Molecular cloning and in silico characterization of knottin peptide, U2-SCRTX-Lit2, from brown spider (Loxosceles intermedia) venom glands

Abstract

Inhibitor cystine knots (ICKs) are a family of structural peptides with a large number of cysteine residues that form intramolecular disulfide bonds, resulting in a knot. These peptides are involved in a variety of biological functions including predation and defense, and are found in various species, such as spiders, scorpions, sea anemones, and plants. The Loxosceles intermedia venom gland transcriptome identified five groups of ICK peptides that represent more than 50 % of toxin-coding transcripts. Here, we describe the molecular cloning of U2-Sicaritoxin-Lit2 (U2-SCRTX-Lit2), bioinformatic characterization, structure prediction, and molecular dynamic analysis. The sequence of U2-SCRTX-Lit2 obtained from the transcriptome is similar to that of μ-Hexatoxin-Mg2, a peptide that inhibits the insect Nav channel. Bioinformatic analysis of sequences classified as ICK family members also showed a conservation of cysteine residues among ICKs from different spiders, with the three dimensional molecular model of U2-SCRTX-Lit2 similar in structure to the hexatoxin from μ-hexatoxin-Mg2a. Molecular docking experiments showed the interaction of U2-SCRTX-Lit2 to its predictable target-the Spodoptera litura voltage-gated sodium channel (SlNaVSC). After 200 ns of molecular dynamic simulation, the final structure of the complex showed stability in agreement with the experimental data. The above analysis corroborates the existence of a peptide toxin with insecticidal activity from a novel ICK family in L. intermedia venom and demonstrates that this peptide targets Nav channels.

Highlights in the knowledge of brown spider toxins

“…The recombinant peptide, named U2-sicaritoxin-Li1b (U2- SCRTX-Li1b), was used as a tool that enabled the demonstration of an antigenic cross-reactivity of antisera raised against crude venom of L. intermedia , L. gaucho, and L. laeta with U2-SCRTX-Li1b. This cross-reactivity corroborates the presence of ICK-like toxin members in these Loxosceles venoms, thus strengthening the idea that this toxin family is widespread throughout the genus [ 105 , 106 ].…”

Loxosceles gaucho Spider Venom: An Untapped Source of Antimicrobial Agents

“…To our knowledge, U 1 -SCRTX-Lg1a is the first antimicrobial peptide isolated from the venom of L. gaucho , an araneomorph spider that belongs to the Sicariidae family. Before this study, the only peptides reported for Loxosceles spiders were ICKs [24,52,53], a family of structural peptides with several cysteine residues that form disulfide bonds that result in a knot, which have generated great interest due to their ability to specifically bind to insect ion channels, conferring them great potential for the development of efficient bioinsecticides for the control of pests that can affect the agricultural sector or insects vectors of infectious diseases [20,21]. Considering this, it is safe to say that U 1 -SCRTX-Lg1a is the newest member of the group of bioactive peptides isolated from the venom of Loxosceles spiders.…”

“…To date, many brown spider toxins have been described and their corresponding biochemical properties have been characterized, providing key information about their great potential for biotechnological purposes such as the design of pharmacological tools, diagnostic and immunotherapeutic reagents, cytotoxicity inducers, and biopesticides [20,21,25]. Included among these molecules are phospholipases D [26,27,28,29,30,31,32,33,34,35], astacins (metalloproteases) [36,37,38,39,40,41], hyaluronidases [42,43,44,45,46], serine proteases [44,47,48,49,50], translationally controlled tumor protein (TCTP) [47,48,51], and inhibitor cystine knot (ICK) peptides [24,52,53].…”

Molecular, Biological and Structural Features of VL CDR-1 Rb44 Peptide, Which Targets the Microtubule Network in Melanoma Cells

“…Hex depicts proteins as rigid bodies and makes a blind search through protein surface while it evaluates the interaction correlation by using the fast Fourier transformation algorithm. As described in Meissner et al (45), solvation and desolvation effects were treated as surface phenomena, since the Hex algorithm models the interaction, excluding volume and complementarity of form. Approximately 350 solutions were found for each combination.…”