Structural analysis of the GPI glycan

被引:7
|
作者
Nakano, Miyako [1 ]
Sabido-Bozo, Susana [2 ,3 ]
Okazaki, Kouta [1 ]
Aguilera-Romero, Auxiliadora [2 ,3 ]
Rodriguez-Gallardo, Sofia [2 ,3 ]
Cortes-Gomez, Alejandro [2 ,3 ]
Lopez, Sergio [2 ,3 ]
Ikeda, Atsuko [1 ]
Funato, Kouichi [1 ]
Muniz, Manuel [2 ,3 ]
机构
[1] Hiroshima Univ, Grad Sch Integrated Sci Life, Higashihiroshima, Hiroshima, Japan
[2] Univ Seville, Hosp Univ Virgen Rocio, Fac Biol, Dept Biol Celular,CSIC, Seville, Spain
[3] Inst Biomed Sevilla IBiS, Seville, Spain
来源
PLOS ONE | 2021年 / 16卷 / 09期
基金
日本学术振兴会;
关键词
ANCHORED PROTEINS; BIOSYNTHESIS; TRANSPORT;
D O I
10.1371/journal.pone.0257435
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页数:6
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