Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

Abstract

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.

Ancient Cytokines, the Role of Astakines as Hematopoietic Growth Factors

“…One study of the venom of two Australian funnel web spiders did focus on the function of astakine-like peptides, the so-called MIT-like atracotoxins (24). Like other invertebrate astakines, these peptides lack the N-terminal motif (AVIT) present in vertebrate prokineticins and accordingly did not affect smooth muscle contractility or restrain contractions induced by human PK1 in guinea pig ileum (23,24).…”

Bv8/Prokineticin proteins and their receptors

“…Intriguing groups of proteins which share the colipase fold and some sequence identity and homology with MIT-Bv8-PKs have been identified in the venom of Australian funnel-web spiders Hadronyche sp. (Szeto et al, 2000;Wen et al, 2005), in the cnidarian allomonal system (Sher et al, 2005) and in hemocytes of shrimps (Soderhall et al, 2005). Because all the invertebrate homologues lack the six conserved N-terminal amino acids, they do not activate the mammalian prokineticin receptors.…”

The Toxicogenomic Multiverse: Convergent Recruitment of Proteins Into Animal Venoms

“…In a longer time frame, Bv8 additionally shows an anorexogenic effect (108), inhibiting the feeding of the affected predator, even in food-deprived situations. Venom of both mygalomorph and araneomorph spider taxa contain peptides (MIT-like atracotoxins, ACTX) that show a similar motif as vertebrate AVIT peptides consisting of 10 conserved cysteine residues (148,162). However, these peptides test negative in assays of PK1/PK2 activity, lack the N-terminal AVIT sequence, and are much more diverse in amino acid composition, sequence length, and scaffold shape.…”