Ks. Hamilton et al., GLYCOSPHINGOLIPID BACKBONE CONFORMATION AND BEHAVIOR IN CHOLESTEROL-CONTAINING PHOSPHOLIPID-BILAYERS, Biochemistry, 32(15), 1993, pp. 4022-4028
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.