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 frog Downloaded from https://academic.oup.com/abbs/article/49/6/550/3573451 by guest on 23 September 2021 species 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 Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, China, 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, Pelophylax 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 amphibians 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 frogs 551 Various novel AOPs are found in the secretions of amphibian 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 genus 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.