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

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.

Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity.

Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of symptoms found in victims after accidents is called loxoscelism and is characterized by two clinical conditions: cutaneous loxoscelism and systemic loxocelism. The only specific treatment is serum therapy, in which an antiserum produced with Loxosceles venom is administered to the victims after spider accidents. Our aim was to improve our knowledge, regarding the immunological relationship among toxins from the most epidemiologic important species in Brazil (Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta). Immunoassays using spider venoms and L. intermedia recombinant toxins were performed and their cross-reactivity assessed. The biological conservation of the main Loxosceles toxins (Phospholipases-D, Astacin-like metalloproteases, Hyaluronidase, ICK-insecticide peptide and TCTP-histamine releasing factor) were investigated. An in silico analysis of the putative epitopes was performed and is discussed on the basis of the experimental results. Our data is an immunological investigation in light of biological conservation throughout the Loxosceles genus. The results bring out new insights on brown spider venom toxins for study, diagnosis and treatment of loxoscelism and putative biotechnological applications concerning immune conserved features in the toxins.

Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins.

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.

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

The venom of a Loxosceles spider is composed of a complex mixture of biologically active components, consisting predominantly of low molecular mass molecules (3-45 kDa). Transcriptome analysis of the Loxosceles intermedia venom gland revealed ESTs with similarity to the previously described LiTx peptides. Sequences similar to the LiTx3 isoform were the most abundant, representing approximately 13.9% of all ESTs and 32% of the toxin-encoding messengers. These peptides are grouped in the ICK (Inhibitor Cystine Knot) family, which contains single chain molecules with low molecular mass (3-10 kDa). Due to their high number of cysteine residues, ICK peptides form intramolecular disulfide bridges. The aims of this study were to clone and express a novel ICK peptide isoform, as well as produce specific hyperimmune serum for immunoassays. The corresponding cDNA was amplified by PCR using specific primers containing restriction sites for the XhoI and BamHI enzymes; this PCR product was then ligated in the pET-14b vector and transformed into E. coli AD494 (DE3) cells. The peptide was expressed by IPTG induction for 4 h at 30 °C and purified by affinity chromatography with Ni-NTA resin. Hyperimmune serum to the recombinant peptide was produced in rabbits and was able to specifically recognize both the purified recombinant peptide and the native form present in the venom. Furthermore, the recombinant peptide was recognized by antisera raised against L. intermedia, L. gaucho and L. laeta whole venoms. The recombinant peptide obtained will enable future studies to characterize its biological activity, as well as investigations regarding possible biotechnological applications.