Clonagem e expressão heterológa de hialuronidase e alérgeno presentes no veneno de aranha marrom (Loxosceles Intermedia)

Co-Orientadora : Drª Andrea Senff RibeiroOrientador: Prof. Silvio Sanches VeigaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 04/03/2010Bibliografia: fls. 81-97Resumo: Loxoscelismo é a denominação do quadro clínico provocado em indivíduos picados por aranhas do gênero Loxosceles. No estado do Paraná, os acidentes com este gênero são problema de saúde pública, em especial na cidade de Curitiba e Região Metropolitana, consideradas áreas endêmicas para o loxoscelismo. O veneno de Loxosceles é composto por uma mistura de toxinas de natureza essencialmente protéica, capazes de causar lesões dermonecróticas com espalhamento gravitacional e manifestações sistêmicas tais como agregação plaquetária, anemia hemolítica, falência renal aguda e rash cutâneo. Nos venenos de aranhas do gênero Loxosceles foram descritas toxinas como fosfolipases-D, serinoproteases, metaloproteases, hialuronidases, entre outras. Estudos bioquímicos revelaram a presença de hialuronidases de 41 e 43KDa no veneno de L. intermedia. Hialuronidases são enzimas que degradam componentes da matriz extracelular facilitando a penetração das outras toxinas em tecidos adjacentes ao local de inoculação do veneno, sendo referida como "fator de espalhamento gravitacional". No veneno de L. intermedia outros componentes além da histamina são relatados como responsáveis pela degranulação de mastócitos e pelos eventos inflamatórios. Para melhor compreensão dos quadros loxoscélicos, este trabalho teve como objetivo a clonagem e expressão de duas novas toxinas do veneno de L. intermedia: hialuronidase e alérgeno. Parte da sequência do alérgeno de L. intermedia era conhecida or meio da análise da biblioteca de cDNA da glândula produtora de veneno. Através de técnicas de biologia molecular e desenhando oligonucleotídeos específicos para esta sequência foi possível obter a sequência completa desta toxina, a qual foi subclonada em plasmídeo de expressão pET- 14b utilizando oligonucleotídeos com sítios de restrição para XhoI e BamHI. No caso da hialuronidase, os oligonucleotídeos iniciadores foram desenhados por meio da análise de sequências de hialuronidase de várias espécies e, com auxílio de técnicas específicas, foi possível obter a sequência completa desta enzima, a qual foi subclonada em pET-14b com auxílio de primers que continham sítios de restrição para as enzimas NdeI e BamHI. Com o teste de indução desta proteína recombinante em cepa E. coli BL21(DE3)pLysS foi padronizado que a condição de expressão desta seria com 0,05mM de indutor (IPTG) por 3,5h. Porém, nas temperaturas de 16ºC a 30ºC a proteína foi expressa e encontrava-se como corpos de inclusão. A expressão em pequena escala com uma cepa alternativa (E. coli AD494(DE3)) demonstrou que na expressão à 30ºC com 0,05mM-0,4mM de IPTG por 5h existe uma fração da hialuronidase recombinante expressa em sua forma solúvel. Os dados do trabalho inauguram a possibilidade de expressão de alérgeno e hialuronidase em grande escala e purificação em cromatografia de afinidade, para que assim sejam realizadas a avaliação de suas atividades biológicas.Abstract: Loxoscelism is the term used to describe the clinical symptoms evoked in victims of accidents with spiders of the Loxosceles genus. In the State of Parana, accidents involving this genus are a public health problem, especially in the city of Curitiba and metropolitan region, which are considered endemic areas for loxoscelism. Loxosceles venom is a mixture of toxins, essentially proteins, capable of causing dermonecrotic lesions with gravitational spreading and systemic manifestations such as platelet aggregation, hemolytic anemia, acute renal failure and cutaneous rash. Toxins as phospholipases-D, serineproteases, metalloproteases, hyaluronidases, among others were already described in the venom of spiders of the genus. Biochemical studies showed hyaluronidase activities at 41 and 43KDa in Loxosceles intermedia whole venom. Hyaluronidases are enzymes that degrade extracellular matrix components and may act increasing the spread of other toxins in adjacent tissues of the inoculation site of venom. Due to this activity, hyaluronidades are popularly known as "spreading factors". In venom of Loxosceles intemedia other components aside from histamine are reported be responsible for mast cell degranulation and inflammatory events. In order to expand the knowledge of loxoscelism this work has aimed the cloning and expression of two new toxins of Loxosceles intermedia venom: hyaluronidase and allergen. Analysis of cDNA library of L. intermedia venom gland identified a partial sequence encoding an allergen. By means of molecular biology techniques and by designing specific primers for this sequence it was possible to obtain the complete sequence of the allergen, which was then subcloned into the expression vector pET-14b using primers with restriction sites for XhoI and BamHI enzymes. In the case of hyaluronidase, primers were designed by analyzing the sequences of hyaluronidases from several species. Then, using specific techniques it was possible to obtain the full sequence of this enzyme, which was cloned with primers with restriction sites for NdeI and BamHI enzymes. After induction trials for this recombinant protein in E. coli BL21(DE3)pLysS it was standardized an expression condition of 0,05mM IPTG during 3,5h. However between 16ºC-30ºC temperatures the protein was expressed in the form of inclusion bodies. A small scale expression in a different strain (E. coli AD494(DE3)) using induction parameters of 0,05mM-0,4mM of IPTG during 5h at 30ºC resulted in part of recombinant hyaluronidase in the soluble form. The data of this work give conditions for expression of hyaluronidase and allergen in large scale as well as purification in affinity chromatography so that will allow the evaluation of their biological activities

