An immunomodulatory peptide related to frenatin 2 from skin secretions of the Tyrrhenian painted frog Discoglossus sardus (Alytidae)
Abstract
Norepinephrine-stimulated skin secretions of the Tyrrhenian painted frog Discoglossus sardus Tschudi, 1837 (Alytidae) did not contain any peptide with antimicrobial or hemolytic activity. However, peptidomic analysis of the secretions revealed the presence of an abundant peptide with structural similarity to frenatin 2, previously isolated from the Australian frog Litoria infrafrenata (Hylidae). The primary structure of the peptide, termed frenatin 2D, was established as DLLGTLGNLPLPFI.NH2 by automated Edman degradation and mass spectrometry with electron-transfer dissociation (ETD)-based fragmentation and confirmed by chemical synthesis. The structure of a second frenatin 2-related peptide, termed frenatin 2.1D, that was present in much lower abundance was established as GTLGNLPAPFPG. Frenatin 2D (20 μg/ml) significantly stimulated production of the proinflammatory cytokines TNF-α (P<0.05) and IL-1β (P<0.01) by mouse peritoneal macrophages but the peptide did not potentiate the stimulation produced by lipopolysaccharide (LPS). The peptide increased IL-12 production in both unstimulated (P<0.01) and LPS-stimulated (P<0.05) cells but stimulatory effects on IL-6 production were not significant. The biological role of frenatin 2D is unknown but it is speculated that the peptide acts on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms.