1. Amphibian peptides that inhibit neuronal nitric oxide synthase. Isolation of lesuerin from the skin secretion of the Australian Stony Creek frog Litoria lesueuri
Jason Doyle, Lyndon E Llewellyn, Craig S Brinkworth, John H Bowie, Kate L Wegener, Tomas Rozek, Paul A Wabnitz, John C Wallace, Michael J Tyler Eur J Biochem. 2002 Jan;269(1):100-9. doi: 10.1046/j.0014-2956.2002.02630.x.
Two neuropeptides have been isolated and identified from the secretions of the skin glands of the Stony Creek Frog Litoria lesueuri. The first of these, the known neuropeptide caerulein 1.1, is a common constituent of anuran skin secretions, and has the sequence pEQY(SO3)TGWMDF-NH2. This neuropeptide is smooth muscle active, an analgaesic more potent than morphine and is also thought to be a hormone. The second neuropeptide, a new peptide, has been named lesueurin and has the primary structure GLLDILKKVGKVA-NH2. Lesueurin shows no significant antibiotic or anticancer activity, but inhibits the formation of the ubiquitous chemical messenger nitric oxide from neuronal nitric oxide synthase (nNOS) at IC(50) (16.2 microm), and is the first amphibian peptide reported to show inhibition of nNOS. As a consequence of this activity, we have tested other peptides previously isolated from Australian amphibians for nNOS inhibition. There are three groups of peptides that inhibit nNOS (IC(50) at microm concentrations): these are (a) the citropin/aurein type peptides (of which lesueurin is a member), e.g. citropin 1.1 (GLFDVIKKVASVIGGL-NH(2)) (8.2 microm); (b) the frenatin type peptides, e.g. frenatin 3 (GLMSVLGHAVGNVLG GLFKPK-OH) (6.8 microm); and (c) the caerin 1 peptides, e.g. caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL-NH(2)) (1.7 microm). From Lineweaver-Burk plots, the mechanism of inhibition is revealed as noncompetitive with respect to the nNOS substrate arginine. When the nNOS inhibition tests with the three peptides outlined above were carried out in the presence of increasing concentrations of Ca(2+) calmodulin, the inhibition dropped by approximately 50% in each case. In addition, these peptides also inhibit the activity of calcineurin, another enzyme that requires the presence of the regulatory protein Ca(2+) calmodulin. It is proposed that the amphibian peptides inhibit nNOS by interacting with Ca(2+)calmodulin, and as a consequence, blocks the attachment of this protein to the calmodulin domain of nNOS.
2. The structures of the frenatin peptides from the skin secretion of the giant tree frog Litoria infrafrenata
M J Raftery, R J Waugh, J H Bowie, J C Wallace, M J Tyler J Pept Sci. 1996 Mar-Apr;2(2):117-24. doi: 10.1002/psc.60.
The granular dorsal glands of the giant tree frog Litoria infrafrenata contain five peptides including caerulein (a known neuropeptide), and four new peptides named franatins 1 (MH+ = 1140 Da), 2 (1423), 3 (2180), 4 (2493). The amino acid sequences of the frenatins are detailed: their structures do not correspond to those of peptides isolated from other amphibians or animals. Frenatin 3, Gly-Leu-Met-Ser-Val-Leu -Gly-His-Ala-Val-Gly-Asn-Val-Leu-Gly-Gly-Leu-Phe-Lys-Ser-(OH), has wide spectrum antimicrobial properties.
3. The solution structure of frenatin 3, a neuronal nitric oxide synthase inhibitor from the giant tree frog, Litoria infrafrenata
Craig S Brinkworth, John A Carver, Kate L Wegener, Jason Doyle, Lyndon E Llewellyn, John H Bowie Biopolymers. 2003 Oct;70(3):424-34. doi: 10.1002/bip.10524.
The peptide frenatin 3 is a major component of the skin secretion of the Australian giant tree frog, Litoria infrafrenata. Frenatin 3 is 22 amino acids in length, and shows neither antimicrobial nor anticancer activity. It inhibits the production of nitric oxide by the enzyme neuronal nitric oxide synthase at a micromolar concentration by binding to its regulatory protein, Ca2+ calmodulin, a protein known to recognize and bind amphipathic alpha-helices. The solution structure of frenatin 3 has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. In trifluoroethanol/water mixtures, the peptide forms an amphipathic alpha-helix over residues 1-14 while the C-terminal eight residues are more flexible and less structured. The flexible region may be responsible for the lack of antimicrobial activity. In water, frenatin 3 exhibits some alpha-helical character in its N-terminal region.
