition. MICs were defined as the lowest con- followed by incubation. Bacteria were incubated in Luria-Bertani centration of peptide that completely inhibits the growth of bacteria (LB) medium for 18 h at optimal temperature (30°C–37°C), while or fungi. DPPH and ABTS free radical scavenging for antioxidant fungi were incubated in Yeast Extract Peptone Dextrose (YPD) activity testing was performed at room temperature in the dark medium for 48 h at 35°C. Fresh pure LB or YPD medium (100 μl) according to a previous report [3]. All experiments were repeated containing the inoculum was used to determine maximal growth. for three times.

Table 2. Structural parameters of peptides from the skin of A. daiyunensis, P. hubeiensis, H. maosuoensis, and N. pleskei

Peptides Number of Net charge Theoretical pI Observed molecular Calculated molecular amino acids at pH 7 masses (Da) masses (Da)

Daiyunin-1 13 2 9.94 – 1519.83 Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021 Daiyunin-2 22 2.1 10.32 – 2473.95 Daiyunin-3 13 3 12.41 – 1471.86 Palustrin-2DY1 29 3 10.38 3062.51 3062.67 Palustrin-2DY2 29 3 10.38 – 3046.63 Temporin-DY1 13 1 10.10 1526.73 1526.91 Brevinin-1DY1 24 3 11.64 2585.01 2585.27 Brevinin-1RTa 24 3 11.64 – 2626.32 Brevinin-2DY1 33 3 10.38 – 3363.99 Brevinin-2HB1 33 4 10.71 – 3349.02 Brevinin-1HB1 24 4 11.01 2575.13 2575.34 Brevinin-1HB2 24 3 10.84 – 2639.34 Brevinin-1HB3 24 4 11.01 – 2653.41 Brevinin-1HB4 24 3 10.84 – 2584.30 Temporin-HB1 13 2 9.88 1445.70 1445.82 Temporin-HB2 13 2 9.88 1468.69 1468.85 Temporin-HB3 13 2 9.88 – 1498.88 Temporin-HB4 13 2 9.88 – 1434.84 Pelophylaxin-HB1 30 3 10.38 3097.56 3097.77 Pelophylaxin-HB2 30 3 10.38 – 2969.61 Pelophylaxin-HB3 30 4 10.54 – 3013.71 Pelophylaxin-1 30 3 10.38 – 2999.64 Esculentin-2HB1 37 4 10.71 – 3743.41 Esculentin-2HB2 37 4 10.71 – 3777.43 Ranacyclin-HB1 20 4 10.63 – 2178.57 Ranacyclin-HB2 20 4 10.63 – 2160.59 Palustrin-2HB1 29 4 10.70 3133.72 3133.82 Maosonensis-1MS1 13 2 10.40 – 1491.67 Maosonensis-1MS2 13 1 9.72 – 1448.64 Esculentin-1MS1 47 7 10.81 – 4998.05 Esculentin-2MS1 37 5.1 10.82 – 3840.54 Temporin-MS1 13 2 9.88 – 1332.72 Temporin-MS2 13 2 9.88 – 1318.69 Temporin-MS3 13 2 9.88 – 1352.71 Temporin-MS4 17 2 9.88 – 1817.30 Temporin-SN1 17 2 9.88 – 1879.37 Temporin-SN5 17 2 9.88 – 1831.32 Brevinin-1LTa 24 4 11.01 – 2562.23 Brevinin-2MS1 33 2 10.05 – 3381.03 Brevinin-2MS2 33 3 10.38 – 3380.04 Odorranaopin-MS1 17 1.1 9.84 – 2108.30 Odorranaopin-MS2 17 1 9.84 – 1977.17 Odorranain-U-MS1 16 2 12.10 – 1817.13 Nigroain-B-MS1 21 4 10.45 – 2443.90 Nigroain-C-MS1 19 4 10.83 – 2135.56 Nigroain-D-SN1 16 1 11.04 – 1748.02 Nigroain-K-SN1 31 4 10.70 – 3376.06 Pleskein-1 16 3 12.41 1861.23 1861.36 Pleskein-2 20 2 10.32 2232.58 2232.79 Pleskein-3 13 2 11.49 – 1427.80

Structural parameters of each peptide calculated using the Innovagen’s peptide calculator. Calculated molecular mass of peptides with hypothetical intramolecular disulfide bridge or C-terminally amidated. 554 Identification novel AOPs and AMPs from four species of frogs

