Ranatuerin-1

The predicted conformation of ranatuerin-1 (SMLSVLKNLG(10)KVGLGFVACK(20)INK QC), an antimicrobial peptide first isolated from the skin of the bullfrog Rana catesbeiana, comprises three structural domains: alpha-helix (residues 1-8), beta-sheet (residues 11-16) and beta-turn (residues 20-25). Circular dichroism studies confirm significant alpha-helical character in 50% trifluoroethanol. Replacement of Cys-19 and Cys-25 by serine resulted only in decreased antimicrobial potency but deletion of either the cyclic heptapeptide region [residues (19-25)] or the N-terminal domain [residues (1-8)] produced inactive analogs. Substitution of the glycine residues in the central domain of the [Ser-19, Ser-25] analog by lysine produced inactive peptides despite increased alpha-helical content and cationicity. The substitution Asn-8-->Lys gave a ranatuerin-1 analog with increased alpha-helicity and cationicity and increased potency against a range of Gram-positive and Gram-negative bacteria and against C. albicans but only a small increase (21%) in hemolytic activity. In contrast, increasing alpha-helicity and hydrophobicity by the substitution Asn-22-->Ala resulted in a 3.5-fold increase in hemolytic activity. Effects on antimicrobial potencies of substitutions of neutral amino acids at positions 4, 18, 22, and 24 by lysine were less marked. Strains of pathogenic E. coli from different groups showed varying degrees of sensitivity to ranatuerin-1 (MIC between 5 and 40 microM) but [Lys-8] ranatuerin-1 showed increased potency (between 2- and 8-fold; P < 0.01) against all strains. The data demonstrate that [Lys-8] ranatuerin-1 shows potential as a candidate for drug development.

Granular glands in the skins of frogs of the genus Rana, a widely distributed group with over 250 species, synthesize and secrete a remarkably diverse array of peptides with antimicrobial activity that are believed to have arisen as a result of multiple gene duplication events. Almost without exception, these components are hydrophobic, cationic and form an amphipathic alpha-helix in a membrane-mimetic solvent. The peptides can be grouped into families on the basis of structural similarity. To date, brevinin-1, esculentin-1, esculentin-2, and temporin peptides have been found in ranid frogs of both Eurasian and North American origin; ranalexin, ranatuerin-1, ranatuerin-2 and palustrin peptides only in N. American frogs; and brevinin-2, tigerinin, japonicin, nigrocin and melittin-related peptides only in Eurasian frogs. It is generally assumed that this structurally diversity serves to protect the organism against a wide range of pathogens but convincing evidence in support of this hypothesis is still required. The possibility that "antimicrobial peptides" fulfill additional or alternative biological functions should not be rejected. The molecular heterogeneity of the peptide families, particularly brevinin-1, brevinin-2 and ranatuerin-2, may be exploited for the purposes of unequivocal identification of specimens and for an understanding of phylogenetic interrelationships between species. The broad-spectrum antibacterial and antifungal activities of certain peptides, for example esculentin-1, ranalexin-1 and ranatuerin, together with their relatively low hemolytic activity, make them candidates for development into therapeutically useful anti-infective agents.

North America is home to anuran species belonging to the families Bufonidae, Eleutherodactylidae, Hylidae, Leiopelmatidae, Ranidae, and Scaphiopodidae but antimicrobial peptides have been identified only in skin secretions and/or skin extracts of frogs belonging to the Leiopelmatidae ("tailed frogs") and Ranidae ("true frogs"). Eight structurally-related cationic alpha-helical peptides with broad-spectrum antibacterial activity, termed ascaphins, have been isolated from specimens of Ascaphus truei (Leiopelmatidae) occupying a coastal range. Characterization of orthologous antimicrobial peptides from Ascaphus specimens occupying an inland range supports the proposal that this population should be regarded as a separate species A. montanus. Ascaphin-8 shows potential for development into a therapeutically valuable anti-infective agent. Peptides belonging to the brevinin-1, esculentin-1, esculentin-2, palustrin-1, palustrin-2, ranacyclin, ranatuerin-1, ranatuerin-2, and temporin families have been isolated from North American ranids. It is proposed that "ranalexins" represent brevinin-1 peptides that have undergone a four amino acid residue internal deletion. Current taxonomic recommendations divide North American frogs from the family Ranidae into two genera: Lithobates and Rana. Cladistic analysis based upon the amino acid sequences of the brevinin-1 peptides provides strong support for this assignment.