Labeling cell surface glycosylphosphatidylinositol-anchored proteins through metabolic engineering using an azide-modified phosphatidylinositol

Glycosylphosphatidylinositol (GPI) anchorage is one of the most common mechanisms to attach proteins to the plasma membrane of eukaryotic cells. GPI-anchored proteins (GPI-APs) play a critical role in many biological processes but are difficult to study. Here, a new method was developed for the effective and selective metabolic engineering and labeling of cell surface GPI-APs with an azide-modified phospha-tidylinositol (PI) as the biosynthetic precursor of GPIs. It was demonstrated that this azido-PI derivative was taken up by HeLa cells and incorporated into the biosynthetic pathway of GPIs to present azide-labeled GPI-APs on the live cell surface. The azido group was used as a molecular handle to install other labels through a biocompatible click reaction to enable various biological studies, e.g., fiuorescent imaging and protein pull-down, which can help explore the functions of GPI-APs and discover new GPI-APs.(c) 2023 Elsevier Inc. All rights reserved.