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

Sayan Kundu,^† Mohit Jaiswal,^† Kendall C. Craig, Jiatong Guo, and Zhongwu Guo^*

University of Florida

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 phosphatidylinositol (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., fluorescent imaging and protein pull-down, which can help explore the functions of GPI-APs and discover new GPI-APs.

In this project, we have developed a novel method for selective labeling, enrichment, and
isolation of GPI-anchored proteins (GPI-APs) and glycoproteins from a complex mixture of
proteins on the cell surface. The technique introduces a bioorthogonal chemical handle on the
appended glycan of GPI-APs, which can be further modified to install an affinity tag to enrich
these proteins from complex protein mixtures. Since the abundance of GPI-APs on the cell
surface is relatively low, this tag-based enrichment method may increase the sensitivity of
proteomics analysis and, optionally, quantification. This method will enable us to monitor changes
in the GPI proteomes under different physiological conditions, e.g., cell differentiation or disease
development, and systematically analyze the glycan moieties attached to GPI-APs. Global
profiling of cell surface GPI-APs in various cancer cell lines using this strategy will facilitate
uncovering of novel protein biomarkers.