Identificação e avaliação de uma isoforma de hialuronidase (Dietrich's Hyaluronidase) do veneno de Loxosceles intermedia (aranha-marrom) : da clonagem molecular à caracterização bioquímica e funcional

Orientador : Prof. Dr. Silvio Sanches VeigaOrientadora : Prof. Dr. Andrea Senff RibeiroTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 25/12/2014Inclui referênciasResumo: Loxoscelismo é o termo utilizado para os sintomas clínicos desencadeados após a picada de aranhas do gênero Loxosceles. As manifestações clínicas incluem necrose da pele com espalhamento gravitacional e complicações sistêmicas. O veneno loxoscélico contém várias enzimas, como por exemplo, fosfolipases-D, serinoproteases, metaloproteases e hialuronidases. As hialuronidases ao degradar glicosaminoglicanos de tecido conjuntivo potencializaria a ação de outros componentes do veneno facilitando a penetração e distribuição dos mesmos a diferentes tecidos. Este trabalho teve como objetivo a clonagem, expressão e caracterização bioquímica e biológica de uma isoforma de hialuronidase do veneno de L. intermedia (aranha-marrom). Através de uma biblioteca de cDNA da glândula de veneno, uma isoforma de hialuronidase (1200 pb) foi clonada e denominada Dietrich's Hyaluronidase (DHAase). Após subclonagem da hialuronidase madura em vetor de expressão pET-14b, a construção resultante foi transformada em Escherichia coli BL21(DE3)pLysS e AD494(DE3). Fora testada a expressão em volume de 50 mL em diferentes concentrações de indutor IPTG (0,05-0,4 mM) e diferentes temperaturas (30º, 22º e 16ºC). Para obtenção da solubilidade da enzima recombinante, a sequência da hialuronidase madura foi também clonada em vetor de expressão pET-SUMO e expressa em E. coli SHuffle a 30ºC sob as mesmas condições de indutor descritas anteriormente. Em um maior volume de cultura (1L) nenhuma das cepas foi eficiente em expressar a DHAase solúvel. Sendo assim a alternativa escolhida para conseguir a hialuronidase recombinante de forma solúvel e ativa foi a técnica de redobramento in vitro. Vários tampões foram testados e a atividade só foi obtida com um tampão contendo glutationas, albumina bovina (BSA) e L-arginina. Depois do redobramento, DHAase foi capaz de degradar ácido hialurônico (HA) e condroitim-4-sulfato (C4S) em ensaios de cinética e zimografia. Por meio de análises de imunodetecção foi visto que anticorpos policlonais produzidos com a DHAase desnaturada e com o veneno reagiram de forma cruzada. Através de experimentos in vivo de dermonecrose em pele de coelho, foi demonstrado que DHAase aumentou a área de necrose produzida por uma isoforma de fosfolipase-D recombinante (toxina dermonecrótica) do veneno de L. intermedia (LiRecDT1). Dados macroscópicos e histológicos suportam a hipótese de que a hialuronidase é um "fator de espalhamento" do veneno de L. intermedia. Adicionalmente, estudando modelos in vitro de células endoteliais de aorta de coelho (RAEC) e células de melanoma murino (B16F1 e B16F10) onde essas células foram expostas por até 24 h com DHAase, foi visto que a hialuronidase recombinante não induziu alteração da morfologia ou desadesão das RAECs. Por outro lado ela alterou a proliferação e a morfologia das células de melanoma murino, sendo que a linhagem mais metastática (B16F10) teve os efeitos mais pronunciados sob a ação de DHAase. A viabilidade celular de B16F10 diminui significativamente com a exposição da hialuronidase recombinante por 16 h. Dietrich's Hyaluronidase foi uma ferramenta útil para um estudo pioneiro no loxoscelismo e se mostrou eficiente em estudar o efeito do catabolismo de HA em linhagens cancerosas de melanoma, embora estudos adicionais se façam necessários. Em conclusão, o presente trabalho tornou evidente a possível utilização de DHAase como uma ferramenta de estudo em processos patológicos relacionados ao ácido hialurônico. Palavras-chave: Loxosceles, aranha-marrom, veneno, ácido hialurônico, hialuronidases, proteínas recombinantes, redobramento in vitro, melanoma.Abstract: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 such as phospholipase-D, serine proteases, metalloproteases and hyaluronidases. Hyaluronidases by degrading glycosaminoglycans from connective tissue may be able to enhance the action from other venom constituents by facilitating the diffusion from local bite to other tissues. This work aimed the cloning, expression and the biochemical and biological characterization of a hyaluronidase isoform from L. intermedia venom (brown spider). Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp) which was named Dietrich's Hyaluronidase (DHAase). The mature sequence was subcloned with a pET-14b plasmid and the expressed construction was inserted into E. coli BL21(DE3)pLysS and AD494(DE3). The induction of recombinant protein expression was tested in different IPTG concentrations (0.05-0.4 mM) and temperatures (30º, 22º and 16ºC). The DHAase mature sequence was also subcloned with pET-SUMO and it was expressed in E. coli SHuffle at 30ºC with the same IPTG concentrations. Neither of E.coli strains tested was able to express DHAase in a soluble form in large scale. The chosen alternative to achieve a soluble and active DHAase was the refolding in vitro technique. Several conditions were tested and the activity was only reached with a buffer containing glutathione, bovine serum albumin (BSA) and L-arginine. After the refolding, the recombinant hyaluronidase was able to degrade hyaluronic acid (HA) and chondroitin-4-sulfate (C4S) in a kinetic assay and zymography. Immunoblot analysis showed that antibodies which recognize the DHAase cross-reacted with native hyaluronidases from the whole venom as well as an anti- whole venom serum reacted with the recombinant protein. Through in vivo experiments of dermonecrosis using rabbit skin, DHAase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin (phospholipase-D) from L. intermedia venom (LiRecDT1). Macroscopic and histologic findings sustain these data. Together, results support the hypothesis that hyaluronidase is a ''spreading factor'' from L. intermedia venom. In addition, studying in vitro culture model of rabbit aortic endhotelial cells (RAEC) and murine melanoma cells (B16F1 e B16F10), which the cells were exposed up to 24 h with DHAase, showed that recombinant hyaluronidase not induced RAEC's morphologic changes neither cell detachment. On the other hand, it caused morphologic and cell proliferation changes in melanoma cells. B16F10, a more metastatic subtype, was more affected by hyaluronidase action than the B16F1. The B16F10 cell viability was significantly reduced by DHAase within 16 h. DHAase provided a useful tool for a pioneering study into loxoscelism and it was efficient in studying the HA metabolites effect in melanoma cancer cells, although further studies are necessary. In conclusion, this work provide evidences of the use of DHAase as a tool for studying HA pathological process. Keywords: Loxosceles, brown spider, venom, hyaluronic acid, hyaluronidases, recombinant protein, refolding in vitro, melanom

MicroRNAs, Hypoxia and the Stem-Like State as Contributors to Cancer Aggressiveness

MicroRNAs (miRNAs) are small non-coding RNA molecules that play key regulatory roles in cancer acting as both oncogenes and tumor suppressors. Due to their potential roles in improving cancer prognostic, predictive, diagnostic and therapeutic approaches, they have become an area of intense research focus in recent years. Several studies have demonstrated an altered expression of several miRNAs under hypoxic condition and even shown that the hypoxic microenvironment drives the selection of a more aggressive cancer cell population through cellular adaptations referred as the cancer stem-like cell. These minor fractions of cells are characterized by their self-renewal abilities and their ability to maintain the tumor mass, suggesting their crucial roles in cancer development. This review aims to highlight the interconnected role between miRNAs, hypoxia and the stem-like state in contributing to the cancer aggressiveness as opposed to their independent contributions, and it is based in four aggressive tumors, namely glioblastoma, cervical, prostate, and breast cancers

