Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process

Autores
Herlax, Vanesa Silvana; Maté, Sabina María; Rimoldi, Omar Jorge; Bakás, Laura Susana
Año de publicación
2009
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
α-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.
Instituto de Investigaciones Bioquímicas de La Plata
Facultad de Ciencias Exactas
Materia
Ciencias Médicas
Bioquímica
Proteínas Hemolisinas
Escherichia coli
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
SEDICI (UNLP)
Institución
Universidad Nacional de La Plata
OAI Identificador
oai:sedici.unlp.edu.ar:10915/82724

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spelling Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization processHerlax, Vanesa SilvanaMaté, Sabina MaríaRimoldi, Omar JorgeBakás, Laura SusanaCiencias MédicasBioquímicaProteínas HemolisinasEscherichia coliα-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.Instituto de Investigaciones Bioquímicas de La PlataFacultad de Ciencias Exactas2009info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionArticulohttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdf25199-25210http://sedici.unlp.edu.ar/handle/10915/82724enginfo:eu-repo/semantics/altIdentifier/issn/0021-9258info:eu-repo/semantics/altIdentifier/doi/10.1074/jbc.M109.009365info:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)reponame:SEDICI (UNLP)instname:Universidad Nacional de La Platainstacron:UNLP2025-09-29T11:15:35Zoai:sedici.unlp.edu.ar:10915/82724Institucionalhttp://sedici.unlp.edu.ar/Universidad públicaNo correspondehttp://sedici.unlp.edu.ar/oai/snrdalira@sedici.unlp.edu.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:13292025-09-29 11:15:36.24SEDICI (UNLP) - Universidad Nacional de La Platafalse
dc.title.none.fl_str_mv Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process
title Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process
spellingShingle Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process
Herlax, Vanesa Silvana
Ciencias Médicas
Bioquímica
Proteínas Hemolisinas
Escherichia coli
title_short Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process
title_full Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process
title_fullStr Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process
title_full_unstemmed Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process
title_sort Relevance of fatty acid covalently bound to Escherichia coli α-hemolysin and membrane microdomains in the oligomerization process
dc.creator.none.fl_str_mv Herlax, Vanesa Silvana
Maté, Sabina María
Rimoldi, Omar Jorge
Bakás, Laura Susana
author Herlax, Vanesa Silvana
author_facet Herlax, Vanesa Silvana
Maté, Sabina María
Rimoldi, Omar Jorge
Bakás, Laura Susana
author_role author
author2 Maté, Sabina María
Rimoldi, Omar Jorge
Bakás, Laura Susana
author2_role author
author
author
dc.subject.none.fl_str_mv Ciencias Médicas
Bioquímica
Proteínas Hemolisinas
Escherichia coli
topic Ciencias Médicas
Bioquímica
Proteínas Hemolisinas
Escherichia coli
dc.description.none.fl_txt_mv α-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.
Instituto de Investigaciones Bioquímicas de La Plata
Facultad de Ciencias Exactas
description α-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.
publishDate 2009
dc.date.none.fl_str_mv 2009
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
Articulo
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://sedici.unlp.edu.ar/handle/10915/82724
url http://sedici.unlp.edu.ar/handle/10915/82724
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/issn/0021-9258
info:eu-repo/semantics/altIdentifier/doi/10.1074/jbc.M109.009365
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
25199-25210
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instname:Universidad Nacional de La Plata
instacron:UNLP
reponame_str SEDICI (UNLP)
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repository.name.fl_str_mv SEDICI (UNLP) - Universidad Nacional de La Plata
repository.mail.fl_str_mv alira@sedici.unlp.edu.ar
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