Abstract
The Tibellus oblongus spider is an active predator that does not spin webs and remains poorly investigated in terms of venom composition. Here, we present a new toxin, named Tbo-IT2, predicted by cDNA analysis of venom glands transcriptome. The presence of Tbo-IT2 in the venom was confirmed by proteomic analyses using the LC-MS and MS/MS techniques. The distinctive features of Tbo-IT2 are the low similarity of primary structure with known animal toxins and the unusual motif of 10 cysteine residues distribution. Recombinant Tbo-IT2 (rTbo-IT2), produced in E. coli using the thioredoxin fusion protein strategy, was structurally and functionally studied. rTbo-IT2 showed insecticidal activity on larvae of the housefly Musca domestica (LD100 200 μg/g) and no activity on the panel of expressed neuronal receptors and ion channels. The spatial structure of the peptide was determined in a water solution by NMR spectroscopy. The Tbo-IT2 structure is a new example of evolutionary adaptation of a well-known inhibitor cystine knot (ICK) fold to 5 disulfide bonds configuration, which determines additional conformational stability and gives opportunities for insectotoxicity and probably some other interesting features.
Highlights
Spiders are the second-largest taxonomic group of terrestrial organisms after insects
We report recombinant toxin production and 3D structure determination of a novel insecticidal 10-Cys polypeptide—rTbo-IT2, which adopted inhibitor cystine knot (ICK) fold with 5 disulfide bridges
As a result of automatic Sanger sequencing of cloned cDNA, a database of expressed sequence tags (EST) corresponding to the peptides encoded in the mRNA was obtained
Summary
Spiders are the second-largest taxonomic group of terrestrial organisms after insects. It has been shown that each species of spider has at least 100 unique peptide toxins in its venom [1]. The total number of individual spider toxins seems to be quite amazing. All this potential diversity of biologically active peptides remains poorly investigated. ArachnoServer (http://www.arachnoserver.org) reports about 1576 spider toxins (on November 2020) from 100 different spider species studied to date, but less than 490 toxins have a described target. It is a tiny part of all spider venom toxins, among which, as believed, molecules that act on almost all molecular targets can be found
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