Biochimie xxx (2011) 1e7 Contents lists available at SciVerse ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi Research paper Host defense peptides in skin secretions of Odorrana tiannanensis: Proof for other survival strategy of the frog than merely anti-microbial Weiyu Hea,1, Feifei Fenga,1, Yong Huangd, Huanhuan Guoa, Songyan Zhangb, Zheng Lib, Jingze Liua, Yipeng Wangc,**, Haining Yua,b,* a College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, China b Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116023, China c Biological Resources Laboratory, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, Shandong 264003, China d Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China article info abstract Article history: Genus Odorrana, among all amphibians studied, is generally reported to have the most abundant and diver- Received 6 August 2011 sified anti-microbial peptides even from a single individual frog. In our previous work, 46 cDNA sequences Accepted 16 September 2011 encoding precursors of 22 different anti-microbial peptides (AMPs) were characterized from the skin of frog, Available online xxx Odorrana tiannanensis. In this work, we reported the purification of three AMPs from skin secretions of O. tiannanensis. Their amino acid sequences matched well with the sequences deduced from cDNAs and they Keywords: were designated as Odorranain-C7HSa, Brevinin-1-OT2 and Odorranain-G-OT, respectively. Furthermore, we Amphibian selected to analyze the four most structurally diversified sequences among the 22 AMPs that are significantly Diversity Anti-microbial peptide (AMP) different from all reported AMPs. By structural characterization, three of them were designated as pleurain-E- fi Antioxidant OT, odorranain-G-OT, odorranain-A-OT, belonging to AMP families already identi ed. The forth one with Odorrana tiannanensis a unique 14-mer sequence of AILTTLANWARKFLa and C-terminal amidation represents the prototypes of a new class of amphibian AMP, and thereby named tiannanensin. Such broad diversity in sequences and structures are consistent with other species in Genus Odorrana. Multi-functions of the synthesized four special AMPs were screened, including anti-microbial, antioxidant, cytotoxic and hemolytic activities. The results suggest that these AMPs may employ sophisticated mechanisms of action in host defense in addition to anti-microbial, although their precise contribution to host defense still seems unclear. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction to the development of antibiotic resistance, they have attracted considerable attentions as a new generation of antibiotics. Over the past several decades, extensive studies have focused on So far, there have been hundreds of AMPs of different families the bioactive compounds present in amphibian skin secretions, characterized from ranid frogs, including gaegurins, brevinins-1 especially some small peptides [1]. Those peptides are very func- and -2, ranalexin, ranatuerins-1 and -2, esculentins-1 and -2, pal- tionally different, and among these functions, the anti-microbial ustrin, japonicin-1 and -2, nigrocin-2, rugosins and temporin [6e8], activities are commonly considered to be the most important for based on their structural characteristics. Besides, there are usually the amphibian staying safe and defending against invasion of more than one anti-microbial peptide families in a single microorganisms in their habitats. Generally, these anti-microbial amphibian species. Most of the AMPs, 10e50 residues in length, peptides (AMPs) are cationic, amphipathic, and central effector have a common highly conserved N-terminal preproregion, fol- molecules of all forms of lives’ innate immunity [2e4]. AMPs can lowed by a markedly different C-terminal domain that corresponds rapidly kill a broad range of bacteria, yeasts, and fungi by forming to mature AMPs [6,7,9]. Functionally, different amphibian anti- pores in the membranes of target organisms, thus disrupting their microbial peptides have different anti-microbial spectrum. metabolic activities [5]. Because these peptides are less susceptible Odorrana is agenusoftrue frogs(Ranidae) from East Asia and surrounding regions. Many of these frogs inhabit in fast-flowing * Corresponding author. College of Life Sciences, Hebei Normal University, Shi- mountain streams. Odorrana tiannanensis, a characteristic species jiazhuang 050016, China. Tel./fax: þ86 311 86268842. of China, is mainly found distributed in Hainan and Yunnan prov- ** þ Corresponding author. Tel./fax: 86 311 86268842. inces of China. Previous work by Li et al purified and characterized E-mail addresses: [email protected] (Y. Wang), [email protected] (H. Yu). 1 These authors have the same contribution to this paper. 