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 (FLGAIAGVAAKFLPKVFCFITKKC) and brevinin-1VLc (FLPVIASVAAKVLPK VFCFITKKC) showed particularly high growth-inhibitory potency (MIC ≤ 3 μM) against a Gram-positive microorganism Staphylococcus aureus and the opportunistic yeast pathogen Candida albicans and potent cytolytic activity (LC₅₀ ≤ 8 μM) against both human erythrocytes and HepG2 hepatoma-derived cells. The peptides were also active against a Gram-negative microorganism Escherichia coli (MIC ≤ 50 μM). Substitutions in brevinin-1VLd (Lys¹¹ → Asn) and brevinin-1VLe (Lys¹¹ → Ser) that decrease cationicity result in loss of activity against E. coli. Ranatuerin-2VLb (GIMDTIKGAAKDLAGQLLDKLKCKITKC) showed relatively weak antimicrobial activity (MIC ≥ 75 μM) but selective cytolytic activity against HepG2 tumor cells (LC₅₀ = 30 μM) compared with erythrocytes (LC₅₀ > 200 μM). 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 μM.