Brevinin-2Ed

Four structurally related peptides (pseudins 1-4) with antimicrobial activity were isolated from an extract of the skin of the paradoxical frog Pseudis paradoxa (Pseudidae). Pseudin-2 (GLNALKKVFQGIHEAIKLINNHVQ) was the most abundant peptide (22 nmol/g tissue) and also the most potent (minimum inhibitory concentrations, MIC = 2.5 microM against Escherichia coli, 80 microM against Staphylococcus aureus, and 130 microM against Candida albicans). The concentration of pseudin-2 producing 50% hemolysis of human erythrocytes was >300 microM. Circular dichroism studies showed that the pseudins belong to the class of cationic, amphipathic alpha-helical antimicrobial peptides but their amino acid sequences are not similar to any previously characterized peptides from frog skin. The pseudins do, however, show sequence similarity with a region at the C-terminus of DEFT, a death effector domain-containing protein expressed in mammalian testicular germ cells that is involved in the regulation of apoptosis.

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Caribbean frog Leptodactylus validus Garman, 1888 led to the identification of three peptides with previously undescribed sequences that were structurally similar to those of antimicrobial peptides isolated from other species of leptodactylid frogs. These paralogs have been termed ocellatin-V1 (GVVDILKGAGKDLLAHALSKLSEKV.NH(2)), ocellatin-V2 (GVLDILKGAGKDLLAHALSKISEKV.NH(2)), and ocellatin-V3 (GVLDILTGAGKDLLAHALSKLSEKV.NH(2)). The very low antimicrobial potency (MIC>200microM) against Escherichia coli and Staphylococcus aureus associated with the peptides is probably a consequence of their lack of amphipathicity and reduced cationicity compared with active members of the ocellatin family from related species.

Neobatrachia is the amphibian suborder with the largest number of species and a most important source of bioactive peptides from frog skin secretions. However, 90% of the studies on this subject have been focused on the frog families Hylidae and Ranidae, while very little is known about peptides of other families, like Leptodactylidae. Our work reports for the first time the isolation and characterization of ocellatin 4 (GLLDFVTGVGKDIFAQLIKQI-NH(2)), a cytolytic peptide from the skin secretion of the South American frog Leptodactylus ocellatus. While most cytolytic amphibian skin peptides are selective for microorganisms and harmless for mammalian cells, the HC(50) of ocellatin 4 against human erythrocytes is 14.3muM. The interaction between ocellatin 4 and zwitterionic phospholipids in mammalian plasma membranes may be favored by its neutral charge at pH 7.0. Ocellatin 4 also shows some antibacterial activity (MICs(E. coli)(and)(S. aureus)=64muM) and its sequence shares similarities with the only six leptodactylid peptides previously known and with four peptides from Australian hylid frogs of the genus Litoria.