ω-Tbo-IT1–New Inhibitor of Insect Calcium Channels Isolated from Spider Venom

Abstract

Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 μg/g in experiments on house fly Musca domestica larvae and with LD50 20 μg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca2+ currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca2+ current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca2+ channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended β-hairpin loop and short β-hairpin loop which are capable of making “scissors-like mutual motions”.

New Insectotoxin from Tibellus Oblongus Spider Venom Presents Novel Adaptation of ICK Fold

“…Earlier, we established a high insectoactivity of the whole venom of the spider T. oblongus [ 17 ]. Crude venom toxicity to M. domestica larvae was estimated as LD 50 10 μg/g for polypeptide fractions and several insectotoxic peptides were determined.…”

“…The previously characterized 41-residue toxin ω-Tbo-IT1 demonstrated significant toxicity to insects. The activity of ω-Tbo-IT1 to M. domestica larvae was LD 50 19 μg/g [ 17 ]. Moreover, the action of ω-Tbo-IT1 on insect presynaptic Ca 2+ channels was demonstrated, which allowed us to attribute the ω-Tbo-IT1 to the ω-group of toxins and inhibited voltage-gated calcium (Ca V ) channels, according to the toxin’s classification [ 41 ].…”

“…T. oblongus , as well as all other species of the Tibellus genus, is an active predator that does not spin webs, but actively pursues its prey. Earlier, we described T. oblongus insectotoxin ω-Tbo-IT1 [ 17 ]—the only studied toxin from the entire Philodromidae family to date. Here, we present the second toxin found in the diversity of T. oblongus spider toxins using transcriptome and proteome analysis methods.…”

Identification of unusual peptides with new Cys frameworks in the venom of the cold-water sea anemone Cnidopus japonicus

“…To test the toxic activity of the peptides, we used aquatic and terrestrial animals: the shrimp 64 Caridina multidentata and fly larva 65 of Musca domestica 66 , respectively. All of the recombinant sea anemone toxins produced neurotoxic effects on the shrimp, ranging from a slight twitching of the walking legs (pereiopods) to immediate and severe paralysis, followed by death.…”

“…7 and Table S10 ). The insecticidal spider toxin ω-Tbo-IT1 (ω-phylotoxin-To1a) 66 and δ-actitoxin-Cgg1a 67 were used as positive controls. Both toxins showed potent toxic activity towards the insects (LD 100 of 5 μg/g for δ-actitoxin-Cgg1a and 30 μg/g for ω-Tbo-IT1, see Fig.…”