The Interface of Cancer, Their Microenvironment and Nanotechnology

Cancer is one of the deadliest diseases with a cure far from being found. Despite the extraordinary advances in the therapy approaches, only a few patients respond to treatments. The tumor microenvironment (TME) plays a crucial role in cancer progression by contributing to the chemoresistance. Thus, emerging efforts are being made in nanotechnology research focusing on nanoparticles' potential role and their application in immune system modulation. Moreover, the omics have contributed to bioengineering and nanotechnology development by elucidating the mechanisms of cancer and specific biomarkers that could be used as new therapeutic targets. Furthermore, the non-coding microRNA as a target for cancer treatment and creation of organoids for the study of new treatments helped for the new therapeutics' era called personalized medicine. Here we will discuss the role played by TME in tumor initiation and progression we will describe the recent nanotechnology applied to cancer treatment. Specifically, we will describe the potential role of nanoparticles (NPs) and their application in immune system modulation, ultimately leading to circumventing tumor cell proliferation.publishersversionpublishe

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

Microglia/Astrocytes–Glioblastoma Crosstalk: Crucial Molecular Mechanisms and Microenvironmental Factors

In recent years, the functions of glial cells, namely, astrocytes and microglia, have gained prominence in several diseases of the central nervous system, especially in glioblastoma (GB), the most malignant primary brain tumor that leads to poor clinical outcomes. Studies showed that microglial cells or astrocytes play a critical role in promoting GB growth. Based on the recent findings, the complex network of the interaction between microglial/astrocytes cells and GB may constitute a potential therapeutic target to overcome tumor malignancy. In the present review, we summarize the most important mechanisms and functions of the molecular factors involved in the microglia or astrocytes–GB interactions, which is particularly the alterations that occur in the cell’s extracellular matrix and the cytoskeleton. We overview the cytokines, chemokines, neurotrophic, morphogenic, metabolic factors, and non-coding RNAs actions crucial to these interactions. We have also discussed the most recent studies regarding the mechanisms of transportation and communication between microglial/astrocytes – GB cells, namely through the ABC transporters or by extracellular vesicles. Lastly, we highlight the therapeutic challenges and improvements regarding the crosstalk between these glial cells and GB

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

Worldwide trends in hypertension prevalence and progress in treatment and control from 1990 to 2019: a pooled analysis of 1201 population-representative studies with 104 million participants

Background Hypertension can be detected at the primary health-care level and low-cost treatments can effectively control hypertension. We aimed to measure the prevalence of hypertension and progress in its detection, treatment, and control from 1990 to 2019 for 200 countries and territories. Methods We used data from 1990 to 2019 on people aged 30–79 years from population-representative studies with measurement of blood pressure and data on blood pressure treatment. We defined hypertension as having systolic blood pressure 140 mm Hg or greater, diastolic blood pressure 90 mm Hg or greater, or taking medication for hypertension. We applied a Bayesian hierarchical model to estimate the prevalence of hypertension and the proportion of people with hypertension who had a previous diagnosis (detection), who were taking medication for hypertension (treatment), and whose hypertension was controlled to below 140/90 mm Hg (control). The model allowed for trends over time to be non-linear and to vary by age. Findings The number of people aged 30–79 years with hypertension doubled from 1990 to 2019, from 331 (95% credible interval 306–359) million women and 317 (292–344) million men in 1990 to 626 (584–668) million women and 652 (604–698) million men in 2019, despite stable global age-standardised prevalence. In 2019, age-standardised hypertension prevalence was lowest in Canada and Peru for both men and women; in Taiwan, South Korea, Japan, and some countries in western Europe including Switzerland, Spain, and the UK for women; and in several low-income and middle-income countries such as Eritrea, Bangladesh, Ethiopia, and Solomon Islands for men. Hypertension prevalence surpassed 50% for women in two countries and men in nine countries, in central and eastern Europe, central Asia, Oceania, and Latin America. Globally, 59% (55–62) of women and 49% (46–52) of men with hypertension reported a previous diagnosis of hypertension in 2019, and 47% (43–51) of women and 38% (35–41) of men were treated. Control rates among people with hypertension in 2019 were 23% (20–27) for women and 18% (16–21) for men. In 2019, treatment and control rates were highest in South Korea, Canada, and Iceland (treatment >70%; control >50%), followed by the USA, Costa Rica, Germany, Portugal, and Taiwan. Treatment rates were less than 25% for women and less than 20% for men in Nepal, Indonesia, and some countries in sub-Saharan Africa and Oceania. Control rates were below 10% for women and men in these countries and for men in some countries in north Africa, central and south Asia, and eastern Europe. Treatment and control rates have improved in most countries since 1990, but we found little change in most countries in sub-Saharan Africa and Oceania. Improvements were largest in high-income countries, central Europe, and some upper-middle-income and recently high-income countries including Costa Rica, Taiwan, Kazakhstan, South Africa, Brazil, Chile, Turkey, and Iran. Interpretation Improvements in the detection, treatment, and control of hypertension have varied substantially across countries, with some middle-income countries now outperforming most high-income nations. The dual approach of reducing hypertension prevalence through primary prevention and enhancing its treatment and control is achievable not only in high-income countries but also in low-income and middle-income settings

