A novel ICK peptide from the Loxosceles intermedia (brown spider) venom gland: Cloning, heterologous expression and immunological cross-reactivity approaches

Matsubara, Fernando Hitomi; Gremski, Luiza Helena; Meissner, Gabriel Otto; Lopes, Eduardo Soares Constantino; Gremski, Waldemiro; Senff‐Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Sílvio Sanches

doi:10.1016/j.toxicon.2013.05.014

Cited by 25 publications

(28 citation statements)

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“…Giving further support to this observation, Matsubara et al . () and Buch et al . () showed antigenic cross‐reactivity between Li1b and antivenom from L. intermedia , L. gaucho and L. laeta , strengthening the idea that knottin peptides constitute a toxin family that is widespread throughout the genus.…”

Section: Discussionmentioning

confidence: 99%

“…The cDNA sequence of Li1b was obtained from Matsubara et al . (). Primers were designed to clone this sequence into the pLicC–NSB2 vector, which is designed to allow the toxin to be expressed as a His 6 ‐tagged MBP fusion protein, with a TEV protease recognition site between the His 6 ‐MBP and Li1b encoding regions, The forward primer was 5′‐G GGT ACC GAA AAC CTG TAT TTT CAG GGA TGC ATT AAA TCT G‐3′; the restriction site for Kpn I is highlighted in italics and the TEV protease site is underlined.…”

Section: Methodsmentioning

confidence: 99%

“…However, the Li1b was not found to be active in any of the in vitro or in vivo assays performed. Moreover, dimers of Li1b were observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and western blotting, even under reducing conditions, suggesting the formation of non‐native intermolecular disulphide bonds (Matsubara et al ., ).…”

Section: Introductionmentioning

confidence: 97%

“…Some identified toxins are well characterized from a biochemical and biological perspective, such as phospholipase D, astacin-like metalloproteases, hyaluronidase and translationally-controlled tumor protein (Chaim et al, 2006;Trevisan-Silva et al, 2010;Sade et al, 2011;Ferrer et al, 2013;Vuitika et al, 2013). Other Loxosceles venom toxins have been identified from transcriptomic analyses, such as knottin peptides, allergens, serine proteases, serine protease inhibitors and Clectins, but there is little information about their activities both in vitro and in vivo (Fernandes-Pedrosa et al, 2008;Gremski et al, 2010;Matsubara et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…The latest report on knottin peptides from Loxosceles spiders was conducted by Matsubara et al (2013). These authors cloned the peptide U 2 -sicaritoxin-Li1b (hereafter Li1b), which is similar to LiTx3.…”

Section: Introductionmentioning

confidence: 99%

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Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus

Matsubara

Meissner

Herzig

et al. 2016

Insect Molecular Biology

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Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.

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Section: Discussionmentioning

confidence: 99%