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...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2021-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0257435 |
| _version_ | 1818993002348019712 |
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| author | Miyako Nakano Susana Sabido-Bozo Kouta Okazaki Auxiliadora Aguilera-Romero Sofia Rodriguez-Gallardo Alejandro Cortes-Gomez Sergio Lopez Atsuko Ikeda Kouichi Funato Manuel Muñiz |
| author_facet | Miyako Nakano Susana Sabido-Bozo Kouta Okazaki Auxiliadora Aguilera-Romero Sofia Rodriguez-Gallardo Alejandro Cortes-Gomez Sergio Lopez Atsuko Ikeda Kouichi Funato Manuel Muñiz |
| author_sort | Miyako Nakano |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-20T20:35:08Z |
| format | Article |
| id | doaj.art-d314bcf6489d4e8b90bacebc982dad0d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-20T20:35:08Z |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-d314bcf6489d4e8b90bacebc982dad0d2022-12-21T19:27:16ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01169e025743510.1371/journal.pone.0257435Structural analysis of the GPI glycan.Miyako NakanoSusana Sabido-BozoKouta OkazakiAuxiliadora Aguilera-RomeroSofia Rodriguez-GallardoAlejandro Cortes-GomezSergio LopezAtsuko IkedaKouichi FunatoManuel MuñizGlycosylphosphatidylinositol (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.https://doi.org/10.1371/journal.pone.0257435 |
| spellingShingle | Miyako Nakano Susana Sabido-Bozo Kouta Okazaki Auxiliadora Aguilera-Romero Sofia Rodriguez-Gallardo Alejandro Cortes-Gomez Sergio Lopez Atsuko Ikeda Kouichi Funato Manuel Muñiz Structural analysis of the GPI glycan. PLoS ONE |
| 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 |
| url | https://doi.org/10.1371/journal.pone.0257435 |
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