Purification of peptides with differential cytolytic activities from the skin secretions of the Central American frog, Lithobates vaillanti (Ranidae)

Peptide-based defenses of ranid frogs from Mexico and Central America have been studied in much less detail than those from North America. Peptides belonging to the brevinin-1 (5 peptides), palustrin-2 (1 peptide), and ranatuerin-2 (3 peptides) families were isolated from norepinephrine-stimulated skin secretions of the Costa Rican frog, Lithobates vaillanti (Ranidae) and characterized structurally. Brevinin-1VLa (FLGAIAG-VAAKFLPKVFCFITKKC) and brevinin-1VLc (FLPVIASVAAKVLPK VFCFITKKC) showed particularly high growth-inhibitory potency (MIC <= 3 mu M) against a Gram-positive microorganism Staphylococcus aureus and the opportunistic yeast pathogen Candida albicans and potent cytolytic activity (LC(50)<= 8 mu M) against both human erythrocytes and HepG2 hepatoma-derived cells. The peptides were also active against a Gram-negative microorganism Escherichia coli (MIC <= 50 mu M). Substitutions in brevinin-1VLd (Lys(11) -> Asn) and brevinin-1VLe (Lys(11) -> Ser) that decrease cationicity result in loss of activity against E. coli. Ranatuerin-2VLb (GIMDTIKGAAKDLAGQLLDKLKCKITKC) showed relatively weak antimicrobial activity (MIC >= 75 mu M) but selective cytolytic activity against HepG2 tumor cells (LC(50)=30 mu M) compared with erythrocytes (LC(50)>200 mu M). In addition, a dodecapeptide (RICYAMWIPYPC) were isolated from the secretions that were devoid of antimicrobial activity. This component contains an Ala-Met bond that constitutes the scissile bond in the selective elastase inhibitor, elafin but the peptide did not inhibit pancreatic elastase at concentrations up to 100 mu M. (C) 2009 Elsevier Ins. All rights reserved.