Heterogeneous contributions of change in population distribution of body mass index to change in obesity and underweight NCD Risk Factor Collaboration (NCD-RisC)

From 1985 to 2016, the prevalence of underweight decreased, and that of obesity and severe obesity increased, in most regions, with significant variation in the magnitude of these changes across regions. We investigated how much change in mean body mass index (BMI) explains changes in the prevalence of underweight, obesity, and severe obesity in different regions using data from 2896 population-based studies with 187 million participants. Changes in the prevalence of underweight and total obesity, and to a lesser extent severe obesity, are largely driven by shifts in the distribution of BMI, with smaller contributions from changes in the shape of the distribution. In East and Southeast Asia and sub-Saharan Africa, the underweight tail of the BMI distribution was left behind as the distribution shifted. There is a need for policies that address all forms of malnutrition by making healthy foods accessible and affordable, while restricting unhealthy foods through fiscal and regulatory restrictions

Identificação e avaliação de uma isoforma de hialuronidase (Dietrich's Hyaluronidase) do veneno de Loxosceles intermedia (aranha-marrom) : da clonagem molecular à caracterização bioquímica e funcional

Orientador : Prof. Dr. Silvio Sanches VeigaOrientadora : Prof. Dr. Andrea Senff RibeiroTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 25/12/2014Inclui referênciasResumo: Loxoscelismo é o termo utilizado para os sintomas clínicos desencadeados após a picada de aranhas do gênero Loxosceles. As manifestações clínicas incluem necrose da pele com espalhamento gravitacional e complicações sistêmicas. O veneno loxoscélico contém várias enzimas, como por exemplo, fosfolipases-D, serinoproteases, metaloproteases e hialuronidases. As hialuronidases ao degradar glicosaminoglicanos de tecido conjuntivo potencializaria a ação de outros componentes do veneno facilitando a penetração e distribuição dos mesmos a diferentes tecidos. Este trabalho teve como objetivo a clonagem, expressão e caracterização bioquímica e biológica de uma isoforma de hialuronidase do veneno de L. intermedia (aranha-marrom). Através de uma biblioteca de cDNA da glândula de veneno, uma isoforma de hialuronidase (1200 pb) foi clonada e denominada Dietrich's Hyaluronidase (DHAase). Após subclonagem da hialuronidase madura em vetor de expressão pET-14b, a construção resultante foi transformada em Escherichia coli BL21(DE3)pLysS e AD494(DE3). Fora testada a expressão em volume de 50 mL em diferentes concentrações de indutor IPTG (0,05-0,4 mM) e diferentes temperaturas (30º, 22º e 16ºC). Para obtenção da solubilidade da enzima recombinante, a sequência da hialuronidase madura foi também clonada em vetor de expressão pET-SUMO e expressa em E. coli SHuffle a 30ºC sob as mesmas condições de indutor descritas anteriormente. Em um maior volume de cultura (1L) nenhuma das cepas foi eficiente em expressar a DHAase solúvel. Sendo assim a alternativa escolhida para conseguir a hialuronidase recombinante de forma solúvel e ativa foi a técnica de redobramento in vitro. Vários tampões foram testados e a atividade só foi obtida com um tampão contendo glutationas, albumina bovina (BSA) e L-arginina. Depois do redobramento, DHAase foi capaz de degradar ácido hialurônico (HA) e condroitim-4-sulfato (C4S) em ensaios de cinética e zimografia. Por meio de análises de imunodetecção foi visto que anticorpos policlonais produzidos com a DHAase desnaturada e com o veneno reagiram de forma cruzada. Através de experimentos in vivo de dermonecrose em pele de coelho, foi demonstrado que DHAase aumentou a área de necrose produzida por uma isoforma de fosfolipase-D recombinante (toxina dermonecrótica) do veneno de L. intermedia (LiRecDT1). Dados macroscópicos e histológicos suportam a hipótese de que a hialuronidase é um "fator de espalhamento" do veneno de L. intermedia. Adicionalmente, estudando modelos in vitro de células endoteliais de aorta de coelho (RAEC) e células