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      Molecular, Immunological, and Biological Characterization of Tityus serrulatus Venom Hyaluronidase: New Insights into Its Role in Envenomation

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          Abstract

          Background

          Scorpionism is a public health problem in Brazil, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. The main toxic components of Ts venom are low-molecular-weight neurotoxins; however, the venom also contains poorly characterized high-molecular-weight enzymes. Hyaluronidase is one such enzyme that has been poorly characterized.

          Methods and principal findings

          We examined clones from a cDNA library of the Ts venom gland and described two novel isoforms of hyaluronidase, TsHyal-1 and TsHyal-2. The isoforms are 83% identical, and alignment of their predicted amino acid sequences with other hyaluronidases showed conserved residues between evolutionarily distant organisms. We performed gel filtration followed by reversed-phase chromatography to purify native hyaluronidase from Ts venom. Purified native Ts hyaluronidase was used to produce anti-hyaluronidase serum in rabbits. As little as 0.94 µl of anti-hyaluronidase serum neutralized 1 LD 50 (13.2 µg) of Ts venom hyaluronidase activity in vitro. In vivo neutralization assays showed that 121.6 µl of anti-hyaluronidase serum inhibited mouse death 100%, whereas 60.8 µl and 15.2 µl of serum delayed mouse death. Inhibition of death was also achieved by using the hyaluronidase pharmacological inhibitor aristolochic acid. Addition of native Ts hyaluronidase (0.418 µg) to pre-neutralized Ts venom (13.2 µg venom+0.94 µl anti-hyaluronidase serum) reversed mouse survival. We used the SPOT method to map TsHyal-1 and TsHyal-2 epitopes. More peptides were recognized by anti-hyaluronidase serum in TsHyal-1 than in TsHyal-2. Epitopes common to both isoforms included active site residues.

          Conclusions

          Hyaluronidase inhibition and immunoneutralization reduced the toxic effects of Ts venom. Our results have implications in scorpionism therapy and challenge the notion that only neurotoxins are important to the envenoming process.

          Author Summary

          In Brazil, accidents with scorpion stings have been a serious public health problem, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. Therefore, efforts have been made to understand the characteristics of the molecules present in scorpion venoms. These venoms are complex mixtures, in which neurotoxins are the main toxic components. Ts venom also contains enzymes, such as hyaluronidase, that have not been well characterized. In this study, we described for the first time two sequences of Ts hyaluronidase isoforms: TsHyal-1 and TsHyal-2. We purified native hyaluronidase from Ts venom and produced anti-hyaluronidase serum in rabbits. This serum neutralized hyaluronidase activity present in Ts venom. In vivo neutralization assays showed that anti-hyaluronidase serum inhibited and delayed mouse death after injection of a lethal dose (50% lethal dose, LD 50) of Ts venom. This work confirms the influence of hyaluronidase in Ts venom lethality and paves the way for the development of new strategies for scorpionism therapy.

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          Most cited references 62

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          Hyaluronan as an immune regulator in human diseases.

          Accumulation and turnover of extracellular matrix components are the hallmarks of tissue injury. Fragmented hyaluronan stimulates the expression of inflammatory genes by a variety of immune cells at the injury site. Hyaluronan binds to a number of cell surface proteins on various cell types. Hyaluronan fragments signal through both Toll-like receptor (TLR) 4 and TLR2 as well as CD44 to stimulate inflammatory genes in inflammatory cells. Hyaluronan is also present on the cell surface of epithelial cells and provides protection against tissue damage from the environment by interacting with TLR2 and TLR4. Hyaluronan and hyaluronan-binding proteins regulate inflammation, tissue injury, and repair through regulating inflammatory cell recruitment, release of inflammatory cytokines, and cell migration. This review focuses on the role of hyaluronan as an immune regulator in human diseases.
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            Hyaluronidases: their genomics, structures, and mechanisms of action.

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              The magic glue hyaluronan and its eraser hyaluronidase: a biological overview.

              Hyaluronan (HA) is a multifunctional high molecular weight polysaccharide found throughout the animal kingdom, especially in the extracellular matrix (ECM) of soft connective tissues. HA is thought to participate in many biological processes, and its level is markedly elevated during embryogenesis, cell migration, wound healing, malignant transformation, and tissue turnover. The enzymes that degrade HA, hyaluronidases (HAases) are expressed both in prokaryotes and eukaryotes. These enzymes are known to be involved in physiological and pathological processes ranging from fertilization to aging. Hyaluronidase-mediated degradation of HA increases the permeability of connective tissues and decreases the viscosity of body fluids and is also involved in bacterial pathogenesis, the spread of toxins and venoms, acrosomal reaction/ovum fertilization, and cancer progression. Furthermore, these enzymes may promote direct contact between pathogens and the host cell surfaces. Depolymerization of HA also adversely affects the role of ECM and impairs its activity as a reservoir of growth factors, cytokines and various enzymes involved in signal transduction. Inhibition of HA degradation therefore may be crucial in reducing disease progression and spread of venom/toxins and bacterial pathogens. Hyaluronidase inhibitors are potent, ubiquitous regulating agents that are involved in maintaining the balance between the anabolism and catabolism of HA. Hyaluronidase inhibitors could also serve as contraceptives and anti-tumor agents and possibly have antibacterial and anti-venom/toxin activities. Additionally, these molecules can be used as pharmacological tools to study the physiological and pathophysiological role of HA and hyaluronidases.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                February 2014
                13 February 2014
                : 8
                : 2
                Affiliations
                [1 ]Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
                [2 ]Programa de Pós-Graduação em Ciências Biológicas: Fisiologia e Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
                [3 ]Departamento de Bioquímica-Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
                Institut de Recherche pour le Développement, Benin
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CCRH BdFM BBROM CCO EK. Performed the experiments: CCRH BdFM BBROM AOdC CGD LFF RAMdA. Analyzed the data: CCRH BdFM BBROM CGD LFF CCO EK. Contributed reagents/materials/analysis tools: LFF CCO EK. Wrote the paper: CCRH BdFM BBROM CGD LFF EK.

                Article
                PNTD-D-13-01361
                10.1371/journal.pntd.0002693
                3923731

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 14
                Funding
                This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; http://www.cnpq.br) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Edital Toxinologia 63/2010; http://www.capes.gov.br), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; http://www.fapemig.br/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Enzymes
                Proteins
                Genetics
                Molecular genetics
                Immunology
                Toxicology

                Infectious disease & Microbiology

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