Crystal Structure of Crotoxin Reveals Key Residues Involved in the Stability and Toxicity of This Potent Heterodimeric β-Neurotoxin

Faure, Grazyna; Xu, Haijin; Saul, F.A.

doi:10.1016/j.jmb.2011.07.027

Cited by 92 publications

(75 citation statements)

References 59 publications

(83 reference statements)

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“…Crotapotins bind specifically to PLA 2 as a natural inhibitor of catalytic activity [41]. We suggest that crotapotin might interact in a less stable way, thus partially avoiding the substrate access to the catalytic site and hiding several key amino acid residues involved in the interfacial binding surface of PLA 2 s. Crotapotin isoforms from Crotalus d. collilineatus (Cdcol F3 and Cdcol F4) and Crotalus d. terrificus (CdtF5 and Cdt F7) significantly inhibit the Btae TX-I activity by approximately 60%.…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

Romero-Vargas¹,

Rocha²,

Cruz‐Höfling

et al. 2014

J Clin Toxicol

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In this research a preliminary identification and biochemical and biological characterization of a PLA 2 (Btae TX-I) from the venom of a viperid snake, Bothriopsis taeniata (Speckled forest pit viper) were obtained. Btae TX-I was purified by two chromatographic steps, molecular exclusion chromatography followed by analytical chromatography reverse phase HPLC. Molecular mass behaved as a homogeneous single chain protein on SDS-PAGE, confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 13889.98 Da. Tryptic peptides were determined in tandem mass spectrometry and showed similarity with other myotoxic PLA 2 s. Btae TX-I belongs to the Asp49 PLA 2 class, is enzymatically active in presence of a synthetic substrate and shows a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45°C. PLA 2 activity in presence of Mn 2+ , Mg 2+ , Cd 2+ and Zn 2+ was reduced either in presence or absence of Ca 2+ , suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca 2+ . Crotalic crotapotins from rattlesnake venom has significantly inhibited (p<0.05) the enzymatic activity of Btae TX-I. In ex vivo experiment, Btae TX-I caused partial blockade of the neuromuscular transmission in chick biventer cervicis preparations in a similar way to other Bothrops species. Btae TX-I also inhibited contractures in the upper concentration (50 µg) to exogenous KCl (20 mM). Histological analysis of the biventer cervicis incubated with Btae TX-I showed that just the highest Btae TX-I PLA 2 dose (50 µg) caused almost 27.4 ± 0.3% damaged fibers. The results give evidence that the main effect of type Asp49 Btae TX-I PLA 2 from Bothriopsis taeniata is at the post-synaptic site.

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Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

Romero-Vargas¹,

Rocha²,

Cruz‐Höfling

et al. 2014

J Clin Toxicol

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“…Recent studies have reported a possible antitumor function for crotoxin [10][11][12][13][14] , although the specific mechanism responsible for this activity remains unclear. However, it is possible that it may act via the release of cytochrome c and the activation of caspase-3, thus destroying the mitochondrial membrane potential of the tumor cells.…”

