RESUMEN
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.
Asunto(s)
Araña Reclusa Parda/genética , Miniproteínas Nodales de Cistina/química , Insecticidas/análisis , Hidrolasas Diéster Fosfóricas/química , Venenos de Araña/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Araña Reclusa Parda/metabolismo , Secuencia Conservada , Miniproteínas Nodales de Cistina/biosíntesis , Miniproteínas Nodales de Cistina/genética , Miniproteínas Nodales de Cistina/aislamiento & purificación , Dípteros , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Datos de Secuencia Molecular , Proteoma , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Pruebas de Toxicidad , TranscriptomaRESUMEN
Malignant hyperthermia is a pharmacogenetic disorder associated with mutations in Ca(2+) regulatory proteins. It manifests as a hypermetabolic crisis triggered by commonly used anesthetics. Malignant hyperthermia susceptibility is a dominantly inherited predisposition to malignant hyperthermia that can be diagnosed by using caffeine/halothane contracture tests. In a multigenerational North American family with a severe form of malignant hyperthermia that has caused four deaths, a novel RYR1 A2350T missense mutation was identified in all individuals testing positive for malignant hyperthermia susceptibility. The same A2350T mutation was identified in an Argentinean family with two known fatal MH reactions. Functional analysis in HEK-293 cells revealed an altered Ca(2+) dependence and increased caffeine sensitivity of the expressed mutant protein thus confirming the pathogenic potential of the RYR1 A2350T mutation.