Glycolipid Fatty Acid Research Articles

Acyl chain length effects related to glycosphingolipid crypticity in phospholipid membranes: probed by 2H-NMR

Wideline 2H-NMR was used to consider the relationships amongst glycosphingolipid and phospholipid fatty acid chain length and glycosphingolipid receptor function, in a system classically associated with crypticity. Galactosyl ceramide (GalCer), having 18−or 24−carbon fatty acid, was deuterium labelled at the conformationally-restricted fatty acid α-carbon (C−2). 2H-NMR spectra of N-[2,2− 2 H 2] stearoyl and N-[2,2− 2 H 2] lignoceroyl GalCer (GalCer with 18−vs. 24−carbon selectively deuterated fatty acid) were then compared over a range of temperatures in phosphatidylcholine/cholesterol membranes in which the host phospholipid had dimyristoyl, dipalmitoyl, or distearoyl fatty acid composition. Findings were evaluated in the light of known sensitivity of antibody interaction with GalCer to temperature and to both glycolipid fatty acid chain length and host matrix fatty acid chain length. Under the conditions of experimentation, spectra were not obtainable for glycolipids having rigid body motions that were slow on the NMR timescale (10 −4-10 −5s)- i.e.. motions typical of non-fluid (gel phase) membranes. The systems, DPPC/cholesterol and DSPC/cholesterol, in which the original observation was made of increased antibody binding to GalCer with long fatty acid, proved to be characterised by receptor motions that were in this slow timescale for both 18:0 and 24:0 GalCer at 22–24δC. Under conditions for which spectra could be obtained, those for GalCer with [2,2− 2H 2]lignoceroyl (24–carbon α-deuterated) fatty acid were qualitatively similar to those of its 18-carbon analogue in all (fluid) membranes examined. However, spectral splittings differed quantitatively between deuterated 18:0 and 24:0 GalCer at a given temperature, dependent upon host matrix. These differences were most marked at lower temperatures and in the longer chain (more ordered) matrices, DPPC/cholesterol and DSPC/cholesterol. This suggests that maximum effects of glycolipid chain length on glycolipid receptor function may be expected to occur in spatially and motionally constrained lipid environments. There was little effect of temperature on spectral splittings seen for a given sample containing deuterated 18:0 GalCer. The small differences seen could be adequately accounted for by relatively minor alterations in glycolipid order and backbone conformation. In contrast, 24:0 GalCer in DPPC/cholesterol and DSPC/cholesterol displayed significant variation in its spectral splittings as the temperature was reduced; and these proved to be the source of the quantitative differences between 18:0 and 24:0 GalCer referred to above. For 18:0 GalCer, the only spectral feature seen to be notably sensitive to temperature and to choice of host membrane phospholipid in the range, 22–65°C, was whole-body motion; which could be abruptly ‘frozen out’ with decreasing membrane fluidity. The 24:0 GalCer analogue was seen to undergo the same loss of motional freedom, in the same temperature range, as 18:0 GalCer. We suggest therefore that several previous observations of GalCer crypticity in phospholipid/cholesterol systems having longer vs. shorter chain phospholipids, may derive most importantly from reduced motional freedom of the glycolipid recognition sites.

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.

Isolation and composition of glycolipids from corn flour

Total lipids extracted according to De Stefanis and Ponte [Biochim. Biophys. Acta, 176 (1969) 198–201] from corn flour (from Cameroon, variety white) amounted to 0·5% of the dry flour weight. The total lipids consisted of 90% neutral lipids, 5% glycolipids and 3% phospholipids. The glycolipids contained glucolipids as major components (4·5%) and minor amounts of a glycerolipidic fraction (0·6%). Glycolipid fatty acids were identified as linoleic, oleic and palmitic acids.

