A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization.
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 an...
Published in: | PLoS Neglected Tropical Diseases |
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ftdoajarticles:oai:doaj.org/article:ffef68a542e1411a99a38218ac139cda 2023-05-15T15:11:49+02:00 A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization. Valéria Pereira Ferrer Thiago Lopes de Mari Luiza Helena Gremski Dilza Trevisan Silva Rafael Bertoni da Silveira Waldemiro Gremski Olga Meiri Chaim Andrea Senff-Ribeiro Helena Bonciani Nader Silvio Sanches Veiga 2013-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0002206 https://doaj.org/article/ffef68a542e1411a99a38218ac139cda EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC3642055?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0002206 https://doaj.org/article/ffef68a542e1411a99a38218ac139cda PLoS Neglected Tropical Diseases, Vol 7, Iss 5, p e2206 (2013) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2013 ftdoajarticles https://doi.org/10.1371/journal.pntd.0002206 2022-12-31T11:26:01Z 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. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 7 5 e2206 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Valéria Pereira Ferrer Thiago Lopes de Mari Luiza Helena Gremski Dilza Trevisan Silva Rafael Bertoni da Silveira Waldemiro Gremski Olga Meiri Chaim Andrea Senff-Ribeiro Helena Bonciani Nader Silvio Sanches Veiga A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
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. |
format |
Article in Journal/Newspaper |
author |
Valéria Pereira Ferrer Thiago Lopes de Mari Luiza Helena Gremski Dilza Trevisan Silva Rafael Bertoni da Silveira Waldemiro Gremski Olga Meiri Chaim Andrea Senff-Ribeiro Helena Bonciani Nader Silvio Sanches Veiga |
author_facet |
Valéria Pereira Ferrer Thiago Lopes de Mari Luiza Helena Gremski Dilza Trevisan Silva Rafael Bertoni da Silveira Waldemiro Gremski Olga Meiri Chaim Andrea Senff-Ribeiro Helena Bonciani Nader Silvio Sanches Veiga |
author_sort |
Valéria Pereira Ferrer |
title |
A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization. |
title_short |
A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization. |
title_full |
A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization. |
title_fullStr |
A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization. |
title_full_unstemmed |
A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization. |
title_sort |
novel hyaluronidase from brown spider (loxosceles intermedia) venom (dietrich's hyaluronidase): from cloning to functional characterization. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2013 |
url |
https://doi.org/10.1371/journal.pntd.0002206 https://doaj.org/article/ffef68a542e1411a99a38218ac139cda |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 7, Iss 5, p e2206 (2013) |
op_relation |
http://europepmc.org/articles/PMC3642055?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0002206 https://doaj.org/article/ffef68a542e1411a99a38218ac139cda |
op_doi |
https://doi.org/10.1371/journal.pntd.0002206 |
container_title |
PLoS Neglected Tropical Diseases |
container_volume |
7 |
container_issue |
5 |
container_start_page |
e2206 |
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1766342607736143872 |