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 Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível 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

Cellular prion protein interaction with vitronectin supports axonal growth and is compensated by integrins

The physiological functions of the cellular prion protein, PrPC, as a cell surface pleiotropic receptor are under debate. We report that PrPC interacts with vitronectin but not with fibronectin or collagen. the binding sites mediating this PrPC-vitronectin interaction were mapped to residues 105-119 of PrPC and the residues 307-320 of vitronectin. the two proteins were co-localized in embryonic dorsal root ganglia from wild-type mice. Vitronectin addition to cultured dorsal root ganglia induced axonal growth, which could be mimicked by vitronectin peptide 307-320 and abrogated by anti-PrPC antibodies. Full-length vitronectin, but not the vitronectin peptide 307-320, induced axonal growth of dorsal root neurons from two strains of PrPC-null mice. Functional assays demonstrated that relative to wild-type cells, PrPC-null dorsal root neurons were more responsive to the Arg-Gly-Asp peptide (an integrin-binding site), and exhibited greater alpha v beta 3 activity. Our findings indicate that PrPC plays an important role in axonal growth, and this function may be rescued in PrPC-knockout animals by integrin compensatory mechanisms.Hosp Alemao Oswaldo Cruz, Ludwig Inst Canc Res, São Paulo, BrazilUniv São Paulo, Inst Quim, Dept Bioquim, BR-05508 São Paulo, BrazilHosp Canc, Ctr Tratamento & Pesquisa, São Paulo, BrazilUniv Fed Parana, Dept Patol Basica, BR-80060000 Curitiba, Parana, BrazilUniv Fed Parana, Dept Biol Celular, BR-80060000 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, INFAR, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, INFAR, BR-04023062 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 Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível 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

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

Proteolitic enzymes in brown spiders venoms(Loxosceles genus): identification, biochemical characterization and its biological significance

Os acidentes envolvendo aranhas marrons (genero Loxosceles) tem sido descritos como causadores de dois sinais clinicos distintos de grande importancia medica. As lesoes na pele, caracterizadas inicialmente por uma reacao inflamatoria que progride para uma lesao dermonecrotica com espalhamento gravitacional, e os efeitos sistemicos compostos por desordens hematologicas como anemia hemolitica, trombocitopenia e coagulacao intravascular disseminada, bem como outros disturbios como falencia renal, febre, fraqueza, nausea e vomito. Apesar dos mecanismos moleculares pelos quais os venenos de aranhas marrons causam estes efeitos patologicos serem complexos e obscuros, eles permanecem sob continua investigacao e muitos relatos na literatura apontam para enzimas proteoliticas como importantes agentes para o desenvolvimento do loxoscelismo. A presente dissertacao e um estudo mais aprofundado da relevancia biologica e das propriedades bioquimicas das enzimas proteoliticas presentes nos venenos de aranhas marrons, bem como uma descricao do aparato produtor de veneno nestes animais. O veneno de Loxosceles intermedia contem duas serino proteases de alta massa molecular, ativadas pelo tratamento com proteases exogenas, com estreita (ou especifica) especificidade de substrato. Tambem e relatado aqui o dano aos vasos sanguineos e os efeitos trombogenicos observados durante o loxoscelismo experimental, alem de uma caracterizacao bioquimica mais detalhada da atividade fibrinogenolitica presente em venenos de Loxosceles. A glandula de veneno de L. intermedia e uma estrutura muscular de formato bulbar, diretamente conectada a quelicera da aranha. Esta glandula possui um epitelio no seu lumen, com um mecanismo holocrino de secrecao do veneno. A glandula exibe, entre o epitelio e os feixes musculares, matriz extracelular conjuntiva e membrana basal. Uma vez que as atividades proteoliticas tambem sao identificadas no extrato de glandula de aranhas marrons, com as mesmas metaloproteases com atividades gelatinolitica, fibronectinolitica e fibrinogenolitica como previamente descrito para o veneno eletroestimulado, fica evidente que estes sao componentes proprios do veneno. Este resultado responde a critica da possivel contaminacao do veneno com enzimas hidroliticas presentes nas secrecoes orais e abdominais da aranha, durante o processo de obtencao do venenoBV UNIFESP: Teses e dissertaçõe

