A chemical approach to unraveling the biological function of the glycosylphosphatidylinositol anchor

The glycosylphosphatidylinositol (GPI) anchor is a C-terminal posttranslational modification found on many eukaryotic proteins that reside in the outer leaflet of the cell membrane. The complex and diverse structures of GPI anchors suggest a rich spectrum of biological functions, but few have been confirmed experimentally because of the lack of appropriate techniques that allow for structural perturbation in a cellular context. We previously synthesized a series of GPI anchor analogs with systematic deletions within the glycan core and coupled them to the GFP by a combination of expressed protein ligation and native chemical ligation [Paulick MG, Wise AR, Forstner MB, Groves JT, Bertozzi CR (2007) J Am Chem Soc 129:11543-11550]. Here we investigate the behavior of these GPI-protein analogs in living cells. These modified proteins integrated into the plasma membranes of a variety of mammalian cells and were internalized and directed to recycling endosomes similarly to GFP bearing a native GPI anchor. The GPI-protein analogs also diffused freely in cellular membranes. However, changes in the glycan structure significantly affected membrane mobility, with the loss of monosaccharide units correlating to decreased diffusion. Thus, this cellular system provides a platform for dissecting the contributions of various GPI anchor components to their biological function.