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

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.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