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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...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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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. |
format | Online Article Text |
id | pubmed-8445438 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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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