Hemolytic assays Table 3. Antioxidant and antimicrobial peptides from the skin and A daiyunensis P hubeiensis H maosuoensis Washed human erythrocytes (2 × 107 cells, 100 μl) were suspended its secretions of . , . , . , and N. pleskei identified by LC–MS/MS in Dulbecco’s phosphate-buffered saline (pH 7.4) as previously described [18]. The erythrocytes were incubated with Triton X-100 Protein name Coveragea (%) Modifications Species (1%) to measure the maximal hemolysis. Serially diluted peptides were incubated with erythrocytes for 1 h at 37°C. Then, the optical Daiyunin-1 76.9 DB A. daiyunensis density of the supernatant was measured at 450 nm after centrifuga- Brevinin-1DY1 79.2 DB A. daiyunensis tion at 12,000 g for 15 s. Palustrin-2DY1 82.8 DB A. daiyunensis Palustrin-2DY2 37.9 NA A. daiyunensis Temporin-DY1 100.0 CA A. daiyunensis Brevinin-2HB1 84.8 DB P. hubeiensis Results Brevinin-1HB1 79.2 DB P. hubeiensis Purification peptides from skin secretions of frogs Brevinin-1HB3 45.8 NA P. hubeiensis Temporin-HB1 100.0 CA P. hubeiensis On Sephadex G-50, the components of skin secretions from A. Temporin-HB2 84.6 NA P. hubeiensis Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021 daiyunensis were separated into three peaks, and antimicrobial Pelophylaxin-HB1 56.7 NA P. hubeiensis activities were detected in peak 3 (Fig. 1A). After being further Pelophylaxin-HB2 56.7 NA P. hubeiensis purified by RP-HPLC, peaks D1–D3 showed antimicrobial activ- Pelophylaxin-1 30.0 NA P. hubeiensis ities (Fig. 1B). The components of skin secretions from P. hubeien- Esculentin-2HB1 86.5 DB P. hubeiensis sis were divided into four peaks (Fig. 1C), and antimicrobial and Esculentin-2HB2 56.8 DB P. hubeiensis antioxidative activities were detected in peak 3. After being further Palustrin-2HB1 100.0 DB P. hubeiensis – purified by RP-HPLC, peaks H1–H5 showed antioxidative or anti- Maosonensis-1MS1 100.0 H. maosuoensis Maosonensis-1MS2 100.0 – H. maosuoensis microbial activities (Fig. 1D). The components of skin secretions Esculentin-2MS1 56.8 DB H. maosuoensis from N. pleskei were separated into 4 peaks, and antimicrobial Temporin-MS1 100.0 CA H. maosuoensis and antioxidative activities were detected in peak 4 (Fig. 1E). After Temporin-MS2 100.0 CA H. maosuoensis fi being further puri ed by RP-HPLC, peaks P1 and P2 showed anti- Temporin-MS4 100.0 CA H. maosuoensis oxidative or antimicrobial activities (Fig. 1F). Because the distribu- Temporin-SN5 88.2 CA H. maosuoensis tion range of H. maosuoensis was narrow and its population was Brevinin-2MS1 54.5 NA H. maosuoensis relatively small, insufficient frogs were collected for the purifica- Brevinin-2MS2 24.2 NA H. maosuoensis tion of skin secretions by RP-HPLC. However, it is still identified Nigroain-K-SN1 54.8 DB H. maosuoensis that the skin secretions of these frogs have antimicrobial and anti- Odorranain-U-MS1 50.0 DB H. maosuoensis – oxidant activities. Using LC–MS method, the structures of these Nigroain-C-MS1 89.5 H. maosuoensis Pleskein-1 87.5 DB N. pleskei peptides were identified. Pleskein-2 65.0 NA N. pleskei

aCoverage is the ratio of the identified tryptic peptide fragment by MS/MS Sequence characterization to the whole length of the each mature antimicrobial peptide. DB: After Edman degradation, sequencing and molecular mass mea- Intramolecular disulfide bridge. CA: C-terminal amidation; NA: not confirm surements, the observed molecular mass of the peptides was modification site by MS/MS. “–”: There is no modification in the peptide. nearly the same as the calculated molecular mass (Table 2), sug- gesting that peptides containing two cysteines had disulfide bond library. All antimicrobial and antioxidative peptide precursors had (brevinin-1DY1, brevinin-1HB1, palustrin-2DY1, pelophylaxin- similar primary structures that were cloned from other frogs. HB1, palustrin-2HB1, pleskein-1, and pleskein-2), and temporin- DY1, temporin-HB1, and temporin-HB2 were amidated with C-terminus. The calculation result of physicochemical properties Free radical scavenging activity in Table 2 showed that all of the peptides were cationic. The pep- The antioxidant activities of peptides from 18 families were tested fi – tides identi ed using LC MS method from skin secretions of by artificial peptide synthesis using free radicals of ABTS and/or A. daiyunensis, P. hubeiensis, H. maosuoensis,andN. pleskei are DPPH (Table 4). The varied primary structures of AOPs from differ- listed in Table 3. ent families led to different antioxidant activity. At a concentration of 50 μM, nigroain-B-MS1 and nigroain-C-MS1 both exhibited rela- tively strong ABTS and DPPH free radical scavenging ability, with cDNA cloning eradication rates of 99.7% and 68.3% (nigroain-B-MS1), and Figure 2 showed the cDNA cloning results. After analyzing 90 99.8% and 58.3% (nigroain-C-MS1), respectively. The eradication clones, we obtained nine novel peptide precursors and deduced nine ability of maosonensis-1MS1, odorranaopin-MS2, daiyunin-1, and ·+ mature peptides from seven families in A. daiyunensis skin cDNA pleskein-2 against ABTS and DPPH under such concentration ·+ library. In addition, after analyzing 120 clones, we obtained 21 within 30 min was low, but their eradication rate against ABTS novel peptide precursors and deduced 18 mature peptides from reached 30.1%, 48.9%, 77.1%, and 20.1%, respectively, when seven families in P. hubeiensis skin cDNA library. Similar analysis reaction time was prolonged to 14 h. of 180 clones was performed on the skin of H. maosuoensis cDNA library. We obtained 21 novel peptide precursors and deduced 20 mature peptides from 12 families. Finally, after analyzing 40 clones, Free radical scavenging kinetics we obtained three novel peptide precursors and deduced three Figure 3A,B shows free radical scavenging kinetics at the concentra- mature peptides from three families in P. hubeiensis skin cDNA tion of 50 μM. Most of these peptides can scavenge free radicals in Identification novel AOPs and AMPs from four species of frogs 555 Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021

Figure 2. Alignments of the peptide precursors from the skin of A. daiyunensis, P. hubeiensis, H. maosuoensis, and N. pleskei Brevinin-1RTa identified from A. daiyunensis was first identified and named from A. ricketti [19]. Pelophylaxin-1 identified from P. hubeiensis was first identified and named from P. plancyi fukienensis [20]. Temporin-SN1, temporin-SN5, nigroain-K-SN1, and nigroain-D-SN1 identified from H. maosuoensis was first identified and named from H. spi- nulosa [21]. Brevinin-1LTa identified from H. maosuoensis was first identified and named from H. latouchii [22]. two phases: initial fast reaction phase and later slow reaction phase Antimicrobial activity [3–5]. For example, nigroain-D-SN1, nigroain-K-SN1, and pleskein- As listed in Table 1, the inhibition of bacteria and fungi by the pep- 2 showed the above characteristics during the eradication of tides from 18 families was investigated. The results showed that the ·+ ·+ ABTS . By contrast, the eradication rate of ABTS by daiyunin-1, antimicrobial spectra of brevinin-1HB1 were broader than that of maosonensis-1MS1, and odorranaopin-MS2 showed linear increase others, and brevinin-1HB1 showed the strongest antimicrobial with time. Moreover, nigroain-B-MS1 and nigroain-C-MS1, which potency, with its MIC against Nocardia asteroides 201118 (IS) showed relatively strong scavenging ability, can scavenge nearly all reaching 1.2 μM. The antimicrobial spectra and antimicrobial ·+ ABTS in a short time (Fig. 3C,D). 556 Identification novel AOPs and AMPs from four species of frogs

Table 4. Antioxidant activities of peptides small portion of them have antioxidative activity. The activity of

·+ peptides is related to their amino acid composition, arrangement Peptides ABTS (ESC%) DPPH (ESC%) and secondary structures [3,4]. Reductive cysteine in the peptide Daiyunin-1 14.9 ± 2.6 8.6 ± 1.4 molecules strongly supports the antioxidative activity of the pep- Ranacyclin-HB1 49.3 ± 3.8 11.6 ± 2.4 tides [4,5]. In contrast to tyrosine, tryptophan, methionine, or pro- Temporin-MS1 21.4 ± 2.2 NA line that may help bring antioxidative function in peptides, the Maosonensis-1MS1 8.8 ± 1.8 NA reductive mercapto group (-SH) in cysteine molecules has stronger Odorranaopin-MS2 9.7 ± 2.2 NA antioxidative activity. Therefore, peptides with free cysteine usually ± ± Nigroain-B-MS1 99.7 0.3 68.3 3.3 have strong and highly efficient antioxidative activity [3–5]. In the ± ± Nigroain-C-MS1 99.8 0.2 58.3 3.8 present study, AOPs nigroain-B-MS1 and nigroain-C-MS1 mole- Nigroain-D-SN1 21.5 ± 2.3 NA cules were found to contain a reduced cysteine, explaining why Nigroain-K-SN1 21.3 ± 2.2 11.3 ± 2.7 they can rapidly scavenge free radicals. Pleskein-2 11.3 ± 2.8 NA It is hypothesized that the production of AOPs may be related to Antioxidant activities of peptides (50 μM) were from the skin secretions of long-time exposure to strong ultraviolet light in oxygen-rich atmos- Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021 A. daiyunensis, P. hubeiensis, N. pleskei, and H. maosuoensis for 30 min. phere [4,5]. The differences in the structure and function of AOPs Values are shown as the mean ± SD of three replicates. NA: no activity. from different amphibians are not only related to their taxonomic status, but also related to their geological distribution, altitude and activities of AMPs in the temporin family, temporin-DY1, temporin- climate. This point was discussed in our previous study [3]. HB1, temporin-HB2, temporin-MS1, and temporin-MS4 are signifi- Consistently, analysis on the types, amounts, and antioxidative activ- cantly varied, possibly due to the high variety of peptide structures ities of AOPs in the skin of amphibians with different geological dis- in this family. Some peptides identified in the present study, such as tributions demonstrates the same point. The frogs H. maosuoensis ranacyclin-HB1, temporin-MS1, nigroain-B-MS1, nigroain-C-MS1, were collected from low altitude (400 m) mountainous region around nigroain-K-SN1, and pleskein-2 AOPs also had certain antimicrobial the border of Guangxi Zhuang Autonomous Region of China and activities. However, some other AOPs such as daiyunin-1, nigroain- Vietnam, which has tropical climate. Sunlight intensity in this region D-SN1, odorranaopin-MS2, and maosonensis-1MS1 did not show is strong, and sunlight time is long. As a result, the exposure time to any antimicrobial activity. sunlight and air for this species is longer than that of the other three species. Because of the threats of this type of living environment, the skin of H. maosuoensis develops more antioxidant materials theoret- Hemolytic activity ically. Indeed, we identified seven AOPs from the skin of H. mao- The hemolytic results were shown in Table 5. Palustrin-2HB1, suoensis, in which nigroain-B-MS1 and nigroain-C-MS1 have brevinin-1DY1, temporin-MS1, and temporin-MS4 showed the stron- extremely strong antioxidative activities. The number and activity of gest hemolytic activity. Especially, brevinin-1DY1 and palustrin-2HB1 AOPs found in the skin of H. maosuoensis are much higher than led to complete hemolysis at 200 μM. Palustrin-2HB1 caused 8.4% those of AOPs identified from the other three species of frogs. hemolysis of red blood cells at 3.1 μM. Comparatively, the hemolytic P. hubeiensis and A. daiyunensis are distributed in subtropical activity of brevinin-1DY1 was stronger, showing 10.3% hemolysis regions. P. hubeiensis is collected from low altitude (50 m) ponds in at the concentration of 1.6 μM. Temporin-DY1, palustrin-2DY1, Wuhan, China, which have few shelters. In the day time, P. hubeien- ranacyclin-HB1, nigroain-C-MS1, nigroain-B-MS1, and nigroain- sis can be observed on lotus leaves. Wuhan is one of the hottest cities D-SN1 showed weak hemolytic activity. Some peptides, such as in China, with relatively long yearly active time for amphibians. daiyunin-1, daiyunin-2, and daiyunin-3, did not show any hemo- Therefore, the living environment of P. hubeiensis enabled them to lytic activity. have longer sunlight time than other amphibians distributed in sub- tropical mountainous regions, and the AOPs in this species have stronger activity. The activity of one AOP, ranacyclin-HB1, identified Discussion in the skin of P. hubeiensis was found to be stronger than that of The skin of amphibians contains abundant types of bioactive pep- daiyunin-1 from A. daiyunensis which lives in the thick forest. tides, including AMPs [23], AOPs [4], protease inhibitors [24], and N. pleskei is collected from the east Qinghai-Tibet Plateau with an bradykinins [25], etc. Although the reason for the production of altitude of 3800 m, which has plateau and mountain climate. bioactive peptides in amphibians is not clear by now, it is generally Because of the long-time low temperature in this region, N. pleskei is hypothesized that the production of AMPs [26] and AOPs [5]is only active in the summer, and has the shortest time for sunlight and related to invasions of biological and non-biological factors in the air among the four species of frogs. Therefore, the activity of AOP in living environment of amphibians. In the present study, we identi- this species is the weakest. Indeed, AOP pleskein-2 which has the fied abundant AMPs and AOPs from four species of frogs, includ- lowest activity was identified only from the skin of N. pleskei. ing some novel peptide families daiyunin-1, daiyunin-2, daiyunin-3, Currently, AMPs are mainly discovered in Anura amphibians. maosonensis-1MS1, pleskein-1, pleskein-2, and pleskein-3, which Few are reported in Caudata [28], while no report is from provided abundant data for further investigations into AMPs and Gymnophiona. AMPs in Anura amphibians are mainly found in AOPs. Since the first AOP was identified from the skin of Bombinatoridae, Hylidae, and Ranidae families. By contrast, only a pleuraden [4], many AOPs have been identified from the skin of few AMPs are identified from Bufonidae, Ceratophryidae, Dicro- several species of amphibians [3,5–7,27]. Similarity analysis of the glossidae, Leptodactylidae, and Rhacophoridae families. In addition, structures of AOPs from different amphibians shows that AOPs there is no report on AMPs identified from Microhylidae, Pelobati- may be common scavengers of reactive oxygen species that exist in dae, Pyxicephalidae, and Scaphiopodidae families. Every now and the skin of amphibians [5]. The skin of amphibians contains abun- then some novel AMPs are identified from species which are not dant peptides with different structures and functions, but only a researched before [3,29]. Nevertheless, there are few reports on Identification novel AOPs and AMPs from four species of frogs 557 Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021

