title: 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 (∼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.
authors: Department of Cell Biology, Federal University of Parana, Curitiba, Parana, Brazil.
authors: Valéria Pereira
authors: VP
authors: Ferrer
authors: Thiago Lopes
authors: TL
authors: de Mari
authors: Luiza Helena
authors: LH
authors: Gremski
authors: Dilza
authors: D
authors: Trevisan Silva
authors: Rafael Bertoni
authors: RB
authors: da Silveira
authors: Waldemiro
authors: W
authors: Gremski
authors: Olga Meiri
authors: OM
authors: Chaim
authors: Andrea
authors: A
authors: Senff-Ribeiro
authors: Helena Bonciani
authors: HB
authors: Nader
authors: Silvio Sanches
authors: SS
authors: Veiga
mesh_terms: D000818:Animals; D001097:Arachnida / Q000201:enzymology*; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000378:metabolism; D003001:Cloning, Molecular; D004195:Disease Models, Animal; D006820:Hyaluronic Acid / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007299:Insect Bites and Stings / Q000473:pathology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014688:Venoms / Q000201:enzymology*
chemical_list: D060829:Arthropod Proteins; D011994:Recombinant Proteins; D014688:Venoms; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D006821:Hyaluronoglucosaminidase
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