A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

Author Ferrer, Valeria Pereira Google Scholar
Mari, Thiago Lopes de Google Scholar
Gremski, Luiza Helena Google Scholar
Silva, Dilza Trevisan Google Scholar
Silveira, Rafael Bertoni da Google Scholar
Gremski, Waldemiro Google Scholar
Chaim, Olga Meiri Google Scholar
Senff-Ribeiro, Andrea Google Scholar
Nader, Helena Bonciani Autor UNIFESP Google Scholar
Veiga, Silvio Sanches Google Scholar
Institution Univ Fed Parana
Univ Estadual Ponta Grossa
Catholic Univ Parana
Universidade Federal de São Paulo (UNIFESP)
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. 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.
Language English
Sponsor 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)
Date 2013-05-01
Published in Plos Neglected Tropical Diseases. San Francisco: Public Library Science, v. 7, n. 5, 12 p., 2013.
ISSN 1935-2735 (Sherpa/Romeo, impact factor)
Publisher Public Library Science
Extent 12
Origin http://dx.doi.org/10.1371/journal.pntd.0002206
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000319994400013
URI http://repositorio.unifesp.br/handle/11600/36249

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