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

Abstract

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.

Fig. 1.

Structures and synthesis of native GPI anchor, GPI anchor analogs, and GPI-protein analogs. (A) Structures of the native GPI anchor from human erythrocyte acetylcholinesterase (structure 1) and GPI anchor analogs 2, 3, and 4. These GPI anchor analogs contain mimetics of the three domains of the GPI anchor: a phosphoethanolamine linker (red), the conserved glycan core (black), and a phospholipid tail (blue). R is a GPI anchor side chain, such as galactose or phosphoethanolamine. (B) Synthesis of GFP-GPI anchor analogs by a combination of expressed protein ligation and native chemical ligation. In this example, the GPI anchor analog 3 is coupled to GFP containing a C-terminal MESNa thioester to generate GFP-3.

Fig. 2.

Incorporation of GFP-2, GFP-3, and GFP-4 onto COS-7 cells. (A) Flow cytometry analysis of COS-7 cells treated with GFP-Cys or GFP-3. Vehicle-treated cells received PBS plus 0.05% β-octyl glucoside in serum-free media. This plot is representative of data obtained from three replicate experiments and from data obtained from cells treated with vehicle, GFP-2, or GFP-4. (B) Mean fluorescence intensity (MFI) in arbitrary units of the cells from A and cells treated with vehicle, GFP-2, or GFP-4. Error bars represent the standard deviation from three replicate experiments.

Fig. 3.

GFP-GPI(DAF), GFP-GPI(FR), GFP-2, GFP-3, and GFP-4 are internalized by CHO cells into similar endocytic compartments, the recycling endosomes. (A) GFP-GPI(DAF) expressed in CHO cells colocalizes with Tf-647, a marker for recycling endosomes. (B) GFP-GPI(FR) expressed in CHO cells colocalizes with Tf-647. (C) After a 4-h incubation with CHO cells, GFP-2 colocalizes with Tf-647. (D) After a 4-h incubation with CHO cells, GFP-3 colocalizes with Tf-647. (E) After a 4-h incubation with CHO cells, GFP-4 colocalizes with Tf-647. Green, GFP fluorescence; red, Tf-647 fluorescence. (Scale bar: 10 μm.) Images are representative of data obtained from three replicate colocalization experiments.

Fig. 4.

Selected images from FRAP experiments of individual HeLa cells at 25°C. (A) GFP-GPI(DAF) expressed in HeLa cells 27 h after transfection. (B) GFP-GPI(FR) expressed in HeLa cells 27 h after transfection. (C) GFP-2 on HeLa cells after a 4-h incubation. (D) GFP-3 on HeLa cells after a 4-h incubation. (E) GFP-4 on HeLa cells after a 4-h incubation. (Scale bar: 10 μm.) Images are representative of data obtained from five to nine cells. The area of the photobleached region is ≈95 μm².

Fig. 5.

GFP-GPI(DAF), GFP-GPI(FR), GFP-2, GFP-3, and GFP-4 are mobile on the HeLa cell surface. (A) Representative average cross-correlation curve (red solid line) with the standard deviation drawn as error bars at every fifth data point for the diffusion of GFP-4 on the surface of an individual HeLa cell at 25°C after a 4-h incubation. By fitting the experimental curves to the theoretical function describing free 2D diffusion (black dashed line) the characteristic correlation time, τ_D, and, consequently, the diffusion coefficient, D, were obtained (38, 48). (B) Calculated τ_D and D values and their standard deviations for GFP-GPI(DAF) and GFP-GPI(FR) endogenously expressed in HeLa cells at 25°C (27 h after transfection) and for GFP-2, GFP-3, and GFP-4 exogenously added to HeLa cells at 25°C (after a 4-h incubation). (C) Graphical representation of the data from B. Error bars represent the standard deviation around the mean values. P values were calculated by using Student's t test.