Kasstasin

Amphibian skin is a rich and unique source of novel bioactive peptides most of which are endowed with either antimicrobial or pharmacological properties. Here, we report the identification and structural characterization of a novel peptide, named senegalin, which possesses both activities. Senegalin is a hexadecapeptide amide (FLPFLIPALTSLISSL-NH2) of unique primary structure found in the skin secretion of the African running frog, Kassina senegalensis. The structure of the biosynthetic precursor of senegalin, deduced from cloned skin cDNA, consists of 76 amino acid residues and displays the typical domain organization of an amphibian skin peptide precursor. Both natural senegalin and its synthetic replicate displayed antimicrobial and myotropic activities. Senegalin was active against Staphylococcus aureus (MIC 50 μM) and Candida albicans (MIC 150 μM) but was non-haemolytic at concentrations up to and including 150 μM. In contrast, senegalin induced a dose-dependent contraction of rat urinary bladder smooth muscle (EC50 2.9 nM) and a dose-dependent relaxation of rat tail artery smooth muscle (EC50 37.7 nM). Senegalin thus represents a prototype biologically active amphibian skin peptide and illustrates the fact that amphibian skin secretion peptidomes continue to be unique sources of such molecules.

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC(50) = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.

We describe the isolation and structural characterization of a family of antimicrobial peptides related to kassinatuerin-2, from the skin secretion of the African hyperoliid frog, Kassina maculata. All four peptides, designated kassinatuerin-2Ma through Md, are C-terminally-amidated 20-mers with the consensus sequence - FX(1)GAIAAALPHVIX(2)AIKNAL - where X(1)=L/F/V/I and X2=S/N. All four peptides are encoded by precursors of 69 amino acids. Synthetic replicates of all kassinatuerin-2 related peptides displayed a potent inhibitory activity against Staphylococcus aureus with a minimal inhibitory concentration of 16microM, at which concentration, however, they effected 18% haemolysis of horse erythrocytes after 2h. Despite obvious membranolytic properties, all peptides were ineffective at inhibiting the growth of Escherichia coli at concentrations up to 200microM and were relatively ineffective against Candida albicans (MIC 120microM). The kassinatuerin-2 related peptides of K. maculata skin secretion thus possess a discrete antimicrobial and weak haemolytic activity in contrast to the prototype kassinatuerin-2 from the skin secretion of Kassina senegalensis.