41 research outputs found
A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization
Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. the venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (similar to 45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in similar to 45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a spreading factor. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.Conselho Nacional de Desenvolvimento CientÃfico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de NÃvel Superior (CAPES)Fundacao Araucaria-PR (FAP)Secretaria de Estado de Ciencia, Tecnologia e Ensino Superior do Parana (SETI)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Fed Parana, Dept Cell Biol, BR-80060000 Curitiba, Parana, BrazilUniv Fed Parana, Clin Hosp, Dept Clin Pathol, BR-80060000 Curitiba, Parana, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc
Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: Endothelial cell membrane phospholipids as targets for toxicity
Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. the mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids. (C) 2010 Elsevier B.V. All rights reserved.Secretaria de Estado de CienciaTecnologia e Ensino Superior (SETI) do ParanaFundacao Araucaria-PRFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento CientÃfico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de NÃvel Superior (CAPES)Univ Fed Parana, Dept Cell Biol, BR-81531990 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc
Alcoholic liver steatosis in mice is aggravated by low-protein diet and reversed by FXR agonist
Autoimmunity in Chronic Chagas Disease: A Road of Multiple Pathways to Cardiomyopathy?
Chagas disease (CD), a neglected tropical disease caused by the protozoan Trypanosoma cruzi, affects around six million individuals in Latin America. Currently, CD occurs worldwide, becoming a significant public health concern due to its silent aspect and high morbimortality rate. T. cruzi presents different escape strategies which allow its evasion from the host immune system, enabling its persistence and the establishment of chronic infection which leads to the development of chronic Chagas cardiomyopathy (CCC). The potent immune stimuli generated by T. cruzi persistence may result in tissue damage and inflammatory response. In addition, molecular mimicry between parasites molecules and host proteins may result in cross-reaction with self-molecules and consequently in autoimmune features including autoantibodies and autoreactive cells. Although controversial, there is evidence demonstrating a role for autoimmunity in the clinical progression of CCC. Nevertheless, the exact mechanism underlying the generation of an autoimmune response in human CD progression is unknown. In this review, we summarize the recent findings and hypotheses related to the autoimmune mechanisms involved in the development and progression of CCC
Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes
Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins
Effects of metalloproteinas from Brotrops leucurus venon and brown spiders venoms on endothelial cell and components of extracellular matrix
A nova metaloprotease fibrinolÃtica isolada do veneno de Bothrops leucurus, denominada leucurolisina-a (23kDa), apresenta-se como um componente de significativo potencial terapêutica para o tratamento de distúrbios de coagulação, pela sua capacidade de dissolução de trombos. A caracterização dessa enzima mostrou similaridade de seqüência com outras metaloproteases de veneno de cabra que pertencem à classe P-I, além de exibir o sÃtio catalÃtico consenso para ligação do Ãon zinco. É destituÃda de atividade hemorrágica. Além de fibrinolÃtica, a enzima mostrou atividade também sobre fibronectina e fibrinogênio. No entanto, a laminina, não foi degradada pela protease, assim como o colágeno I. A protease inibiu a agregação plaquetária induzida por ADP de maneira dose dependente. O efeito citotóxico da leuc¬a sobre células endoteliais em cultura pôde ser observado apenas quando a exposição ocorreu em tempos prolongados e com altas concentrações da metaloprotease. Quando nessas condições, as células expostas alteram a sua morfologia por uma diminuição da adesão, tornando-se arredondadas e culminando no desprendimento lotal do substrato. Não houve redução da viabilidade das células endoteliais expostas à leuc-a em concentrações moderadas. Quando a sua concentração e tempo de exposição foram aumentados de maneira significativa, as células desprendidas perderam sua estabilidade estrutural, o que resultou na lise de parte delas. A atividade do veneno total de B. leucurus sobre as células endoteliais foi similar à leuc-a, porém numa intensidade muito maior. Outra metaloprotease purificada do veneno de Bothrops leucurus pelo grupo foi uma enzima pertencente à classe P-III das metaloproteases de venenos de serpentes (55 kDa). Resultados preliminares mostram uma atividade hemorrágica intensa por parte dessa protease. Análises do perfil proteolÃtico dessa nova metaloprotease caracterizaram-na com uma potente α-fibrinogenase. A metaloprotease de 55 kDa não mostrou atividade proteolÃtica sobre laminina e degradou apenas parcialmente colágeno IV. No entanto, mostrou atividade bastante potente sobre a entactina. Análises da atividade dessa protease sobre os colágenos do tipo I e IV mostraram apenas degradação parcial. No entanto, quando testada sobre gelatina essa protease de massa molecular aproximada de 55 kDa comporta-se com uma gelatinase. Diversas metaloproteases também já foram isoladas e caracterizadas nos venenos de aranhas marrons (gênero Loxosceles). Quando células endoteliais em cultura foram expostas ao veneno do L. intermedia observou-se uma profunda alteração na morfologia dessas células, tornando-se arredondadas e com longas filopodia sendo projetadas da periferia da célula. A marcação dos filamentos de actina e da vinculina por fluorescência, mostrou a desorganização do citoesqueleto e reorganização dos pontos de adesão focal, eventos estes que acontecem simultaneamente com as mudanças morfológicas observadas. Houve diminuição do perfil de adesão das células sobre fibronectina após exposição ao veneno de L. intermedia. Além disso, a imunomarcação da fibronectina na matriz extracelular das células em cultura expostas ao veneno confirmou a atividade fibronectinolÃtica do veneno. Esse resultado, junto com a observação da ligação direta do veneno à superfÃcie celular, pode explicar a citotoxicidade induzida pelo veneno sobre as células endoteliais.BV UNIFESP: Teses e dissertaçõe
The effect of brown spider venom on endothelial cell morphology and adhesive structures
Spiders of the Loxosceles genus have been responsible for severe clinical cases of envenomation worldwide. Accidents involving brown spiders can cause dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of animals treated by venom have shown subendothelial blebs, vacuoles and endothelial cell membrane degeneration of blood vessel walls, as well as fibrin and thrombus formation. the mechanisms by which the venom causes these disorders are poorly understood. in this work, with an endothelial cell line derived from rabbit aorta, we were able to demonstrate that venom binds to the cell surface and the extracellular matrix. Moreover, we observed that the venom also induced morphological alterations, such as cell retraction, homophilic disadhesion and an increasing in filopodia projections. We also demonstrated that toxins present in the venom disorganized focal adhesion points and actin microfilaments of endothelial cells. Nevertheless, endothelial cell viability showed no alterations compared to controls. Additionally, venom treatment changed the fibronectin matrix profile synthesized by these cells as well as cell adhesion to fibronectin. These results suggest that the deleterious effects of venom on blood vessel walls could be a consequence of the direct effect on the endothelial cell surface and adhesive structures involved in blood vessel stability. These effects indirectly lead to leukocyte and platelet activation, disseminated intravascular coagulation and an increase in vessel permeability. (c) 2006 Elsevier B.V. All rights reserved.Univ Fed Parana, Dept Cell Biol, BR-81531990 Curitiba, Parana, BrazilUNIFESP, Dept Med, Med Clin Discipline, São Paulo, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUNIFESP, Dept Biochem, São Paulo, BrazilUNIFESP, Dept Med, Med Clin Discipline, São Paulo, BrazilUNIFESP, Dept Biochem, São Paulo, BrazilWeb of Scienc