Hylin-b1

RP-HPLC fractionation of the electrically stimulated skin secretion of the arboreal South American frog Hypsiboas albopunctatus ("spotted treefrog") led to the isolation of a cytolytic C-terminally amidated peptide. This novel peptide, named hylin a1 (Hy-a1), consists of 18 amino acid residues (IFGAILPLALGALKNLIK-NH(2)). The alpha-helical structure of the synthetic hylin a1 peptide was confirmed by CD spectroscopy in the presence of 60% (v/v) TFE. The synthetic peptide displayed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa and also against fungi (Candida albicans, C. krusei, C. parapsilosis and Cryptococcus neoformans). Hylin a1 was also able to disrupt human erytrocytes (HC(50)=18 microM). Similarity analysis using PSI-BLAST revealed 50-44% of identity to maximins Hv, H16, H15 and H10 from Bombina maxima and also to hylins b1 and b2 (Hy-b1 and Hy-b2) from Hypsiboas lundii (synonym: Hyla biobeba).

Amphibian skin secretions are known to contain numerous peptides with a large array of biological activities. Bombinins are a group of amphibian-derived peptides with broad spectrum antimicrobial activities that have been only identified from the ancient toad species, Bombina. In this study, we described the identification and characterization of a novel bombinin precursor which encoded a bombinin-like peptide (BLP-7) and a novel bombinin H-type peptide (named as Bombinin H-BO) from the skin secretion of Oriental fire-bellied toad, Bombina orientalis. The primary structures of both mature peptides were determined by combinations of molecular cloning of peptide precursor-encoding cDNAs and mass spectrometry techniques. Secondary structure prediction revealed that both peptides had cationic amphipathic α-helical structural features. The synthetic replicate of BLP-7 displayed more potent antimicrobial activity than Bombinin H-BO against Gram-positive and Gram-negative bacteria and yeast. Also, in vitro antitumour assay showed that both peptides possessed obvious antiproliferative activity on three human hepatoma cells (Hep G2/SK-HEP-1/Huh7) at the non-toxic doses. These results indicate the peptide family of bombinins could be a potential source of drug candidates for anti-infection and anticancer therapy.

We describe the isolation and structural characterization of a family of antimicrobial peptides related to kassinatuerin-2, from the skin secretion of the African hyperoliid frog, Kassina maculata. All four peptides, designated kassinatuerin-2Ma through Md, are C-terminally-amidated 20-mers with the consensus sequence - FX(1)GAIAAALPHVIX(2)AIKNAL - where X(1)=L/F/V/I and X2=S/N. All four peptides are encoded by precursors of 69 amino acids. Synthetic replicates of all kassinatuerin-2 related peptides displayed a potent inhibitory activity against Staphylococcus aureus with a minimal inhibitory concentration of 16microM, at which concentration, however, they effected 18% haemolysis of horse erythrocytes after 2h. Despite obvious membranolytic properties, all peptides were ineffective at inhibiting the growth of Escherichia coli at concentrations up to 200microM and were relatively ineffective against Candida albicans (MIC 120microM). The kassinatuerin-2 related peptides of K. maculata skin secretion thus possess a discrete antimicrobial and weak haemolytic activity in contrast to the prototype kassinatuerin-2 from the skin secretion of Kassina senegalensis.