Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs.
Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the "shotgun" cloning of novel brevinins by means of 3'-RACE, using a "universal" degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.
Chen
Tianbao
T
Institute of Biotechnology, Fuzhou University, 523 Gongye Road, Fuzhou 350002, Fujian Province, People's Republic of China.
Li
Long
L
Zhou
Mei
M
Rao
Pingfan
P
Walker
Brian
B
Shaw
Chris
C
D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000737:chemistry* / Q000235:genetics*; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011898:Ranidae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012867:Skin / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029845:Amphibian Proteins; D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary; C078413:brevinin-1 protein, Rana