Molecular Cloning and Characterization of cDNAs Encoding Biosynthetic Precursors for the Antimicrobial Peptides Japonicin-1Ja, Japonicin-2Ja, and Temporin-1Ja in the Japanese Brown Frog,Rana japonica

Abstract

Using a combination of reverse-transcription polymerase chain reaction and the 5'- and/or 3'-rapid amplification of cDNA ends, we cloned, from a Japanese brown frog (Rana japonica) skin total RNA preparation, cDNAs encoding biosynthetic precursors for the antimicrobial peptides (AMPs) japonicin-1Ja (FFPIGVFCKIFKTC), japonicin-2Ja (FGLPMLSILPKALCILLKRKC), and temporin-1Ja (ILPLVGNLLNDLL.NH2). These peptides were previously isolated from an extract of R. japonica skin. The present study is the first report to describe the molecular cloning of the cDNA encoding a japonicin-2 family peptide. The nucleotide and deduced amino acid sequence analyses revealed that the hypothetical precursor protein of japonicin-2Ja, as well as japonicin-1Ja and temporin-1Ja, is organized similarly to those of typical amphibian AMP precursors, with a highly conserved signal peptide, a relatively well conserved intervening sequence, and a hypervariable AMP mature region. Antimicrobial assays for synthetic replicates of cyclic and linear japonicin-2Ja revealed that the intramolecular disulfide bond is necessary for activity. A semi-quantitative analysis by real-time RTPCR using TaqMan probes revealed that the relative values of preprojaponicin-2Ja mRNA expression levels in the skin, skeletal muscle of hind leg, kidney, testis, small intestine, and stomach total RNA sample specimens in adult R. japonica were 6.5×10(5), 9.6, 2.0, 1.6, 1.6, and 1.0, respectively. The presence of preprojaponicin-2Ja mRNAs in the cytoplasm of glandular cells in R. japonica dorsal skin glands was demonstrated by means of in situ hybridization using digoxigenin-labeled cRNA probes for the precursor.