GPI-anchored proteins in reconstituted lipid bilayers: Structure, function, and cleavage by PI-specific phospholipase C

Many eukaryotic proteins are anchored to the cell surface by a glycosylphosphatidylinositol (GPI) moiety. One of these proteins, the enzyme ecto-5'-nucleotidase (5'-NTase), was purified from porcine lymphocytes and reconstituted into defined lipid bilayers. The GPI anchor was removed from all 5'-NTase molecules following cleavage by PI-specific phospholipase C from Bacillus thuringiensis (Bt-PI-PLC). Anchor cleavage was modulated by the composition of the membrane bilayer, suggesting that lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation of 5'-NTase was observed following Bt-PI-PLC cleavage from lipid bilayers. The degree of activation depended on the lipid composition of the reconstituted vesicles. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5' -NTase, possibly via conformational changes in the enzyme, and activity is restored upon release from the membrane.; A kinetic study of the release of 5'-NTase from membrane bilayers by Bt-PI-PLC indicated that lipid fluidity and bilayer packing were the most important factors influencing cleavage activity. Very high rates of cleavage were observed in fluid lipids with a low phase transition temperature (Tm), lymphocyte plasma membrane, and in lipid mixtures that form rafts. Arrhenius plots of the rate of anchor cleavage in various lipids showed a characteristic break at the Tm of the bilayer. The introduction of charged species into the membrane bilayer had little effect on anchor cleavage, indicating that membrane surface charge is less important in the regulation of Bt-PI-PLC activity.; The GPI-anchored protein placental alkaline phosphatase (PLAP) was labelled with 7-dimethylamino-coumarin-4 acetic acid (DMACA) or Oregon Green 488 (OG488) (fluorescent donors) and reconstituted into lipid bilayer vesicles containing increasing mole fractions of (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn-glycero-3-phosphoethanolamine (NBD-PE) or octadecyl rhodamine B (C18RhoB) (acceptors), respectively. Resonance energy transfer between the two donor/acceptor pairs was analysed to estimate the distance between the fluorescent label on PLAP and the membrane surface. The results indicated that the protein portion of PLAP is located at a distance of 8-12 A from the bilayer surface, suggesting that the protein lies close to the membrane, possibly resting on the surface.