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

Valéria Pereira Ferrer,Silvio Sanches Veiga,Andrea Senff-Ribeiro,Rafael Bertoni Da Silveira,Dilza Trevisan Silva,Helena Bonciani Nader,Waldemiro Gremski,Thiago Lopes De Mari,Olga Meiri Chaim,Luiza Helena Gremski

doi:10.1371/journal.pntd.0002206

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 (∼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 ∼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.

Highlights

Bites involving brown spiders are characterized by skin injuries at the venom inoculation site, including swelling, erythema, hemorrhage, dermonecrosis, and the hallmark of loxoscelism: gravitational spreading of cutaneous lesions [1,2]
A previous study identified the activity of L. intermedia venom upon glycosaminoglycans which are abundant components in the extracellular matrix of many tissues
Disclosing a little more about the role of hyaluronidases within this venom, we investigated the activities of a recombinant hyaluronidase from L. intermedia venom

Summary

Introduction

Bites involving brown spiders are characterized by skin injuries at the venom inoculation site, including swelling, erythema, hemorrhage, dermonecrosis, and the hallmark of loxoscelism: gravitational spreading of cutaneous lesions [1,2]. The gravitational spread of skin lesions is a distinct characteristic of loxoscelism, described after experimental venom exposure in the skin of rabbits and in real cases. It appears hours or days after venom inoculation. The lesion is reported as a collection of inflammatory cells in and around the blood vessels and diffusely distributed in the dermis. The wound is described as aseptic coagulative necrosis [1,2,6,7,8]

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