Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles.
The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium; D002417:Cattle; D004910:Erythrocyte Membrane / Q000378:metabolism; D006021:Glycophorins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D037102:Lectins; D008081:Liposomes; D010718:Phosphatidylserines / Q000302:isolation & purification; D012795:Sialoglycoproteins / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D006021:Glycophorins; D037102:Lectins; D008081:Liposomes; D010718:Phosphatidylserines; D012795:Sialoglycoproteins; D014909:Wheat Germ Agglutinins; D002118:Calcium