Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe

Autores
Valko, Ayelén; Gallo, Giovanna Lucrecia; Weisz, Ariel D.; Parodi, Armando José A.; D'alessio, Cecilia
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N-glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [14C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms.
Fil: Valko, Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina
Fil: Gallo, Giovanna Lucrecia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Ministerio de Producción y Trabajo. Secretaría de Gobierno de Agroindustria. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Centro de Virología Humana y Animal. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Virología Humana y Animal; Argentina
Fil: Weisz, Ariel D.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina
Fil: Parodi, Armando José A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: D'alessio, Cecilia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
CHROMATOGRAM DECONVOLUTION
ENDOPLASMIC RETICULUM
LIPID-LINKED OLIGOSACCHARIDE
METABOLIC LABELING
N-GLYCOSYLATION
SCHIZOSACCHAROMYCES POMBE
YEAST
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/212945
Acceder
id CONICETDig_6d959224e8acd216878357e685381b15
oai_identifier_str oai:ri.conicet.gov.ar:11336/212945
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombeValko, AyelénGallo, Giovanna LucreciaWeisz, Ariel D.Parodi, Armando José A.D'alessio, CeciliaCHROMATOGRAM DECONVOLUTIONENDOPLASMIC RETICULUMLIPID-LINKED OLIGOSACCHARIDEMETABOLIC LABELINGN-GLYCOSYLATIONSCHIZOSACCHAROMYCES POMBEYEASThttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N-glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [14C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms.Fil: Valko, Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; ArgentinaFil: Gallo, Giovanna Lucrecia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Ministerio de Producción y Trabajo. Secretaría de Gobierno de Agroindustria. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Centro de Virología Humana y Animal. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Virología Humana y Animal; ArgentinaFil: Weisz, Ariel D.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; ArgentinaFil: Parodi, Armando José A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: D'alessio, Cecilia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaBio-protocol LLC2022-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/212945Valko, Ayelén; Gallo, Giovanna Lucrecia; Weisz, Ariel D.; Parodi, Armando José A.; D'alessio, Cecilia; Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe; Bio-protocol LLC; Bio-protocol; 12; 18; 9-2022; 1-152331-8325CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://bio-protocol.org/e4508info:eu-repo/semantics/altIdentifier/doi/10.21769/BioProtoc.4508info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:50:51Zoai:ri.conicet.gov.ar:11336/212945instacron: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:34982025-09-03 09:50:51.891CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe
title Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe
spellingShingle Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe
Valko, Ayelén
CHROMATOGRAM DECONVOLUTION
ENDOPLASMIC RETICULUM
LIPID-LINKED OLIGOSACCHARIDE
METABOLIC LABELING
N-GLYCOSYLATION
SCHIZOSACCHAROMYCES POMBE
YEAST
title_short Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe
title_full Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe
title_fullStr Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe
title_full_unstemmed Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe
title_sort Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe
dc.creator.none.fl_str_mv Valko, Ayelén
Gallo, Giovanna Lucrecia
Weisz, Ariel D.
Parodi, Armando José A.
D'alessio, Cecilia
author Valko, Ayelén
author_facet Valko, Ayelén
Gallo, Giovanna Lucrecia
Weisz, Ariel D.
Parodi, Armando José A.
D'alessio, Cecilia
author_role author
author2 Gallo, Giovanna Lucrecia
Weisz, Ariel D.
Parodi, Armando José A.
D'alessio, Cecilia
author2_role author
author
author
author
dc.subject.none.fl_str_mv CHROMATOGRAM DECONVOLUTION
ENDOPLASMIC RETICULUM
LIPID-LINKED OLIGOSACCHARIDE
METABOLIC LABELING
N-GLYCOSYLATION
SCHIZOSACCHAROMYCES POMBE
YEAST
topic CHROMATOGRAM DECONVOLUTION
ENDOPLASMIC RETICULUM
LIPID-LINKED OLIGOSACCHARIDE
METABOLIC LABELING
N-GLYCOSYLATION
SCHIZOSACCHAROMYCES POMBE
YEAST
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N-glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [14C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms.
Fil: Valko, Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina
Fil: Gallo, Giovanna Lucrecia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Ministerio de Producción y Trabajo. Secretaría de Gobierno de Agroindustria. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Centro de Virología Humana y Animal. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Virología Humana y Animal; Argentina
Fil: Weisz, Ariel D.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina
Fil: Parodi, Armando José A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: D'alessio, Cecilia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N-glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [14C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms.
publishDate 2022
dc.date.none.fl_str_mv 2022-09
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/11336/212945
Valko, Ayelén; Gallo, Giovanna Lucrecia; Weisz, Ariel D.; Parodi, Armando José A.; D'alessio, Cecilia; Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe; Bio-protocol LLC; Bio-protocol; 12; 18; 9-2022; 1-15
2331-8325
CONICET Digital
CONICET
url http://hdl.handle.net/11336/212945
identifier_str_mv Valko, Ayelén; Gallo, Giovanna Lucrecia; Weisz, Ariel D.; Parodi, Armando José A.; D'alessio, Cecilia; Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe; Bio-protocol LLC; Bio-protocol; 12; 18; 9-2022; 1-15
2331-8325
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://bio-protocol.org/e4508
info:eu-repo/semantics/altIdentifier/doi/10.21769/BioProtoc.4508
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
dc.publisher.none.fl_str_mv Bio-protocol LLC
publisher.none.fl_str_mv Bio-protocol LLC
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
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score 13.13397

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