Glycosphingolipid interdigitation in phospholipid bilayers examined by deuterium NMR and EPR

Glycosphingolipid fatty acids commonly have up to eight methylene carbons more than do their surrounding phospholipid-attached counterparts. The resultant ‘extra’ segment may very well modulate glycosphingolipid function as receptor and structural element. As part of an investigation of this phenomenon, galactosylceramide was prepared with a deuterated 18-carbon fatty acid chain. Deuterium-labelled galactosylceramide was assembled at 10 mol% into unsonicated phosphatidylcholine bilayers having all 14-carbon or all 18-carbon saturated fatty acid chains (DMPC and DSPC, respectively). The systems were studied by 2H-NMR spectroscopy above and below the phase transition temperatures, T m , of the host matrices. At comparable reduced temperatures in fluid membranes the degree of motional order exhibited by the glycolipid fatty acid was significantly higher in the phospholipid host matrix that was four carbons shorter. The fatty acid chain segment least affected by the change from long to short chain host matrix was the terminal (deutero)methyl group (an increase of 8% in quadrupolar splitting for the terminal methyl vs. 16% for deuterons at C 17 and 23–28% for the remainder of the chain). Order parameter profiles for galactosylceramide were qualitatively very similar in the two host membranes, arguing against any major conformational difference between the arrangement of the 18-carbon glycolipid fatty acid in the 18-carbon vs. 14-carbon host matrices. Similarly a nitroxide spin probe covalently attached to carbon-12 of the galactosylceramide fatty acid gave clear indication of greater order in the fluid 14-carbon fatty acid phospholipid bilayer. These results are consistent with ‘tethering’ of the extra length of fatty acid via interdigitation into the opposing monolayer. There was no spectroscopic evidence of any intrinsic difference in glycolipid behaviour in the two fluid host matrices. 2H-NMR spectra of galactosylceramide at comparable reduced temperatures below T m of the phospholipid bilayer were very different for 14-carbon vs. 18-carbon host matrices. The glycolipid fatty acid showed evidence of relatively reduced mobility in the shorter chain matrix.

Fatty acid profile of the digestive gland and mantle in the bivalve Macoma balthica

1. 1. Digestive gland and mantle fatty acids were studied in spring and summer in the bivalve Macoma balthica off the southern coast of Finland. The presence of lipids was also examined histochemically in various clam tissues. 2. 2. the neutral lipid content of the digestive gland increased ca 4.5-fold during the annual growth period. 3. 3. Neutral lipid fatty acids of the digestive gland, of which palmitoleic, eicosapentaenoic and palmitic acids were predominant, were clearly distinguished from phospho- and glycolipid fatty acids. 4. 4. The degree of unsaturation of phospholipid fatty acids was higher in the cold season both in the digestive gland and mantle, mainly due to the titer of eicosapentaenoic acid.

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.

Globoside with spin-labelled fatty acid: bilayer lateral distribution and immune recognition

We have critically addressed the question of lateral distribution of glycolpids in bilayer membranes, and the effect of glycolipid fatty acid chain length upon such distribution. For this purpose we synthesised the complex neutral glycosphingolipid, globoside, with spin-labelled fatty acid. Base hydrolysis to remove the natural fatty acid was found to deacetylate the GalNAc residue concomitantly, necessitating application of the synthetic route described for gangliosides by Neuenhofer et al. (Biochemistry 24, 525–532 (1985)). Globosides were produced with 18-carbon and 24-carbon fatty acids bearing a spin label at the C-16 position. Spin-labelled globosides were incorporated at 2 and 10 mol% into rigid, highly cooperative bilayer matrices of 1,2-dipalmitoylglycerophosphocholine (DPPC) and also into semi-fluid, non-cooperative membranes of DPPC/cholesterol. Recorded electron paramagnetic resonance (EPR) spectra were analysed by comparison with a library of standards representing samples of known composition. Spectra were manipulated using a computer program which permitted linear combination of standards to simulate coexistence of laterally separated domains of different composition. The most important conclusions ere as follows: (1) at least 80% of the globoside was definitely not confined to domains highly enriched in glycolipid, although there was evidence of binary-phase separation in the rigid DPPC/globoside matrix; (2) the presence of 33 mol% cholesterol reduced the evidence of globoside phase separation; (3) there was remarkably little difference in results whether the globoside fatty acid chain length was similar to that of the phospholipid host matrix or eight carbons longer. Temperature profiles derived over the phase-transition region of DPPC using spin-labelled globoside or an unattached amphiphilic spin label were consistent with these findings. The same systems lent themselves to consideration of the role of glycolipid fatty acid chain length and cholesterol in determining glycolipid crypticity in membranes: (1) polyclonal anti-globoside IgG bound to globoside in DPPC liposomes without inducing agglutination. (2) The same antibodies did agglutinate DPPC/cholesterol liposomes bearing globoside. (3) The effect of cholesterol probably was upon glycolipid dynamics or attitude in the membrane, rather than upon distribution. (4) These observations were basically unaffected by the choice of 18-carbon vs. 24-carbon glycolipid fatty acid. (5) It appears that the (tetrasaccharide) globoside sugar chain is sufficiently long that the importance of fatty acid chain length in determining headgroup ‘accessibility’ to antibodies is greatly reduced relative to the situation of monosaccharide species such as galactosylceramide.

