Insertion of a Glycosylphosphatidylinositol-Anchored Enzyme into Liposomes

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of ³H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance—about 2.5 nm—between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein.Abbreviations: AP, alkaline phosphatase; AP-GPI, glycosylphosphatidylinositol-linked alkaline phosphatase; EM, electron microscopy; EPA, egg phosphatidic acid; GPI, glycosylphosphatidylinositol; OctGlc, n-octyl -D-glucoside; PtdCho, egg yolk phosphatidylcholine; PtdIns-PLC, glycosylphosphatidylinositol-specific phospholipase C. Enzymes: Alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (EC 3.1.3.1); glycosylphosphatidylinositol-specific phospholipase C (EC 3.1.4.10).