Abbreviations: ACh, acetylcholine; ACTX, atracotoxin; BCA, bicinchoninic acid; Bv8, 8kDa protein from the skin secretions of toads (Bombina variegata); CHO, Chinese hamster ovary; DDH, disulfidedirected -hairpin; EST, expressed sequence tag; FLIPR, fluorometric imaging plate reader; HEK, human embryonic kidney; ICK, inhibitory cystine-knot; MIT1, mamba intestinal toxin 1 from the venom of the black mamba snake Dendroaspis p. polylepis; PK, prokineticin (also known as endocrine-gland vascular endothelial growth factor or EG-VEGF); PKR, prokineticin receptor; RACE, rapid amplification of cDNA ends; TCEP, Tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid. Atracinae), that appear to undergo N-and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 GM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1).
KeywordsThe peptide also lacked activity on other isolated smooth muscle preparations including rat aorta.Furthermore, a FLIPR Ca 2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed -hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.