107 novel AMPs belonging to 30 different families, including 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.09.017 Please cite this article in press as: W. He, et al., Host defense peptides in skin secretions of Odorrana tiannanensis: Proof for other survival strategy of the frog than merely anti-microbial, Biochimie (2011), doi:10.1016/j.biochi.2011.09.017 2 W. He et al. / Biochimie xxx (2011) 1e7 24 novel families from Odorrana grahami [9]. Totally 372 different protocols. After the cleavage and deprotection of side-chain, the cDNAs encoding these anti-microbial peptides were identified in crude synthetic peptide was purified on a Vydac C18 RP-HPLC that work. In our previous work, 46 cDNA sequences encoding column (25 cm  1 cm), eluting at a flow rate of 1 ml/min by precursors of 22 different AMPs from skin cDNA library of frog, a linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water. O. tiannanensis were successfully cloned. In current work, the Identity of the peptide was confirmed by automated Edman structures and properties of the four most structurally diversified degradation with a protein sequencer and MALDI-TOF-MS analysis. AMPs present in the skin of O. tiannanensis were investigated, and The synthesized peptides containing two cysteins were further furthermore the actual contributions of the general AMPs to the subject to oxidation to form an intrapeptide disulphide bridge. host innate system were discussed to a certain extent. 2.5. Anti-microbial assay 2. Materials and methods Standard and clinical-isolated drug-resistant strains of bacteria 2.1. Collection of frog skin secretions and fungi used in assays were listed in Table 1. The assay was conducted as described in our previous paper [10]. Minimal Adult specimens of O. tiannanensis (n ¼ 20) were captured in inhibitory concentration (MIC) was determined in 96-well micro- Sanya, Hainan Province, China. They were put into a cylinder titer plate by a standard dilution method. Bacteria were incubated container and stimulated gently with an electrical device (10 V, with in MuellereHinton broth (MH) at 37 C to exponential phase of pulse duration of 3 ms). The skin secretions were collected by washing growth and diluted with fresh MH broth to 106 CFU/ml 50 mlof the dorsal region with 0.9% Sodium chloride solution. Totally 200 ml serial dilutions of the peptides in MH were prepared in 96-well solution was collected and centrifuged at 12,000 rpm for 20 min. microtiter plates and mixed with 50 ml of bacteria inoculum. The supernatant was removed, lyophilized and stored at À20 C. Plates were incubated at 37 C for 18 h and the minimal concen- tration at which no visible growth occurred was recorded. 2.2. Peptide purification and sequencing 2.6. Hemolysis assay Lyophilized sample (0.8 g, total OD280 nm of 200) was dissolved in 10 ml 0.1 M phosphate buffer (PB), pH 6.0, and then applied to Hemolysis assay was conducted as previously reported [11]. The a Sephadex G-50 (Superfine, Amersham Biosciences, 1.6 cm  serial dilution of peptides were incubated with washed human 90 cm) column equilibrated with 0.1 M PB, pH 6.0 buffer (Na2H- erythrocytes at 37 C for 30 min and centrifuged at 2000 rpm for PO4$12H2O 4.41 g, NaH2PO4$2H2O 13.69 g, H2O 1000 ml). Elution 5 min. The supernatant was removed and the absorbance at 540 nm was performed using the same buffer with collecting fractions of was measured. 1% v/v Triton X-100 was used to determine the 3.0 ml/10 min, and monitored at 220 nm. The anti-microbial maximal hemolysis. activity of fractions was screened, and interesting peaks were fi further puri ed by C18 reversed phase high performance liquid 2.7. Cytotoxic activity chromatography (RP-HPLC, Hypersil BDS C18, 30 cm  0.46 cm) column. Complete peptide sequencing was determined by Edman In vitro cytotoxic activity of odorranain-G-OT, odorranain-A-OT degradation method on an Applied Biosystems pulsed liquid-phase and tiannanensin were examined using tumor cell lines, SGC7901, fi fi sequencer, model 491. Mass ngerprints (MFPs) of puri ed AMPs Hela and MCF-7. The cells were cultured in Dulbecco’s Modified were obtained using electrospray ionization, quadrupole orthog- Eagle’s Medium (DMEM, 11960-044, Gibco, USA) supplemented fl onal time-of- ight mass spectrometry (ESI-QTOF-MS Applied Bio- with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 U/ml of systems/MDS Sciex Toronto, Canada) instrument. streptomycin in a humidified 5% CO2 atmosphere at 37 C. Cells (2  104 per well) were seeded in 96-well plates and cultured 2.3. Construction of cDNA library and screening of cDNAs overnight until adhered to the plate. Various concentrations of encoding AMPs AMPs dissolved in the corresponding culture medium were added to the wells and the plates were incubated at 37 C for 48 h. The dorsal skin of frog was removed and cut into small pieces, Cytotoxicity of three AMPs were measured by the MTT (3-(4,5- which then were quickly frozen with liquid nitrogen and grinded to dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method.