Isolation, characterization and immunolocalization of phosphorylcholine-substituted glycolipids in developmental...

Caenorhabditis elegans displays three neutral glycosphingolipids with structural homology to glycosphingolipids from the porcine nematode parasite, Ascaris suum. The present findings extend the degree of structural conservation between the two nematode species to glycosphingolipids with a phosphodiester substitution. Using a combination of hydrofluoric acid pretreatment, immunochemical characterization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, three zwitterionic, phosphorylcholine-substituted glycosphingolipids could be identified in the neutral glycolipid fraction of C. elegans. The components were isolated as their zwitterionic, phosphorylcholine-substituted, pyridylaminated oligosaccharides by HPLC. Structural analysis was performed using hydrofluoric acid treatment, partial acid hydrolysis, methylation analysis, gas chromatography-mass spectrometry, cleavage with exoglycosidases and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures are proposed as: component Nz1, GalNAc(beta1-4)[phosphorylcholine]GlcNAc(beta1-3)Man(beta1-4)Glc-cera mide; component Nz2, Gal(alpha1-3)GalNAc(beta1-4)[phosphorylcholine]-GlcNAc(beta1-3)Man(be ta1-4)Glc-ceramide; and component Nz3, Gal(beta1-3)- Gal(alpha1-3)GalNAc(beta1-4)[phosphorylcholine]GlcNAc(beta1-3)Man(bet a1-4)Glc-ceramide. The oligosaccharide core is characteristic of the biosynthetic arthro-carbohydrate series of protostomial glycosphingolipids. The ceramide moiety was specified by a d17 : 1 sphingoid-base with iso-branching and anteiso-branching, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids. Analysis of the spatial and temporal expression of the phosphorylcholine epitope, during embryonic and postembryonic development, showed it to be localized predominantly in seam cells and basement membranes, respectively. In early embryonic ontogenesis the phosphorylcholine epitope was only lipid bound, while in late embryonic and postembryonic development this epitope was both lipid bound and protein bound.