Anti-infection Peptidomics of Amphibian Skin*□S

Peptidomics and genomics analyses were used to study an anti-infection array of peptides of amphibian skin. 372 cDNA sequences of antimicrobial peptides were characterized from a single individual skin of the frog Odorrana grahami that encode 107 novel antimicrobial peptides. This contribution almost triples the number of currently reported amphibian antimicrobial peptides. The peptides could be organized into 30 divergent groups, including 24 novel groups. The diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal. The patterns of diversification suggest that point mutations as well as insertion, deletion, and “shuffling” of oligonucleotide sequences were responsible for the diversity. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms. These diverse peptides exhibited both diverse secondary structure and “host defense” properties. Such extreme antimicrobial peptide diversity in a single amphibian species is amazing. This has led us to reconsider the strong capability of innate immunity and molecular genetics of amphibian ecological diversification and doubt the general opinion that 20–30 different antimicrobial peptides can protect an animal because of the relatively wide specificity of the peptide antibiotics. The antimicrobial mechanisms of O. grahami peptides were investigated. They exerted their antimicrobial functions by various means, including forming lamellar mesosome-like structures, peeling off the cell walls, forming pores, and inducing DNA condensation. With respect to the development of antibiotics, these peptides provide potential new templates to explore further. Molecular & Cellular Proteomics 6:882–894, 2007. Vertebrate immune system genes exhibit exceptionally high levels of polymorphism that is driven by selective pressure to detect a diversity of quickly evolving pathogens (1–8). Innate immunity uses gene-encoded antimicrobial peptides to form a first line of host defense against noxious microorganisms (4, 9). Granular glands in the skin of anuran amphibians, particularly those belonging to the families Pipidae, Hylidae, Hyperoliidae, Pseudidae, and Ranidae, synthesize and secrete a remarkably diverse array of antimicrobial peptides, 10–50 residues in length, that are released onto the outer layer of the skin to provide an effective and fast acting defense against harmful microorganisms (1, 2). Extensive studies have been conducted on amphibian antimicrobial peptides of frogs belonging to the genus Rana. Members of the Rana genus comprise more than 250 species and are distributed worldwide except for the polar regions, southern South America, and most of Australia. About 160 antimicrobial peptides have been identified from more than 20 ranid amphibians (10). The ranid frogs synthesize and secrete multiple active components. The representative case is Rana palustris: 22 antimicrobial peptides belonging to eight different families were found in its skin secretions (11). Each of these antimicrobial peptides differs in size, charge, hydrophobicity, conformation, and spectrum of action. Based on these discoveries, it has been suggested that 20–30 different antimicrobial peptides are required to provide ranids with an adequate antimicrobial defense (7). On the basis of broad structural characteristics, amphibian antimicrobial peptides have been grouped into various families including gaegurins (24–37 residues), brevinin-1 (17–24 residues) and -2 (30–34 residues), ranalexin (20 residues), ranatuerin-1 (25 residues) and -2 (33 residues), esculentin-1 (46 residues) and -2 (37 residues), palustrin (31 residues), japonicin-1 (14 residues) and -2 (21 residues), nigrocin-2 (21 residues), rugosins (33–37 residues), and temporin (10–14 residues) (1, 2, 10). Most amphibian antimicrobial peptides from ranid frogs share a conserved disulfide-bridged heptapeptide segment at the carboxyl-terminal end. The ranid antimicrobial peptides have a common aminoterminal preproregion, which is highly conserved both intraand interspecifically, followed by a markedly different carboxyl-terminal domain that corresponds to the mature antimicrobial peptides. The conserved preproregion comprises a hydrophobic signal peptide of 22 residues followed by a 16–25residue acidic propiece that terminates by a typical From the Biotoxin Units and Laboratory of Molecular Immunopharmacology, Key Laboratory of Animal Models and Human Disease Mechanisms and Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China, Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Life Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu, China, School of Biological Sciences University of Liverpool, Liverpool L69 7ZB, United Kingdom, College of Life Sciences of Hebei Normal University, Shijiazhuang 050016, Hebei, China, Beijing Institute of Biomedicine, 15 Xinjiangongmen Rd., Beijing 100091, China, and Graduate School of the Chinese Academy of Sciences, Beijing 100009, China Received, August 29, 2006, and in revised form, January 29, 2007 Published, MCP Papers in Press, January 31, 2007, DOI 10.1074/ mcp.M600334-MCP200 Research