Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry
- Autores
- Vacchina, Paola; Tripodi, Karina Eva Josefina; Escalante, Andrea Marta; Uttaro, Antonio Domingo
- Año de publicación
- 2012
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2 mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2 mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time.
Fil: Vacchina, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Tripodi, Karina Eva Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Escalante, Andrea Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Orgánica. Área Farmacognosia; Argentina
Fil: Uttaro, Antonio Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina - Materia
-
Trypanosoma
Leishmania
Phytosphingosine
Sphingosine
Dihydroceramide desaturase
Hydroxylase - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/268845
Ver los metadatos del registro completo
id |
CONICETDig_f27485da59d297a425b3af0411fb6b78 |
---|---|
oai_identifier_str |
oai:ri.conicet.gov.ar:11336/268845 |
network_acronym_str |
CONICETDig |
repository_id_str |
3498 |
network_name_str |
CONICET Digital (CONICET) |
spelling |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometryVacchina, PaolaTripodi, Karina Eva JosefinaEscalante, Andrea MartaUttaro, Antonio DomingoTrypanosomaLeishmaniaPhytosphingosineSphingosineDihydroceramide desaturaseHydroxylasehttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2 mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2 mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time.Fil: Vacchina, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Tripodi, Karina Eva Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Escalante, Andrea Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Orgánica. Área Farmacognosia; ArgentinaFil: Uttaro, Antonio Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaElsevier Science2012-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/268845Vacchina, Paola; Tripodi, Karina Eva Josefina; Escalante, Andrea Marta; Uttaro, Antonio Domingo; Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry; Elsevier Science; Molecular and Biochemical Parasitology; 184; 1; 7-2012; 29-380166-6851CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0166685112000989info:eu-repo/semantics/altIdentifier/doi/10.1016/j.molbiopara.2012.04.005info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:13:55Zoai:ri.conicet.gov.ar:11336/268845instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:13:56.03CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry |
title |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry |
spellingShingle |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry Vacchina, Paola Trypanosoma Leishmania Phytosphingosine Sphingosine Dihydroceramide desaturase Hydroxylase |
title_short |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry |
title_full |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry |
title_fullStr |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry |
title_full_unstemmed |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry |
title_sort |
Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry |
dc.creator.none.fl_str_mv |
Vacchina, Paola Tripodi, Karina Eva Josefina Escalante, Andrea Marta Uttaro, Antonio Domingo |
author |
Vacchina, Paola |
author_facet |
Vacchina, Paola Tripodi, Karina Eva Josefina Escalante, Andrea Marta Uttaro, Antonio Domingo |
author_role |
author |
author2 |
Tripodi, Karina Eva Josefina Escalante, Andrea Marta Uttaro, Antonio Domingo |
author2_role |
author author author |
dc.subject.none.fl_str_mv |
Trypanosoma Leishmania Phytosphingosine Sphingosine Dihydroceramide desaturase Hydroxylase |
topic |
Trypanosoma Leishmania Phytosphingosine Sphingosine Dihydroceramide desaturase Hydroxylase |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2 mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2 mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time. Fil: Vacchina, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Tripodi, Karina Eva Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Escalante, Andrea Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Orgánica. Área Farmacognosia; Argentina Fil: Uttaro, Antonio Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina |
description |
Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2 mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2 mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time. |
publishDate |
2012 |
dc.date.none.fl_str_mv |
2012-07 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 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://hdl.handle.net/11336/268845 Vacchina, Paola; Tripodi, Karina Eva Josefina; Escalante, Andrea Marta; Uttaro, Antonio Domingo; Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry; Elsevier Science; Molecular and Biochemical Parasitology; 184; 1; 7-2012; 29-38 0166-6851 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/268845 |
identifier_str_mv |
Vacchina, Paola; Tripodi, Karina Eva Josefina; Escalante, Andrea Marta; Uttaro, Antonio Domingo; Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry; Elsevier Science; Molecular and Biochemical Parasitology; 184; 1; 7-2012; 29-38 0166-6851 CONICET Digital CONICET |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0166685112000989 info:eu-repo/semantics/altIdentifier/doi/10.1016/j.molbiopara.2012.04.005 |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf application/pdf application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Elsevier Science |
publisher.none.fl_str_mv |
Elsevier Science |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
reponame_str |
CONICET Digital (CONICET) |
collection |
CONICET Digital (CONICET) |
instname_str |
Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.name.fl_str_mv |
CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
_version_ |
1844614061823623168 |
score |
13.070432 |