No abstract

Galactosylceramides bearing a variety of different pure fatty acid chains were 2H labeled in the carbohydrate headgroup at C6 of the terminal galactose residue, for study by 2H NMR. Fatty acids investigated included the 24-carbon saturated lignoceric acid, 18-carbon saturated stearic acid, cis-9,10-unsaturated oleic acid, and D- and L-stereoisomers of alpha-hydroxystearic acid. Headgroup-deuterated glycolipids were incorporated at 10 mol % into unsonicated bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and 2H NMR spectra were recorded at 65 and 40 degrees C. Under these experimental conditions, the membranes studied were primarily in the liquid-crystalline phase. At a given temperature, spectra for deuterated galactosylceramides dispersed in the fluid phase were remarkably similar, regardless of the nature of the fatty acid attached to the glycolipid sphingosine backbone. In each case, the spectrum consisted of a superposition of two quadrupolar powder patterns of approximately equal intensity. The spectra may be interpreted as arising from equal populations of two stereoisomers (pro-R and pro-S) of the deuterated galactose hydroxymethyl function, which is undergoing rapid (greater than 10(6) s-1) interconversion among the possible rotamers about the C5-C6 bond of the sugar ring. Within experimental error, the only fatty-acid-induced spectral difference detected among these glycosphingolipids deuterated in the carbohydrate headgroup was in the species with alpha-hydroxy-substituted fatty acids. At 65 degrees C, N-(D-alpha-hydroxy)stearoyl- and N-(L-alpha-hydroxy)stearoylgalactosylceramide gave rise to the same quadrupole splittings, but these differed marginally from the splittings observed for the other glycolipids studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Wideline 2H NMR of model membranes was used to consider the molecular consequences of factors often suggested as modulators of complex glycosphingolipid oligosaccharide arrangement and motional characteristics at cell surfaces. GM1, asialo-GM1, and globoside were studied as examples of plasma membrane recognition sites. The experimental approach involved substitution of deuterons (D) for protons at specific locations within the carbohydrate chains. Deuterated glycolipids were then dispersed at 7-10 mol% in unsonicated bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine. Factors tested for their significance to carbohydrate chain conformation and dynamics included glycolipid natural alkyl and acyl chain variability, membrane fluidity, and the presence of cholesterol and a charged sugar residue (neuraminic acid). Effects of Ca2+ and membrane-associated protein were briefly considered. Two distinct strategies were employed in substituting deuterons for selected protons of carbohydrate residues. Neither approach necessitated alteration of the glycolipid natural fatty acid composition. (i) Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of GM1 and asialo-GM1, and on the terminal N-acetylgalactosamine (GalNAc) residue of globoside, were replaced with deuterium (producing -CDHOH) by an enzymatic oxidation/reduction cycle. This represents the first application of such an approach to deuteration of complex neutral glycolipids. Spectral results were compared to those obtained for the similarly-deuterated monoglycosyl lipid, galactosylceramide (GalCer), with natural fatty acid composition. Efficacy of this labeling method may in principle be influenced by structural variations within a given glycolipid family. Also, asymmetric rotation of the deuterated group made it less attractive than the second method for relating spectral features to receptor geometry. (ii) A general synthetic, nonenzymatic method was investigated for replacing amino sugar N-acetyl groups with deuterated acetate (-COCD3). The acetate group of the GalNAc residue of globoside, GM1, and asialo-GM1, as well as that on neuraminic acid in GM1, was replaced with -COCD3. This second method afforded better signal-to-noise--an important consideration for 2H NMR. The NMR technique employed had the potential for detecting changes of as little as 10% in oligosaccharide orientation or motional order. Each glycolipid demonstrated clear evidence of preferred average oligosaccharide conformations in all (fluid) membrane environments examined. The most striking observation was that, in fluid matrices, conformation and motional order of the complex oligosaccharide chains were only modestly influenced by factors tested, including natural variation in the glycolipid hydrocarbon chains, membrane fluidity, temperature, and the presence of cholesterol or the N-acetylneuraminic acid (NeuAc) residue on GM1.(ABSTRACT TRUNCATED AT 400 WORDS)

Deuterium wide line NMR spectroscopy was used to study cholesterol effects on the ceramide portions of two glycosphingolipids (GSLs) distributed as minor components in fluid membranes. The common existence of very long fatty acids on GSLs was taken into account by including one glycolipid species with fatty acid chain length matching that of the host matrix, and one longer by 6 carbons. N-stearoyl and N-lignoceroyl galactosyl ceramide with perdeuterated fatty acid (18:0[d35] GalCer and 24:0[d47] GalCer) were prepared by partial synthesis. They were dispersed in bilayer membranes having the 18-carbon-fatty-acid phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), as major component. Glycolipid fatty acid chain behavior and arrangement were analyzed using order profiles derived from their 2H-NMR spectra. Cholesterol effects on order parameter profiles for 18:0[d35] GalCer, with chain length equal to that of the host matrix, followed the pattern known for acyl chains of phospholipids. The presence of sterol led to restriction of trans/gauche isomerization along the length of the chain, with the largest absolute increase in order parameters being toward the surface, but somewhat greater relative effect just below the "plateau" region. In cholesterol-containing membranes, order parameter profiles for the long chain species, 24:0[d47] GalCer, showed a characteristic secondary "plateau" associated with carbon atoms C14 to C23, a feature also present in SOPC bilayers without cholesterol and in pure hydrated 24:0[d47] GalCer. Cholesterol-induced ordering effects on the long chain glycolipid were similar to those described for the shorter chain species, but were minimal at the methyl terminus. Within a given membrane,SCD profiles for 1 8:O[d3] GalCer and 24:0[d47] GalCer were quantitatively similar to a membrane depth of C13 to C14. SCD values at C16 and C17 were about 15% and 28% higher, respectively, for the long chain GSL than for its short chain analogue inSOPC/cholesterol (compared to 21 and 31%, respectively, in membranes without cholesterol). Nitroxide spin labels attached rigidly to C16 of the long chain glycolipid gave EPR order parameters that were twice as high as for the same spin label at C16 on the shorter chain glycolipid in both matrices. It would appear that the above factors impose a tendency for the "extra" portion of the 24-carbon chain to cross the bilayer midplane where it may interact with terminal portions of acyl chains in the opposing monolayer; however, steric constraints, and probably collision events associated with lateral diffusion, induce wide orientation fluctuations in the segment involved.

Deuterium-labeled glycosphingolipids, N-lignoceroyl[d47]galactosylceramide (24:0 fatty acid form) and N-stearoyl[d35]galactosylceramide (18:0 fatty acid form) were prepared by partial synthesis. These probe-labeled species, differing only in the lengths of their fatty acids, were compared via 2H NMR with regard to arrangement and behavior in bilayers of the monounsaturated phospholipid 1-stearoyl-2-oleoylphosphatidylcholine (SOPC). Results were used to consider the physical significance of the great range of common acyl chain lengths that is a frequent feature of cell membrane glycosphingolipids. N-lignoceroyl[d47]- and N-stearoyl[d35]galactosylceramide were incorporated at concentrations ranging from 5 to 50 mol % into unsonicated phospholipid liposomes, and their spectra were analyzed in the range +73 to -14 degrees C. For the 18:0 fatty acid derivative, first spectral moments, M1, were calculated and plotted as a function of temperature for each sample composition. Spectral inspection for regions of phase coexistence, in conjunction with consideration of M1 curves, permitted derivation of phase diagram boundaries which were then refined using spectral subtraction techniques. The phase diagram for galactosylceramide with short fatty acid in SOPC was compared to the corresponding phase diagram for its long-chain analogue, derived previously in the same fashion [Morrow, M. R., Singh, D., Lu, D., & Grant, C. W. M. (1992) Biochim. Biophys. Acta 1106, 85-93]. The binary phase diagrams referred to above, which reflect the behavior of short- and long-chain glycolipids in a common phospholipid host matrix, displayed important similarities and differences. In fluid membranes, the behavior appeared to be remarkably alike, as reflected in superimposable fluidus curves over the concentration range studied.(ABSTRACT TRUNCATED AT 250 WORDS)