Figure 3. Free radical scavenging kinetics of AOPs identified from A. daiyunensis, P. hubeiensis, H. maosuoensis, and N. pleskei (A,B) ABTS·+ and DPPH free radical scavenging kinetics of all AOPs at 50 μM. (C,D) Free radical scavenging kinetics of nigroain-B-MS1 at different concentrations. (E,F) Free radical scaven- ging kinetics of nigroain-C-MS1 at different concentrations.

AMPs identified from genus Nanorana which belongs to the Dicro- activity. By sharing the same precursors with antimicrobial or AOP, glossidae family [6,30]. In the present study, some new families of pleskein-3 may possess other functions which can, in combination peptides pleskein-1, pleskein-2, and pleskein-3 from N. pleskei were with antimicrobial or AOP, help frogs to adapt to their living envir- identified, among which pleskein-1 and pleskein-2 have relatively onments [4,31]. weak antimicrobial activities, and pleskein-2 has certain antioxidant In summary, we have identified 50 novel peptides with antioxi- activity. Pleskein-3 does not display any antimicrobial or antioxidant dant and/or antimicrobial capacity from four species of amphibians 558 Identification novel AOPs and AMPs from four species of frogs

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Temporin-HB2 78.0 77.8 77.4 64.8 33.3 7.9 NA NA research.amnh.org/vz/herpetology/amphibia/index.php.st. Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021 Brevinin-1HB1 61.8 61.3 60.6 46.0 38.1 10.2 NA NA 9. Wang G, Wang Y, Ma D, Liu H, Li J, Zhang K, Yang X, et al. ve novel Pelophylaxin-HB1 40.9 23.1 11.7 NA NA NA NA NA antimicrobial peptides from the Kuhl’s wart frog skin secretions, Ranacyclin-HB1 8.5 6.8 NA NA NA NA NA NA Limnonectes kuhlii. Mol Biol Rep 2013, 40: 1097–1102. Palustrin-2HB1 100.0 68.4 45.4 35.2 22.8 13.0 8.4 NA 10. Wu Y, Wang L, Zhou M, Ma C, Chen X, Bai B, Chen T, et al. Temporin-MS1 99.8 95.3 59.0 13.0 NA NA NA NA Limnonectins: a new class of antimicrobial peptides from the skin secre- Temporin-MS4 92.4 84.8 39.9 15.9 NA NA NA NA tion of the Fujian large-headed frog (Limnonectes fujianensis). Biochimie Maosonensis-1MS1 NA NA NA NA NA NA NA NA 2011, 93: 981–987. Odorranaopin-MS1 NA NA NA NA NA NA NA NA 11. 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