Effects of temperature and light on the lipids of Sphagnum magellanicum

The growth, lipid, protein and chlorophyll contents and fatty acid composition changes in polar lipids were studied in the moss Sphagnum magellanicum cultured aseptically under different light and temperature conditions. In dim light, changes in growth and the amounts of the studied organic components were larger than in darkness. The lipid and chlorophyll a and b amounts increased in dim light, while the actual growth declined. The chlorophyll a/b ratios increased slightly when the light decreased. In normal light, temperature decrease was accompanied by a decrease in the amounts of linolenic acid and a corresponding increase in the amounts of palmitic acid in the glycolipids. When the change in temperature was combined with a change in light, decreasing light and temperature was accompanied by a decrease in linolenic and a reverse reaction for palmitic acids in the glycolipids. Often no linear pattern of change in the glycolipid fatty acids could be seen, and no profound unsaturation/saturation changes occurred. When the temperature change was combined with a corresponding change in the length of the light period (in normal light), the optimum conditions for the formation of linolenic acid appeared to be 15° and 12 hr/light, although no clear patterns of change were seen regardless of the glyco- or phospholipid studied. The largest deviations from the ‘normal’ fatty acid composition under these conditions were seen generally at lower temperatures (0–5°) and shorter periods of light (–6 hr light/day).

The glycolipid fatty acids of Porphyridium purpureum cultured in the presence of detergents

The glycolipid fatty acid composition of Porphyridium purpureum on a solid medium was studied in the presence of Triton X-100 (TX), sodium desocycholate (SDC), sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB). TLC and GC/MS were used in determining the relative fatty acid compositions of mono-and digalactosyl diglycerides (MGDG and DGDG) and in assessing the MGDG/DGDG ratio. The most common fatty acids were palmitic (16:0), stearic (18:0), linoleic (18:2 ω6), arachidonic (20:4 ω6) and eicosapentaenoic (20:5 ω3) acids, the long-chain polyunsaturated acids being more abundant in DGDG; α-linolenic acid (18:3 ω3) was absent. TX and SDC in particular caused an increase in the saturation grade of both MGDG and DGDG fatty acids at very low concentrations (5–15 ppm). With a detergent concentration of 20 ppm a reversion of this tendency was sometimes found, and the fatty acid composition approached the controls again. The effects of SDS and CTAB were not as prominent. All the detergents studied increased the normal MGDG/DGDG ratio (0.3) to a maximum of ∼ 1. The effect of increasing detergent concentration is not linear. The results suggest that in some cases very low detergent concentrations can be more effective than higher ones, a fact which may be important in algae growing in polluted waters.

Metabolism of brain glycolipid fatty acids

The metabolism of the fatty acid moieties of brain cerebrosides, sulfatides, and gangliosides is reviewed and discussed. The methodology involved in the isolation of the fatty acids is described briefly. It seems clear now that most of these acids are made by chain elongation of intermediate length fatty acids by addition of acetate residues. The unsaturated acids are made by desaturation of the intermediate length acids (palmitic, heptadecanoic, stearic) followed by chain elongation. The hydroxy acids are made directly from the corresponding nonhydroxy acids, saturated, unsaturated, and odd-numbered. All the hydroxy acids undergo oxidative decarboxylation to yield fatty acids containing one less carbon atom. The odd-numbered acids are also made from propionate, which is elongated to intermediate length acids and then to longer acids. The major intermediate length "primer" acid seems to be palmitate, but there is evidence that the stearate used for cerebroside synthesis is also madede novo from acetate. The ganglioside fatty acids were found to turn over somewhat faster than the other fatty acids. Two metabolic pools for the cerebroside acids were found, one with a very high turnover rate, the other with a very low turnover rate.