Abstract
The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupiennius salei venom.
Highlights
With more than 48,000 species of terrestrial arthropods, spiders are the most diverse group after insects [1] and this success is often explained by their unique combination of silk and venomToxins 2020, 12, 250; doi:10.3390/toxins12040250 www.mdpi.com/journal/toxinsToxins 2020, 12, 250 glands [2]
In bioassays with Drosophila flies, we demonstrated a comparable peptide–peptide interaction of CsTx-13 when co-injected with CsTx-1 or CsTx-9
With the exclusion of the C-terminal α-helix of CsTx-13 alone as a driving force for the observed strong cytolytic effects between CsTx-13 and CsTx-1/9, we focused on the inhibitor cystine knot (ICK) motif of both peptide types
Summary
With more than 48,000 species of terrestrial arthropods, spiders are the most diverse group after insects [1] and this success is often explained by their unique combination of silk and venom. In the venom of Cupiennius salei, 76% of all cysteine containing neurotoxin-like transcripts exhibit, besides the N-terminally ICK motif, C-terminally an α-helical motif [6] They are composed of three disulfide bridges as C1-C4, C2-C5, and C3-C8 and an additional fourth disulfide bridge C6-C7 (Figure 1). The second structural type possesses, besides a two-domain architecture, a further post-translational modification in which the loop between the disulfide bridge C6-C7 is post-translationally opened by a specific protease This protease recognizes a PQM and an inverted PQM motif within this loop, which results in a heterodimeric peptide as reported for CsTx-13 [6,8,9]. We show that remarkable interactions between structural type 1 (or 3) and type 2 toxins, described as neurotoxin merging, lead to a strong increase of venom toxicity This result offers new access to the high diversity of venom compounds.
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