Glycosphingolipid Fatty Acid Research Articles

Glycosphingolipids from bovine milk and milk fat globule membranes: a comparative study. Adhesion to enterotoxigenic Escherichia coli strains

Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.

Glycosphingolipid fatty acid arrangement in phospholipid bilayers: cholesterol effects

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.

Effect of glycosphingolipid fatty acid chain length on behavior in unsaturated phosphatidylcholine bilayers: A deuterium NMR study

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)

Behaviour of a glycosphingolipid with unsaturated fatty acid in phosphatidylcholine bilayers

N-(Oleoyl)galactosylceramide with perdeuterated acyl chain was prepared by partial synthesis, and studied by wide line 2H-NMR in phospholipid liposomes. Spectra were obtained for low glycolipid concentrations in bilayers of dimyristoyl-, distearoyl-, and 1-palmitoyl-2-oleoylphosphatidylcholines. In an attempt to isolate the effects of glycosphingolipid fatty acid cis unsaturation on glycolipid behaviour in membranes, spectral findings related to the above species were compared to literature NMR data for pure 1-palmitoyl-2-oleoylphosphatidylcholine bilayers in which the oleoyl chain of the phospholipid had been deuterated, and to analogously deuterated glycerol based lipids in Acholeplasma laidlawii membranes. The results for N-(oleoyl- d 33)galactosylceramide proved to be qualitatively and quantitatively very similar to published data dealing with glycerol based lipids at comparable temperatures. In addition, the results were strikingly similar for glycolipids dispersed in saturated and unsaturated phospholipid host matrices. It would appear that the primary effects of cis 9,10 fatty acid unsaturation in glycosphingolipids (at low concentration in fluid phospholipid membranes) are the same as those of fatty acid cis unsaturation in glycerolipids. It further appears that the overall dynamic behaviour of N-(oleoyl)galactosylceramide in fluid phospholipid membranes is very similar to that of glycerolipids with comparable acyl chains.

Effects of fatty acid alpha-hydroxylation on glycosphingolipid properties in phosphatidylcholine bilayers

The role of glycosphingolipid fatty acid α-hydroxylation as a modulator of glycolipid organization and dynamics was considered by 2H-NMR in bilayer membranes. For these experiments, galactosylceramides were prepared in which the natural fatty acid mixture was replaced with perdeuterated 18-carbon hydroxylated or non-hydroxylated stearic acid. The l-stereoisomer of N- α-(OH-stearoyl- d 34)galactosylceramide and its naturally-occurring d-α-OH analogue, were isolated for independent study. Bilayers were formed using 10 mol% galactosylceramide in a shorter chain phospholipid, dimyristoylphosphatidylcholine, in an attempt to reproduce several features of glycolipid-phospholipid interactions typical of cell membranes. Spectra of deuterated galactosylceramide in gel phase phospholipid membranes indicated that α-hydroxylation led to greater motional freedom and/or conformational disorder, with no measurable difference between d- and l-α-OH fatty acid derivatives. In fluid phosphatidylcholine bilayers the effects were modest. Glycolipid fatty acid hydroxylation led to broadening of the range of order parameters associated with methylene groups near the membrane surface (frequently referred to as the ‘plateau region’) - this effect being more marked for the naturally-occurring ( d) stereoisomer. The degree of overall molecular order sensed by the glycolipid fatty acid chain in a fluid host matrix was minimally affected by α-hydroxylation; although the plateau region of the d isomer was slightly more ordered than that of the l isomer and the non-hydroxylated species. These results suggest that a significant aspect of the α-hydroxy group effect on glycosphingolipid behaviour in bilayer membranes with low glycolipid content was interference with glycolipid packing amongst host phospholipids in the upper portion of the acyl chains. For the d stereoisomer, there was some evidence that the hydroxy group led to strengthening of interlipid interaction near the membrane surface.

A comparison of physical behaviour amongst four glycosphingolipid families

In the general search for information regarding glycosphingolipid behaviour and arrangement in membranes there has arisen the question of whether the answers may be very different for different glycosphingolipid families. We show here that in fact considerable basic similarity exists at least amongst a certain number of families as sensed by a spin label probe covalently attached to the glycosphingolipid fatty acid. This potential for similarity may be as important to take account of as is the clearly demonstrated potential for differences based on headgroup structure (Maggio, B., Albert, J. and Yu, R.K. (1988) Biochim. Biophys. Acta 945, 145–160). The ganglioside, G M1, was examined, as were the neutral species, galactosyl ceramide, lactosyl ceramide, and globoside, in phospholipid bilayers at 2 mol% and 10 mol%. In each case EPR spectra of a spin probe at carbon 16 of the single glycolipid fatty acid chain were compared. This was done for both 18-carbon and 24-carbon species in order to test the phenomenon over the range of common glycosphingolipid fatty acid chain length. The most telling result was that spectra of these four families were identical within experimental error in both rigid gel phase and semi-fluid non-cooperative bilayer matrices as sensed by a nitroxide spin probe near the centre of the membrane hydrophobic interior. No glycolipid family showed a detectable difference in organization either laterally or vertically within the membranes, nor in membrane dynamics. In addition, the membrane phase transition sensed by each family fell within the same narrow range. Freeze-etch visualization of glycolipid lateral distribution was attempted using a variety of native lectins and antibodies as markers. Success was severely limited by low affinity of marker attachment to neutral species; however, where testable, no differences were observed amongst families.