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
Leucurolysin-a (leuc-a), a 23 kDa non-hemorrhagic metalloproteinase, is found in venom of the viper Bothrops leucurus. Here, we examine the biological consequences of leuc-a, including thrombolytic activity, direct effects on endothelial cells in culture and edematogenic activity in vivo. We demonstrate fibrinolytic activity of leuc-a, in which the protease specifically degrades alpha, beta, and gamma-gamma chains. While not causing hemorrhaging, leuc-a does cause thrombolytic activities in whole blood clots. Endothelial cells are highly resistant to leuc-a in culture. Cell viability suffered only when cells were exposed to large quantities of the protease. Nevertheless, leuc-a induces changes in cell morphology. The impact of leuc-a on cell adhesion was confirmed by an adhesion assay, in which cell adhesion to fibronectin decreased due to leuc-a. This mild cellular impact is unlike that of crude venom, where lower concentrations triggered cell death and a greater reduction in cell adhesion. Also, leuc-a increased microvessel permeability with marked edema in mice peritoneum and foot pads. These effects are similar to those of other P-I class SVPMs. These in vivo effects were weaker when crude venom was tested. In conclusion, albeit not showing significant hemorrhagic activity, leuc-a can induce a prominent edema which appears to be significant in the local effects observed after B. leucurus venom accidents.
Asunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/toxicidad , Fibrinólisis/efectos de los fármacos , Metaloproteasas/toxicidad , Análisis de Varianza , Animales , Adhesión Celular/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/metabolismo , Edema , Fibrina/metabolismo , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Metaloproteasas/aislamiento & purificación , Metaloproteasas/metabolismo , Microvasos/metabolismo , Conejos , Trombina/metabolismoRESUMEN
In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.
Asunto(s)
Bothrops , Venenos de Crotálidos/enzimología , Endopeptidasas , Secuencia de Aminoácidos , Animales , Plaquetas/metabolismo , Venenos de Crotálidos/genética , Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Endopeptidasas/metabolismo , Estabilidad de Enzimas , Fibrina/metabolismo , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Humanos , Laminina/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Plasminógeno/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
This is the first study on the hemolymph from a spider of the Loxosceles genus. These animals are responsible for a great number of envenomation cases worldwide. Several studies on Loxosceles venoms have been published, and the knowledge about the venom and its toxins is considerable, not only regarding the biological and biochemical characterization, but also regarding structural, genetic and phylogenetic approaches. However, the literature on Loxosceles hemolymph is nonexistent. The main goal of the present study was to characterize biochemically the hemolymph content, and especially, to identify its different hemocytes. Moreover, many papers have already shown molecules whose source is the hemolymph and their very interesting activities and biomedical applications, for example, antifungal and antibacterial activities. A 2D-SDS-PAGE of brown spider hemolymph showed approximately 111 spots for pH 3-10 and 150 spots for pH 4-7. A lectin-blotting assay showed that hemolymph carbohydrate residues were similar to those found in venom. Several types of TAG and DAG phospholipids were found in the hemolymph and characterized by HPTLC and mass spectrometry. Four different hemocytes were characterized in Loxosceles intermedia hemolymph: prohemocyte, plasmatocyte, granulocyte and adipohemocyte. This paper opens new possibilities on toxinology, studying an unknown biological material, and it characterizes a source of molecules with putative biotechnological applications.