Abstract
Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure—GMRPPWF-NH2—was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μM). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.
Highlights
Amphibians represent the most ancient group of terrestrial vertebrates and are widely distributed globally, occurring on all continents with the exception of Antarctica [1, 2]
Frogs are difficult to see as they press themselves against leaves and pull their limbs tightly towards their bodies, becoming a green blob on the leaf surface
Several peptides with a range of biological effects have been reported from these skin secretions and these include both antimicrobial and pharmacologically active peptides [1, 2, 16]
Summary
Amphibians represent the most ancient group of terrestrial vertebrates and are widely distributed globally, occurring on all continents with the exception of Antarctica [1, 2]. The unlocking of the molecular secrets of amphibian defensive skin secretions was predominantly initiated by two, twentieth-century pioneer pharmacological chemists— Vittorio Erspamer (1909–1999) (endogenous peptides and biogenic amines) and John Daly (1933–2008) (exogenous alkaloids) Their combined efforts resulted in the unravelling of the molecular structures and biological actions of several thousands of such molecules representing many classes of biochemical, studies on which have served to reveal many novel regulatory systems and potential drug targets in mammalian systems [1,2,3,4]. We describe the identification, structural, and functional characterization of a novel Type-2 tryptophyllin, named AcT-2, from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas This peptide was first identified through pharmacological screening for myotropic activity and was subsequently, in addition, found to have a broad spectrum of antimicrobial activity, albeit relatively low potency. The peptide was found to possess a higher potency against the yeast, Candida albicans, than against the model Gram-positive and Gram-negative bacteria employed
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