Biologically active peptides from Australian amphibians.

Abstract

Amphibians secrete potent host defence compounds from dorsal glands onto the skin when stressed, sick or under attack by predators and microbials. Many of these defence compounds, such as biologically active peptides, provide potential targets for new biotechnological and therapeutic investigation. The research presented in this study focuses on the isolation and investigation of peptides from Australian frogs of the genera Litoria and Crinia and endeavours to determine the biological activity and important structural and mechanistic features of these biological compounds. Isolation and identification of the skin peptide profile of the Eastern Dwarf Tree Frog Litoria fallax has revealed a number of novel peptides named fallaxidins. This frog species is quite unique in that it does not secrete a peptide that displays potent broad spectrum antimicrobial activity nor a peptide that inhibits nitric oxide formation through the enzyme neuronal nitric oxide synthase. Instead it secretes several narrow spectrum antimicrobial peptides, including fallaxidin 3.1. In addition, there are numerous small peptides displaying unique primary structures with unknown biological function. Interestingly, L. fallax produces a skin peptide profile that is quite distinct from the skin peptide profiles of other related Litoria species. The majority of anurans from the Litoria genus contain at least one peptide in their skin secretion that inhibits the enzyme neuronal nitric oxide synthase. These peptides exert this action by preventing the association of the regulatory cofactor Ca²⁺ calmodulin to the enzyme binding site. The non-covalent binding of the potent neuronal nitric oxide synthase inhibitor dahlein 5.6 (L. dahlii) to calmodulin in the presence of Ca²⁺ is confirmed by electrospray ionisation mass spectrometry. A peptide-protein complex was observed in the gas-phase with a 1:1:4 calmodulin/dahlein 5.6/Ca²⁺ stoichiometry. In addition, the structure and binding interactions have been investigated by means of nuclear magnetic resonance spectroscopy. These experiments illustrated that upon binding dahlein 5.6, Ca²⁺ calmodulin undergoes a substantial conformational transition towards a globular complex with the helical dahlein 5.6 engulfed in a hydrophobic channel. Typically, the granular secretion of amphibians contains numerous peptides that exert activities in the central nervous system, termed neuropeptides. The biological activities, in particular smooth muscle action, proliferation of lymphocytes and opioid action are investigated to provide insight into the role of these peptides in the host defence. The structure activity relationships of disulfide peptides, caerulein peptides, tryptophyllins, rothein 1 and its related synthetically modified peptides has identified several important structural features essential for their corresponding biological function. Peptides from the granular secretion of anurans are synthesized within and released from larger precursors molecules. The genes that encode for the skin peptides of Crinia riparia and several Litoria species were isolated and identified. The cDNA sequence of the precursors provides a mechanism by which the evolution of amphibian species can be traced and information about the relationships existing among closely or distantly related species be obtained. All prepropeptides isolated from the Litoria species illustrated sequence homology to those isolated from numerous ranid and hylid frogs and demonstrate that the skin prepropeptides originated from a common ancestral gene. The precursors of peptides from C. riparia are significantly diverse and suggest that these prepropeptides either originated from the same common ancestral gene but have undergone substantial divergent evolution relative to the ranid and hylid frogs or that they have originated from distinct ancestral genes.