Cloning and expression of genes enocoding antimicrobial peptides and bradykinin from the skin and brain of Oki Tago's brown frog, Rana tagoi okiensis

Previous studies led to the isolation from skin extracts of Oki Tago's brown frog, Rana tagoi okiensis of five antimicrobial peptides belonging to the brevinin-1 (brevinin-1TOa), temporin (temporin-TOa and -TOb), and ranatuerin-2 (ranatuerin-2TOa and -2TOb) families, and bradykinin (BK) identical to mammalian BK. Using the reverse-transcription polymerase chain reaction (RT-PCR), we have now cloned from skin total RNA preparations cDNAs encoding biosynthetic precursors of brevinin-1TOa and brevinin-1TOb (containing the substitution Gly1 → Val), temporin-TOa and -TOb, and ranatuerin-2TOa and -2TOb. In addition, three cDNA clones encoding preprobradykinins were obtained that contained either one, two, or three tandem repeats of the sequence of BK followed by the sequence of [Thr6]-BK. In tissue expression analyses, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain compared with peripheral tissues (heart, small intestine, kidney, liver lung, skeletal muscle, stomach, and testis). RT-PCR of brain RNA resulted in the amplification of cDNAs encoding ranatuerin-2TOc and ranatuerin-2TOd that contained the amino acid substitutions Lys6 → Arg and Ala14 → Thr, respectively compared with ranatuerin-2TOb. cDNAs encoding preprobrevinin-1TOa and preprotemporin-TOa were amplified from brain RNA as well as a second preprotemporin cDNA that contained a 10-nucleotide insertion that introduced a frame shift resulting in a premature stop codon. A cDNA encoding a novel peptide, DK25 (DVNDLKNLCAKTHNLLPMCAMFGKK) was amplified from brain RNA but neither DK25 nor its putative post-translationally modified form, DF22-amide (DVNDLKNLCAKTHNLLPMCAMF·NH2) displayed antimicrobial or hemolytic activities.