Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
Facultad de Biologia, Departamento de Biologia Celular, Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Universidad de Sevilla e Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain.
PLoS One. 2021 Sep 16;16(9):e0257435. doi: 10.1371/journal.pone.0257435. eCollection 2021.
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.
糖基磷脂酰肌醇（GPI）锚定蛋白是所有真核生物中一种重要的翻译后修饰，发生在内质网（ER），并将 GPI-锚定蛋白（GPI-APs）递送到细胞表面，在那里它们发挥着广泛的重要生理作用。本文描述了一种在酵母中纯化和结构分析单个 GPI-AP 的 GPI 聚糖的特殊方法。该方案涉及 GFP 标记的特定 GPI-AP 的表达，从细胞膜部分酶释放，免疫纯化，电泳分离和胰蛋白酶消化后通过质谱分析携带 GPI 聚糖的肽。我们特别使用此方案来解决特定 GPI-AP 的 GPI 聚糖在其运输到细胞表面过程中经历的结构重塑。该方法也可用于研究 GPI-AP 生物合成途径，并直接确认单个蛋白质的预测 GPI 锚定。
