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Structural analysis of the GPI glycan

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological rol...

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Autores principales: Nakano, Miyako, Sabido-Bozo, Susana, Okazaki, Kouta, Aguilera-Romero, Auxiliadora, Rodriguez-Gallardo, Sofia, Cortes-Gomez, Alejandro, Lopez, Sergio, Ikeda, Atsuko, Funato, Kouichi, Muñiz, Manuel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445438/
https://www.ncbi.nlm.nih.gov/pubmed/34529709
http://dx.doi.org/10.1371/journal.pone.0257435
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author Nakano, Miyako
Sabido-Bozo, Susana
Okazaki, Kouta
Aguilera-Romero, Auxiliadora
Rodriguez-Gallardo, Sofia
Cortes-Gomez, Alejandro
Lopez, Sergio
Ikeda, Atsuko
Funato, Kouichi
Muñiz, Manuel
author_facet Nakano, Miyako
Sabido-Bozo, Susana
Okazaki, Kouta
Aguilera-Romero, Auxiliadora
Rodriguez-Gallardo, Sofia
Cortes-Gomez, Alejandro
Lopez, Sergio
Ikeda, Atsuko
Funato, Kouichi
Muñiz, Manuel
author_sort Nakano, Miyako
collection PubMed
description Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.
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spelling pubmed-84454382021-09-17 Structural analysis of the GPI glycan Nakano, Miyako Sabido-Bozo, Susana Okazaki, Kouta Aguilera-Romero, Auxiliadora Rodriguez-Gallardo, Sofia Cortes-Gomez, Alejandro Lopez, Sergio Ikeda, Atsuko Funato, Kouichi Muñiz, Manuel PLoS One Lab Protocol Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins. Public Library of Science 2021-09-16 /pmc/articles/PMC8445438/ /pubmed/34529709 http://dx.doi.org/10.1371/journal.pone.0257435 Text en © 2021 Nakano et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Lab Protocol
Nakano, Miyako
Sabido-Bozo, Susana
Okazaki, Kouta
Aguilera-Romero, Auxiliadora
Rodriguez-Gallardo, Sofia
Cortes-Gomez, Alejandro
Lopez, Sergio
Ikeda, Atsuko
Funato, Kouichi
Muñiz, Manuel
Structural analysis of the GPI glycan
title Structural analysis of the GPI glycan
title_full Structural analysis of the GPI glycan
title_fullStr Structural analysis of the GPI glycan
title_full_unstemmed Structural analysis of the GPI glycan
title_short Structural analysis of the GPI glycan
title_sort structural analysis of the gpi glycan
topic Lab Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445438/
https://www.ncbi.nlm.nih.gov/pubmed/34529709
http://dx.doi.org/10.1371/journal.pone.0257435
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