Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii Li-Li Jin a,b , Qiang Li a , Shu-Sen Song a , Kai Feng a , Dian-Bao Zhang a , Qiu-Yu Wang a, ⁎, Yu-Hua Chen b, ⁎ a School of Life Science, Liaoning University, 66# Chong Shan Zhong Lu, Shenyang,110036, China b Department of Developmental Biology, Key Laboratory of Cell Biology, Ministry of Public Health of China, China Medical University, Shenyang,110001, China abstract article info Article history: Received 6 February 2009 Received in revised form 27 May 2009 Accepted 28 May 2009 Available online 17 June 2009 Keywords: Rana dybowskii Skin secretions ESI-MS/MS cDNA cloning Sequence analysis Novel antimicrobial peptides The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics. © 2009 Elsevier Inc. All rights reserved. 1. Introduction Antimicrobial peptides are evolutionarily conserved components of the innate immune system which defend the body against invading of bacteria, viruses, and fungi (Boman, 1995; Nicolas and Mor, 1995). Such polypeptides are attractive as potential therapeutic agents since they may be effective against a growing number of pathogenic microorganisms that have become resistant to conventional anti- biotics (Thompson et al., 2007). Amphibians were among the first groups of organisms to form a connecting link between land and water and were thus forced to adapt and survive in a variety of conditions. Although their denudate and moist skin surface represents a potentially ideal habitat for microbial growth, they evolved an excellent chemical defense system consisting of pharmacological inhibitors and antimicrobial peptides (Nicolas and Mor, 1995; Zasloff, 2002). To adapt diversiform environmental habitat the skin secretions of Ranid frogs, like other anuran species, contain peptides with distinctive amino acid sequences and broad-spectrum antimicrobial activity (Simmaco et al., 1998). Extensive studies have been conducted on amphibian antimicrobial peptides of frogs belonging to the genus Rana. Analysis of skin secretions from different species of Ranid frogs has led to the characterization of distinct families of structurally-related peptides with antimicrobial activity. On the basis of amino acid sequence similarity, 13 such families have been identified (Conlon et al., 2004a,b); including brevinin-1 and brevinin-2 (Morikawa et al., 1992), esculentin-1 and esculentin-2 (Simmaco et al., 1994), ranatuerin-1 and ranatuerin-2 (Goraya et al., 1998), ranalexin and palustrin (Clark et al., 1994), temporin (Simmaco et al., 1990, 1996), tigerinins (Sai et al., 2001), japonicin-1 and japonicin -2 (Isaacson et al., 2002), nigrocin-2 (Park et al., 2001) and melittin-related peptides (Conlon et al., 2003). Each of these antimicrobial peptides differs in size, charge, hydrophobicity, con- formation, and spectrum of action. It is speculated that this molecular diversity may provide a broader spectrum of defense against pathogenic microorganisms encountered in the environment (Conlon et al., 2009). Based on these discoveries, it has been suggested that 20–30 different antimicrobial peptides are required to provide ranids with an adequate antimicrobial defense (Barra et al., 1998). However the identification of 372 cDNA sequences for antimicrobial peptides in a single skin from the frog species Odorrana grahami emphasizes the extraordinary capacity of amphibian skin for diversification with respect to both molecular genetics and innate immunity (Li et al., 2007). Rana dybowskii lives in valley groves of the northeast of China Korea and Japan. It is often confused with the Chinese wood frog (R. chensinensis), however Xie and colleagues have recommended the name R. dybowskii based on their study of its distribution in various Comparative Biochemistry and Physiology, Part B 154 (2009) 174–178 ⁎ Corresponding authors. Chen is to be contacted at Tel./fax: +86 24 2326 0246. Wang, Tel.: +86 24 6220 2074; fax: +86 24 8685 2421. E-mail addresses: qiuyuwang@lnu.edu.cn (Q.-Y. Wang), yhchen@mail.cmu.edu.cn (Y.-H. Chen). 1096-4959/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2009.05.015 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb