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

H-2 NMR spectroscopy was used to consider correspondence between exist ing single-crystal X-ray data for glycosphingolipids and their ceramid e backbone conformation in fluid phospholipid membranes. A monoglycosy lated sphingolipid, glucosylceramide (GlcCer), which represents the co re structure of many important glycosphingolipids, was derived by part ial synthesis through replacement of all native fatty acids with the 1 8-carbon species, stearic acid, deuterated at C2. N-[2,2-H-2(2)]]stear oyl-GlcCer was used to probe glycosphingolipid orientation and motion at low concentration in ''fluid'' phospholipid bilayers composed of di myristoylphosphatidylcholine (DMPC), with and without physiological am ounts of cholesterol. Spectral analysis, aided by stereoselective mono deuteration of the GlcCer fatty acid at C2, demonstrated that glycosph ingolipid average acyl chain backbone conformation in fluid phospholip id membranes, with or without cholesterol, is likely closely related t o 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 obse rvation, specific comparisons were made involving galactosylceramide ( GalCer) and globoside. GalCer provided a glycolipid differing only in monosaccharide stereochemistry (galactose vs glucose). Globoside permi tted isolation of the effect of headgroup size, since it is derived fr om GlcCer via extension of the carbohydrate portion by the oligosaccha ride, GalNAcbeta1 --> 3Galalpha1 --> 4Gal attached in beta1 --> 4 link age to the Glc residue. Spectra obtained for N-[2,2-H-2(2)]stearoyl-Ga lCer in the range 30-degrees-C to 45-degrees-C were within experimenta l error of those seen for N-[2,2-H-2(2)]stearoyl-GlcCer uniformly disp ersed in fluid membranes, indicating that orientational properties at the level of the fatty acid are extremely similar for these two specie s. Spectra obtained from N-[2,2-H-2(2)]stearoylgloboside consistently showed quadrupole splittings that were up to 10% smaller than those se en for the monoglycosyl lipids in a given membrane, likely reflecting somewhat greater orientational disorder. The presence of 33 mol % chol esterol accentuated spectral differences between glycolipids having lo ng vs short carbohydrate headgroups. An important characteristic featu re of glycolipids in eukaryotes is their common possession of very lon g 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) fat ty acid was synthesized to address this issue. At 10 mol % in DMPC/cho lesterol matrices, its spectra proved to be remarkably similar to thos e for the 18-carbon analogue described above; suggesting that in cell membranes the same conclusions apply regarding orientation at C2.