de melanoma murino (B16F1 e B16F10) onde essas células foram expostas por até 24 h com DHAase, foi visto que a hialuronidase recombinante não induziu alteração da morfologia ou desadesão das RAECs. Por outro lado ela alterou a proliferação e a morfologia das células de melanoma murino, sendo que a linhagem mais metastática (B16F10) teve os efeitos mais pronunciados sob a ação de DHAase. A viabilidade celular de B16F10 diminui significativamente com a exposição da hialuronidase recombinante por 16 h. Dietrich's Hyaluronidase foi uma ferramenta útil para um estudo pioneiro no loxoscelismo e se mostrou eficiente em estudar o efeito do catabolismo de HA em linhagens cancerosas de melanoma, embora estudos adicionais se façam necessários. Em conclusão, o presente trabalho tornou evidente a possível utilização de DHAase como uma ferramenta de estudo em processos patológicos relacionados ao ácido hialurônico. Palavras-chave: Loxosceles, aranha-marrom, veneno, ácido hialurônico, hialuronidases, proteínas recombinantes, redobramento in vitro, melanoma.Abstract: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 such as phospholipase-D, serine proteases, metalloproteases and hyaluronidases. Hyaluronidases by degrading glycosaminoglycans from connective tissue may be able to enhance the action from other venom constituents by facilitating the diffusion from local bite to other tissues. This work aimed the cloning, expression and the biochemical and biological characterization of a hyaluronidase isoform from L. intermedia venom (brown spider). Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp) which was named Dietrich's Hyaluronidase (DHAase). The mature sequence was subcloned with a pET-14b plasmid and the expressed construction was inserted into E. coli BL21(DE3)pLysS and AD494(DE3). The induction of recombinant protein expression was tested in different IPTG concentrations (0.05-0.4 mM) and temperatures (30º, 22º and 16ºC). The DHAase mature sequence was also subcloned with pET-SUMO and it was expressed in E. coli SHuffle at 30ºC with the same IPTG concentrations. Neither of E.coli strains tested was able to express DHAase in a soluble form in large scale. The chosen alternative to achieve a soluble and active DHAase was the refolding in vitro technique. Several conditions were tested and the activity was only reached with a buffer containing glutathione, bovine serum albumin (BSA) and L-arginine. After the refolding, the recombinant hyaluronidase was able to degrade hyaluronic acid (HA) and chondroitin-4-sulfate (C4S) in a kinetic assay and zymography. Immunoblot analysis showed that antibodies which recognize the DHAase cross-reacted with native hyaluronidases from the whole venom as well as an anti- whole venom serum reacted with the recombinant protein. Through in vivo experiments of dermonecrosis using rabbit skin, DHAase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin (phospholipase-D) from L. intermedia venom (LiRecDT1). Macroscopic and histologic findings sustain these data. Together, results support the hypothesis that hyaluronidase is a ''spreading factor'' from L. intermedia venom. In addition, studying in vitro culture model of rabbit aortic endhotelial cells (RAEC) and murine melanoma cells (B16F1 e B16F10), which the cells were exposed up to 24 h with DHAase, showed that recombinant hyaluronidase not induced RAEC's morphologic changes neither cell detachment. On the other hand, it caused morphologic and cell proliferation changes in melanoma cells. B16F10, a more metastatic subtype, was more affected by hyaluronidase action than the B16F1. The B16F10 cell viability was significantly reduced by DHAase within 16 h. DHAase provided a useful tool for a pioneering study into loxoscelism and it was efficient in studying the HA metabolites effect in melanoma cancer cells, although further studies are necessary. In conclusion, this work provide evidences of the use of DHAase as a tool for studying HA pathological process. Keywords: Loxosceles, brown spider, venom, hyaluronic acid, hyaluronidases, recombinant protein, refolding in vitro, melanom