ABSTRACT
Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.
ABSTRACT
Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.
ABSTRACT
The phospholipases D (PLDs) of the venom of the spider Loxosceles gaucho are the main toxins responsible for the observed effects in the poisoning. These toxins can exert a high hydrolytic activity on sphingomyelins, releasing ceramide 1-phosphate (C1P) and cyclic phosphates (cPA). cPA affects numerous cellular functions, including inhibition of tumor cell invasion and metastasis. This feature could be interesting for biotechnological use, however, PLDs promote strong platelet aggregation. On the other hand, the disintegrins present in Viperidae venom venoms can inhibit platelet aggregation and can bind to tumor cells because of their ability to interact with the highly expressed integrins (αvβ3) in these cells. Thus, a fusion of a snake disintegrin with a spider PLD may be an interesting option to eliminate unwanted platelet aggregation caused by PLD, and further act as a toxin carrier for tumor cells. Thus, in order to exploit the antitumor potential of a L. gaucho PLD called LgRec1 and the particularity of an E. carinatus snake disintegrin called Echistatin to bind to tumor cells, we designed a hybrid toxin composed by the fusion of these two molecules which was called Rechistatin. This sequence was cloned into the pAE vector and transformed into BL21 (DE3) bacterium for protein expression, which was then purified by IMAC. Mass spectrometry and circular dichroism analyzed the molecular mass and secondary structure of the hybrid molecule, respectively. Platelet aggregation was measured by aggregometry assay. The cytotoxicity of Rechistatin was analyzed in tumor cells. The results showed that the hybrid molecule was cloned and successfully expressed in the soluble form. Rechistatin showed an expected band size of 38 kDa as confirmed by mass spectrometry. Circular dichroism revealed an expected pattern of conformation. Fusion of Echistatin to LgRec1 was effective to abolish the aggregation properties of LgRec1 PLD and ELISA tests on cells showed that Echistatin was efficient in directing LgRec1 to tumor cells U87-MG and RD. Additionally, the results showed that Rechistatin exerts cytotoxicity on U87-MG and RD cells. Taken together, these results indicate that the fusion of Echistatin with LgRec1 did not impair the biological activity of the chimeric toxin and was effective in abolishing the strong platelet aggregation of LgRec1. In addition, Echistatin was efficient in delivery LgRec1 to tumor cells, promoting cytotoxic activity.
As fosfolipases D (FLDs) do veneno da aranha Loxosceles gaucho são as principais toxinas responsáveis pelos efeitos observados no envenenamento. Estas toxinas podem exercer uma elevada atividade hidrolítica em esfingomielinas, liberando ceramida 1-fosfato (C1P) e fosfatos cíclicos (cPA). O cPA afeta numerosas funções celulares, incluindo a inibição da invasão de células tumorais e metástases. Esta caracteristica poderia ser interessante para utilização biotenológica, no entanto, FLDs promovem forte agregação plaquetária. Por outro lado, as disintegrinas presentes nos venenos de serpentes Viperidae são capazes de inibir a agregação plaquetária e podem se ligar às células tumorais devido à sua capacidade de interagir com as integrinas (αvβ3) altamente expressas nestas células. Assim, a fusão de uma disintegrina de serpente com uma FLD de aranha pode ser uma abordagem interessante para eliminar a agregação plaquetária indesejada provocada pela FLD, e ainda funcionar como uma carreadora desta toxina para células tumorais. Desta forma, visando explorar o potencial antitumoral de uma FLD de L. gaucho denominada LgRec1 e a particularidade de uma disintegrina de serpente E. carinatus denominada Echistatina de se ligar as células tumorais, realizamos a construção de uma toxina híbrida composta pela fusão dessas duas moléculas que foi denominada de Rechistatina. Esta sequência foi clonada no vector pAE e transformada em bactéria BL21 (DE3) para expressão proteica, que foi então purificada por IMAC. A massa molecular e a estrutura secundária da molécula híbrida foram analisadas por espectrometria de massas e dicroísmo circular, respectivamente. A agregação plaquetária foi medida por ensaio de agregometria, e a citotoxicidade da Rechistatina foi analisada em células tumorais. Os resultados mostraram que a molécula híbrida foi clonada e expressa com êxito na forma solúvel. A Rechistatina mostrou uma massa esperada de 38 kDa como confirmado por espectrometria de massa. O dicroísmo circular revelou um padrão de conformação esperado. A fusão de Echistatin a LgRec1 foi eficaz para abolir as propriedades de agregação da FLD LgRec1 e testes de ELISA em células mostraram que a Echistatina foi eficiente em direcionar a LgRec1 à celulas tumorais U87-MG e RD. Adicionalmente, os resultados mostraram que a Rechistatina exerce citotoxicidade nas células U87-MG e RD dose dependente. Em conjunto, estes resultados indicam que a fusão de Echistatina com LgRec1 não prejudicou a atividade biológica da toxina quimérica e foi eficaz para abolir a forte agregação plaquetária da LgRec1. Além disso, a Echistatina foi eficiente em direcionar a LgRec1 às células tumorias, promovendo atividade citotóxica.
