Brevinin-2ISb

We recently reported the primary structures, antimicrobial activities and cDNA precursors of nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorranaishikawae. Their cDNA clones revealed a highly conserved approximately 60 bp region upstream of the start codon. This conserved region was used in the "shotgun" cDNA cloning method to reveal additional cDNAs encoding novel antimicrobial peptides of O.ishikawae. After sequencing 344 clones, we identified novel 13 cDNAs encoding dermal peptides in addition to the previously identified nine antimicrobial peptides. These 13 unique cDNAs encoded precursor proteins each containing a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg/Lys processing site and a dermal peptide at the C-terminus. The dermal peptides were members of the palustrin-2 (two peptides; termed palustrin-2ISc and palustrin-2ISd), nigrocin-2 (one peptide; nigrocin-2ISc), brevinin-1 (one peptide; brevinin-1ISa), odorranain-M (one peptide; odorranain-MISa) and entirely novel peptides (eight peptides; ishikawain-1-8). Although palustrin-2ISd and odorranain-MISa had few antimicrobial activities, palustrin-2ISc and nigrocin-2ISc possessed a broad-spectrum of growth inhibition against bacteria. Brevinin-1ISa had the most potent antimicrobial activities against the Gram-positive bacteria and the fungus but not the Gram-negative bacterium, Escherichiacoli. However, eight novel peptides showed no growth inhibition against these microorganisms.

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.

A novel antimicrobial peptide named odorranain-NR was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLSGILGAGKHIVCGLTGCAKA. Odorranain-NR was classified into a novel family of antimicrobial peptide although it shared similarity with amphibian antimicrobial peptide family of nigrocin. Odorranain-NR has an unusual intramolecular disulfide-bridged hexapeptide segment that is different from the intramolecular disulfide-bridged heptapeptide segment at the C-terminal end of nigrocins. Furthermore, the -AKA fragment at the C-terminal of odorranain-NR is also different from nigrocins. Three different cDNAs encoding two odorranain-NR precursors and only one mature odorranain-NR was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms except Escherichia coli (ATCC25922). Its antimicrobial mechanisms were investigated by transmission electron microcopy. Odorranain-NR exerted its antimicrobial functions by various means depending on different microorganisms.