Reconstitution into liposomes of highly purified PO glycoprotein from avian peripheral nerve myelin

Abstract

To explore the adhesive, possibly fusogenic properties of the major protein of peripheral nerve myelin (PO protein), three methods were employed for the reconstitution of this glycoprotein into liposomes of various lipid compositions. The most successful technique for the preparation of proteoliposomes was a detergent solubilization that employed Bio-Beads for the removal of Triton X-100 from the PO protein – lipid – detergent mixture. This method yielded a similar level of PO protein relative to lipids in the liposomes to that found in myelin. The protein to lipid ratio of the reconstituted proteoliposomes was only 1.4-fold lower than the initial protein to lipid ratio employed to prepare the liposomes. In contrast, the solvent evaporation and reverse-phase evaporation methods provided liposomes with 30 times lower protein to lipid ratio when compared with PO protein to lipid ratio initially used for the preparation of proteoliposomes. The incorporation of PO protein was dependent on the lipid composition of the vesicles and on the degree of purification of the protein. Proteolytic enzyme treatment showed that proteoliposomes containing the highly purified form of PO protein were partially or completely sensitive to trypsin treatment, whereas in liposomes containing a partially purified form of PO protein, a 22.4-kilodalton fragment was completely protected from trypsin digestion, indicating a transmembrane orientation of the partially purified polypeptide. Endoglycosidase F treatment of the liposomes established that all of the oligosaccharide moieties were removed from the liposome-associated protein, indicating the oligosaccharide moiety was exposed to the outside surface of the liposomes. Taken together, the results of limited proteolysis and endoglycosidase treatments did not provide strong evidence for the transmembrane orientation of highly purified PO in the liposomes, but were consistent with a surface of partial hairpin configuration, where the extracellular domains are facing the outside milieu. Electron microscopic examinations revealed highly convoluted, elongated shapes arid extensive fusion of PO liposomes containing lipids capable of adopting the hexagonal (H11) phase. Proteoliposomes prepared with the simple lipid composition of dipalmitoyl phosphatidylcholine -cholesterol (10:1 molar ratio) yielded stable, unilamellar liposomes of 80 nm average diameter, that encapsulated water-soluble drugs efficiently into the intravesicular compartment.Key words: liposomes, PO glycoprotein, myelin, peripheral nerve.