Cloning and funcional analysis of toxin brown spiders

Os acidentes com aranhas marrons (gênero Loxosceles) são responsáveis por sério danos na pele, caracterizados por dermonecrose com espalhamento gravitacional e, em alguns casos, por manifestações de nível sistêmico tais como hemólise intravascular, coagulação intravascular disseminada e insuficiência renal aguda. Os mecanismos pelos quais o veneno da aranha marrom causa seus efeitos tóxicos não são totalmente conhecidos, embora muitas de suas toxinas têm sido bem caracterizadas do ponto de vista bioquímico e biológico. Várias evidências na literatura mostram a existência de múltiplas isoformas de diferentes toxinas no veneno, especialmente para as fosfolipases D. O estudo do veneno da aranha marrom é particularmente difícil devido às pequenas quantidades de veneno bruto obtidas nas extrações. Dessa forma, a clonagem e a expressão de proteínas recombinantes são ferramentas muito úteis que viabilizam estudos de cunho estrutural e biológico das toxinas do veneno. Além disso, a produção de toxinas recombinantes biologicamente ativas também oferece possibilidades biotecnológicas, tais como: o uso destas proteínas como antígenos substitutos na produção de antisoros, ou ainda a utilização destas toxinas como ferramentas de pesquisa em bioquímica e biologia celular. Esta tese teve como objetivos a produção e a análise funcional de toxinas recombinantes, a partir de uma biblioteca plasmidial de cDNA das glândulas produtoras de veneno de Loxosceles intermedia. Os resultados comprovam a nefrotoxicidade direta exercida pela toxina dermonecrótica, bem como mostram a funcionalidade diferencial de pelo menos cinco isoformas desta mesma toxina, de maneira proporcional à atividade fosfolipásica. O rastreamento da biblioteca de cDNA resultou também no isolamento de três isoformas de metaloproteases, classificadas como membros da família das astacinas para metaloendopeptidases dependentes de zinco. Embora a relevância biológica deste último grupo de enzimas no veneno seja ainda desconhecida, a existência de múltiplas isoformas para diferentes toxinas na glândula de veneno reforça a idéia de que estas moléculas atuem de maneira sinergística, resultando no quadro patológico provocado pelo veneno da aranha marrom, além do fato destes resultados representarem os primeiros relatos de enzimas semelhantes às astacinas em venenos de animais peçonhentos.BV UNIFESP: Teses e dissertaçõe

Biotechnological applications of brown spider (Loxosceles genus) venom toxins

Loxoscelisin (the term used to define accidents by the bite of brown spiders) has been reported worldwide. Clinical manifestations following brown spider bites are frequently associated with skin degeneration, a massive inflammatory response at the injured region, intravascular hemolysis, platelet aggregation causing thrombocytopenia and renal disturbances. the mechanisms by which the venom exerts its noxious effects are currently under investigation. the whole venom is a complex mixture of toxins enriched with low molecular mass proteins in the range of 5-40 kDa. Toxins including alkaline phosphatase, hyaluronidase, metalloproteases (astacin-like proteases), low molecular mass (5.6-7.9 kDa) insecticidal peptides and phospholipases-D (dermonecrotic toxins) have been identified in the venom. the purpose of the present review is to describe biotechnological applications of whole venom or some toxins, with especial emphasis upon molecular biology findings obtained in the last years. (C) 2007 Elsevier Inc. All rights reserved.Univ Fed Parana, Dept Cell Biol, BR-80060000 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Med Clin Discipline, São Paulo, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, Parana, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniv Fed Parana, Dept Physiol, BR-80060000 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Med Clin Discipline, São Paulo, BrazilWeb of Scienc

Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from Brown spider (Loxosceles intermedia) venom: From cloning to functional characterization

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. the venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. the recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(-)(2+)chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research. (c) 2007 Elsevier Masson SAS. All rights reserved.Univ Fed Parana, Dept Cell Biol, BR-81531990 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc