RESUMO
In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.
Assuntos
Venenos de Aranha , Aranhas , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos/química , Antivenenos/química , Reações Cruzadas , Miniproteínas Nó de Cistina/química , Fosfolipase D/química , Venenos de Aranha/química , Aranhas/química , Epitopos/químicaRESUMO
Phα1ß (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1ß administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1ß and its 15N-labeled analogue. Spatial structure and dynamics of Phα1ß were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1ß structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1ß has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1ß significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1ß as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.
Assuntos
Miniproteínas Nó de Cistina , Venenos de Aranha , Aranhas , Toxinas Biológicas , Ratos , Animais , Canal de Cátion TRPA1/genética , Aranhas/química , Neurotoxinas , Espectroscopia de Ressonância Magnética , Dissulfetos , Venenos de Aranha/farmacologia , Venenos de Aranha/químicaRESUMO
Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.
Assuntos
Miniproteínas Nó de Cistina/química , Proteínas de Plantas/química , Proteômica , Proteínas Recombinantes , Solanum tuberosum/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Animais , Carboxipeptidases/antagonistas & inibidores , Bovinos , Clonagem Molecular , Miniproteínas Nó de Cistina/análise , Miniproteínas Nó de Cistina/genética , Miniproteínas Nó de Cistina/isolamento & purificação , Ativação Enzimática/efeitos dos fármacos , Cinética , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Inibidores de Proteases/análise , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Proteômica/métodos , Análise de Sequência de DNA , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , SuínosRESUMO
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 U2 -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 U2 -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.
Assuntos
Aranha Marrom Reclusa/genética , Miniproteínas Nó de Cistina/química , Inseticidas/análise , Diester Fosfórico Hidrolases/química , Venenos de Aranha/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Aranha Marrom Reclusa/metabolismo , Sequência Conservada , Miniproteínas Nó de Cistina/biossíntese , Miniproteínas Nó de Cistina/genética , Miniproteínas Nó de Cistina/isolamento & purificação , Dípteros , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Dados de Sequência Molecular , Proteoma , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Testes de Toxicidade , TranscriptomaRESUMO
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.