GLYCOSPHINGOLIPID BACKBONE CONFORMATION AND BEHAVIOR IN CHOLESTEROL-CONTAINING PHOSPHOLIPID-BILAYERS

H-2 NMR spectroscopy was used to consider correspondence between existing single-crystal X-ray data for glycosphingolipids and their ceramide backbone conformation in fluid phospholipid membranes. A monoglycosylated sphingolipid, glucosylceramide (GlcCer), which represents the core structure of many important glycosphingolipids, was derived by partial synthesis through replacement of all native fatty acids with the 18-carbon species, stearic acid, deuterated at C2. N-[2,2-H-2(2)]]stearoyl-GlcCer was used to probe glycosphingolipid orientation and motion at low concentration in ''fluid'' phospholipid bilayers composed of dimyristoylphosphatidylcholine (DMPC), with and without physiological amounts of cholesterol. Spectral analysis, aided by stereoselective monodeuteration of the GlcCer fatty acid at C2, demonstrated that glycosphingolipid average acyl chain backbone conformation in fluid phospholipid membranes, with or without cholesterol, is likely closely related to that predicted from single crystal X-ray studies [Pascher, I. (1976) Biochim. Biophys. Acta 455, 433-451; Pascher, I., & Sundell, S. (1977) Chem. Phys. Lipids 20, 175-191]. To test the generality of this observation, specific comparisons were made involving galactosylceramide (GalCer) and globoside. GalCer provided a glycolipid differing only in monosaccharide stereochemistry (galactose vs glucose). Globoside permitted isolation of the effect of headgroup size, since it is derived from GlcCer via extension of the carbohydrate portion by the oligosaccharide, GalNAcbeta1 --> 3Galalpha1 --> 4Gal attached in beta1 --> 4 linkage to the Glc residue. Spectra obtained for N-[2,2-H-2(2)]stearoyl-GalCer in the range 30-degrees-C to 45-degrees-C were within experimental error of those seen for N-[2,2-H-2(2)]stearoyl-GlcCer uniformly dispersed in fluid membranes, indicating that orientational properties at the level of the fatty acid are extremely similar for these two species. Spectra obtained from N-[2,2-H-2(2)]stearoylgloboside consistently showed quadrupole splittings that were up to 10% smaller than those seen for the monoglycosyl lipids in a given membrane, likely reflecting somewhat greater orientational disorder. The presence of 33 mol % cholesterol accentuated spectral differences between glycolipids having long vs short carbohydrate headgroups. An important characteristic feature of glycolipids in eukaryotes is their common possession of very long fatty acids-for which X-ray data, and NMR data for C2, do not exist. GalCer with deuterium-labeled 24-carbon ([2,2-H-2(2)]lignoceroyl) fatty acid was synthesized to address this issue. At 10 mol % in DMPC/cholesterol matrices, its spectra proved to be remarkably similar to those for the 18-carbon analogue described above; suggesting that in cell membranes the same conclusions apply regarding orientation at C2.