Section: Discussionmentioning

confidence: 99%

Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice

Wang

et al. 2012

Acta Pharmacol Sin

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Aim: To investigate the antitumor actions of the Crotalus durissus neurotoxin (crotoxin) on human esophageal carcinoma (Eca-109) cells in vitro and transplanted esophageal Eca-109 tumors in nude mice. Methods: The growth-inhibitory effect was analyzed in Eca-109 cells using MTT assay. Cell morphology changes in nuclei were observed using Hoechst 33342 staining, while apoptosis and cell cycle distribution were examined by flow cytometry. RT-PCR was used to measure the Bcl-2, p15, and caspase-3 p17 gene expression levels. A tumor transplantation model was established by inoculation of Eca-109 cells were into female Balb/c nude mice. Crotoxin (25, 50, and 100 mg/kg) was subcutaneously injected into the transplanted tumors every 2 d for a total of 10 injections. Tumor size and weight were measured. Bcl-2, p15, and caspase-3 p17 protein expression in transplanted tumors was analyzed using Western blotting. Results: Crotoxin (25, 50, and 100 µg/mL) inhibited the growth of Eca-109 cells in a dose-dependent manner with inhibition rates of 22.9%, 35.8%, and 57.2%, respectively. Hoechst 33342 staining revealed apoptotic cells with pyknotic nuclear chromatin after crotoxin treatment. In Eca-109 cells, crotoxin induced apoptosis and G1 block, significantly upregulated the expression of p15 and caspase-3 p17 genes and downregulated the expression of Bcl-2 gene. Furthermore, crotoxin inhibited the growth of Eca-109 tumors in nude mice in a dose-dependent manner. Western blotting showed that crotoxin increased p15 and caspase-3 p17 protein levels and reduced Bcl-2 protein level in tumor specimens. Conclusion: Crotoxin inhibits the growth of Eca-109 cells in vitro via apoptosis induction and G 1 block. Local administration of crotoxin inhibits the growth of subcutaneously transplanted Eca-109 cells in nude mice, possibly via increasing p15 and caspase-3 p17 protein expression and reducing Bcl-2 protein expression.

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“…En este trabajo se aisló e identificó la crotoxina B del veneno de C. d. cumanensis. Esta fosfolipasa A2 hace parte del complejo crotoxina, formado por la crotoxina A (subunidad ácida) y la crotoxina B (subunidad básica) unidas de forma covalente (29), y es responsable de la neurotoxicidad del veneno de las cascabeles suramericanas (30,31).…”

Section: Análisis Estadísticounclassified

Efecto citotóxico de fosfolipasas A2 del veneno de Crotalus durissus cumanensis de Colombia

Quintana-Castillo¹,

Ávila-Gómez²,

Ceballos-Ruiz³

et al. 2017

Revista Investig. Salud Univ. Boyacá

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RESUMENIntroducción. Los venenos de serpientes representan una fuente importante de proteínas y péptidos, los cuales exhiben diversas actividades biológicas, tales como antibacterianas, antiparasitarias, antivirales, antitumorales, antifúngicas y contra la agregación plaquetaria, entre otras.Las fosfolipasas A2 presentes en los venenos de serpientes son las proteínas más estudiadas en estos modelos. Se ha demostrado que las fosfolipasas A2, activas e inactivas, poseen actividad catalítica contra células tumorales.Objetivo. Aislar, purificar y caracterizar la fosfolipasa A2 del veneno de Crotalus durissus cumanensis para evaluar su actividad antitumoral in vitro.Materiales y métodos. El aislamiento, la purificación y la identificación de la crotoxina B se hizo mediante la cromatografía de exclusión molecular, la cromatografía líquida de alto rendimiento de fase inversa (Reversed Phase High-Performance Liquid Chromatography, RP-HPLC) y la espectrometría de masas. El efecto citotóxico sobre células tumorales (K562) y células normales (células mononucleares de sangre periférica) se determinó utilizando la técnica de MTT.Resultados. La separación y posterior identificación de la crotoxina B del veneno de C. d. cumanensis de Colombia, permitieron evidenciar que esta fosfolipasa A2 posee efecto citotóxico sobre las células mononucleares de sangre periférica con una dosis de 18,23 ± 0,57 µg/ml, mientras que, para las células K562, fue de 2,34 ± 0,199 µg/ml.Conclusiones. Los resultados sugieren la posibilidad de utilizar la crotoxina B aislada del veneno de C.d. cumanensis como un posible recurso terapéutico para su aplicación en humanos.

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Crystal Structure of Crotoxin Reveals Key Residues Involved in the Stability and Toxicity of This Potent Heterodimeric β-Neurotoxin

Cited by 92 publications

References 59 publications

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

Biochemical Characterization of a PLA2 Btae TX-I Isolated from Bothriopsis taeniata Snake Venom: A Pharmacological and Morphological Study

Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice

Efecto citotóxico de fosfolipasas A2 del veneno de Crotalus durissus cumanensis de Colombia

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