RESUMO
Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.
Assuntos
Reações Cruzadas/imunologia , Metaloendopeptidases/imunologia , Diester Fosfórico Hidrolases/imunologia , Venenos de Aranha/imunologia , Aranhas/imunologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , Epitopos/imunologia , Epitopos/metabolismo , Immunoblotting , Metaloendopeptidases/classificação , Metaloendopeptidases/genética , Modelos Moleculares , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Filogenia , Domínios Proteicos , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Aranhas/genética , Aranhas/metabolismoRESUMO
Canova is a Brazilian homeopathic medication with immunomodulatory properties, recommended for patients where the immune system is depressed. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix and progenitor cells that differentiate into mature blood cells. We now report the effect of in vitro administration of the medication on the mononuclear differentiation of the bone marrow cell. Swiss mice femurs were dissected cleaned and the cells of the marrow were flushed. The cells were plated, treated or not, incubated for different times and processed for light, transmission and scanning electron, and confocal microscopy analysis. Bone marrow cells showed an enhanced proliferation in vitro in response to Canova medication and Canova plus M-CSF and an increase was also observed in the numbers of the cell niches and ring-shaped nuclei cells. Confocal and transmission and scanning electron microscopy showed the stages of monocyte maturation, with resting and activated cells. With Canova treatment there was a marked increase in cell size, which is mainly attributable to the augmented cytoplasm, an increase in the number of mitochondria, expansion of the RER and an enlarged Golgi. The response to Canova treatment indicates that it influences mononuclear differentiation and activation of bone marrow progenitor and stromal cells.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Extratos Vegetais/farmacologia , Animais , Células da Medula Óssea/ultraestrutura , Formulários Homeopáticos como Assunto , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Ativação de Macrófagos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de TransmissãoRESUMO
Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.