title: Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes.
abstract: The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.
authors.affiliation: Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC, Américo Vespucio 49, 41092 Sevilla, Spain.
authors.forename: Julia B
authors.initials: JB
authors.lastname: Bonilla
authors.forename: M Belén
authors.initials: MB
authors.lastname: Cid
authors.forename: F-Xabier
authors.initials: FX
authors.lastname: Contreras
authors.forename: Félix M
authors.initials: FM
authors.lastname: Goñi
authors.forename: Manuel
authors.initials: M
authors.lastname: Martín-Lomas
mesh_terms: D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D015394:Molecular Structure; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000737:chemistry*; D054801:Phosphoinositide Phospholipase C; D010743:Phospholipids / Q000737:chemistry; D010738:Type C Phospholipases / Q000737:chemistry*
chemical_list: D017261:Glycosylphosphatidylinositols; D008051:Lipid Bilayers; D008081:Liposomes; D010743:Phospholipids; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase