The different conformations of the glycerol region of crystalline acylglycerols.

From nearly 40 crystal structures of glycerolipids, accumulated during three decades, preferred structural features are now emerging regarding favored conformations of lipid molecules and the molecular arrangement at the surface of lipid assemblages. Comparisons with conformations of glycerolipid molecules in lipid-protein complexes and in dynamic systems, studied by spectroscopic methods and molecular modeling, show the general validity of these preferred solid-state conformations.

Steric presentation and recognition of the saccharide chains of glycolipids at the cell surface: favoured conformations of the saccharide-lipid linkage calculated using molecular mechanics (MM3).

The orientation of the saccharide moiety of glycolipids at the membrane surface is determined by an interplay of different steric factors, e.g. the conformation of the saccharide chain, the conformation of the saccharide-lipid linkage and restrictions due to the membrane surface. In the present study the preferred conformations of the saccharide-lipid linkages of glucosylceramides with normal and hydroxy fatty acids and glucosyldiglycerides with acyl and alkyl chains were studied using molecular mechanics (MM3). The populations of different conformers were calculated on the basis of relaxed energy maps. Calculations on glucosylceramides at a dielectric constant (epsilon) of 4 showed three dominating conformers: phi/psi/theta 1 = +sc/ap/-sc (global energy minimum), /-sc/ap and +sc/ap/ap, respectively. In sphingolipids the +sc rotamer of theta 1 is disfavoured due to a Hassel-Ottar interaction involving the sphingosine O1 and O3 oxygen atoms. alpha-O Hydroxylation of the fatty acid does not significantly affect the conformational preferences of the saccharide-ceramide linkage at epsilon-values relevant for biomembranes. In glycoglycerolipids the global energy minimum is shifted to the phi/psi/theta 1 = +sc/ap/ap conformation. For glycolipids located in membranes additional steric restrictions are imposed by the surrounding lipid layer. These restrictions in the steric presentation appear to be of crucial significance for the selective recognition and crypticity of glycolipids in membranes.

Orientation of the saccharide chains of glycolipids at the membrane surface: conformational analysis of the glucose-ceramide and the glucose-glyceride linkages using molecular mechanics (MM3).

Preferred conformations of the saccharide-ceramide linkage of glucosylceramides with different ceramide structures (normal and hydroxy fatty acids) were investigated by molecular mechanics (MM3) calculations and compared with conformational features obtained for glucosylglycerolipids (diacyl and dialkyl analogues). Relaxed energy map calculations with MM3 were performed for the three bonds (C1'-O1-C1-C2, torsion angles phi, psi, and theta 1) of the glucose-ceramide/diglyceride linkage at different values of the dielectric constant. For the phi torsion of the glycosidic C1'-O1 bond the calculations show a strict preference for the +sc range whereas the psi/theta 1 energy surface is dependent on the structure of the lipid moiety as well as on the dielectric constant (epsilon). Calculations performed on glucosylceramide with normal and hydroxy fatty acids at epsilon = 4 (bilayer subsurface conditions) show three dominating conformers (psi/theta 1 = ap/-sc, -sc/ap, and ap/ap). The ap/-sc conformer, which represents the global energy minimum, is stabilized by polar interactions involving the amide group. The +sc rotamer of theta 1 is unfavored in sphingolipids due to a Hassel-Ottar effect involving the sphingosine O3 and O1 oxygen atoms. Comparative calculations on glycosylglycerolipid analogues (ester and ether derivatives) show a distinct preference for the ap rotamer of theta 1. An evaluation of the steric hindrance imposed by the surrounding membrane surface shows that in a bilayer arrangement the range of possible conformations for the saccharide-lipid linkage is considerably reduced. The significance of preferred conformations of the saccharide-ceramide linkage for the presentation and recognition of the saccharide chains of glycosphingolipids at the membrane surface is discussed.

Characterisation of the anti-A antibody response following an ABO incompatible (A2 to O) kidney transplantation.

Anti-A,B antibodies produced in a blood group OLe(a-b-) recipient receiving a kidney graft from a blood group A2Le(a-b+) donor have been analysed for their ability to bind to different glycosphingolipid antigens. Solid-phase RIA using pure glycosphingolipid antigens and a chromatogram binding assay using total nonacid glycosphingolipid fractions from erythrocytes of different human blood group phenotypes together with pure glycolipid antigens were used as assay systems. Serum antibodies were shown to bind equally well to A (types 1, 2, 3 and 4) and B (types 1 and 2) antigenic structures but no binding to H antigens (types 1, 2 and 4) was detected. After adsorption of serum antibodies on A1 Le(a-b+) erythrocytes there was a residual anti-A antibody activity which could not be adsorbed by synthetic A-trisaccharides coupled to crystalline silica (Synsorb-A). These residual antibodies, which are not present in a pretransplant serum sample, had a specificity for the A antigen with type 1 core saccharide chain and the binding epitope obviously included both the N-acetylgalactosamine and the N-acetylglucosamine. The fucose residue was apparently not obligate for binding. The conformation of the sugar units involved in the binding epitope was determined.

Saccharide orientation at the cell surface affects glycolipid receptor function.

Three allelic variants of P-pilus-associated G-adhesins (lectins) with different cell-binding properties were recently described. Here we have analyzed Escherichia coli HB101 strains expressing the recombinant G-adhesin variants for their ability to agglutinate erythrocytes from various species as this relates to the glycosphingolipid (GSL) composition in the erythrocyte membranes. All three variants exhibit similar specificities for the globo-series GSLs affixed to artificial surfaces. However, only the PapGJ96 adhesin induces agglutination of erythrocytes having globotriaosylceramide (GbO3) [Gal(alpha 1-4)LacCer] as the major GSL. Furthermore, only PapGAD110 induces strong agglutination of erythrocytes having globotetraosylceramide (GbO4) [GalNAc(beta 1-3)Gal(alpha 1-4)LacCer] as the major GSL, while PrsGJ96 alone agglutinates those containing globopentaosylceramide (GbO5) [GalNAc(alpha 1-3)GalNAc(beta 1-3)Gal(alpha 1-4)LacCer]. Molecular modeling of these globo-GSLs demonstrates different saccharide orientations to the membrane surface for these isoreceptors. We suggest that the differential binding of the three G-adhesin variants results from differences in epitope presentation at the membrane among these globo-GSLs.

The effect of hydrogen bonds on the conformation of glycosphingolipids. Methylated and unmethylated cerebroside studied by X-ray single crystal analysis and model calculations.

The conformation and molecular packing of permethylated beta-D-galactosyl-N-octadecanoyl-D-spingosine (cerebroside) was determined by X-ray single crystal analysis at 185 K (R = 0.16). The lipid crystallizes in the orthorhombic space group P2(1)2(1)2(1) with the unit cell dimensions a = 8.03, b = 7.04 and c = 88.10 A. The four molecules in the unit cell pack in a bilayer arrangement with tilting (48 degrees) hydrocarbon chains. The direction of the chain tilt alternates in the two bilayer halves and in adjacent bilayers. In order to define the effect of hydrogen bonds on the molecular conformation the structural features of the permethylated cerebroside are compared with that of unsubstituted cerebroside (I. Pascher and S. Sundell (1977) Chem. Phys. Lipids 20, 179). It is shown that methylation of the hydrogen donor groups does not affect the conformation of the ceramide part. However, by abolishing the intramolecular hydrogen bond between the amide N--H group and the glycosidic oxygen the galactose ring changes its orientation from layer-parallel to layer-perpendicular. Calculations using molecular mechanics, MM2(87), show that in natural cerebroside the intramolecular hydrogen bond stabilizes the theta 1 = -syn-clinal conformation about the C(1)--C(2) sphingosine bond by 2-2.5 kcal/mol compared to other staggered conformations. The significance of the L shape of the native cerebroside, making both the carbohydrate and polar ceramide groups accessible as a binding epitope in recognition processes, is discussed.

Conformational analysis of blood group A-active glycosphingolipids using HSEA-calculations. The possible significance of the core oligosaccharide chain for the presentation and recognition of the A-determinant.

Conformational analysis of four different A-active glycosphingolipids, A types 1-4, was carried out using HSEA-calculations with the GESA-program. In their minimum energy conformations the oligosaccharide chains are more or less curved; in particular the type 3 and 4 have a strongly bent shape. When the carbohydrate structures are linked to ceramide, using the conformational features predominantly observed in crystal structures of membrane lipids, rather drastic differences in the orientation of the oligosaccharide chains are obtained. For the type 1 glycosphingolipid the model study indicates that the A-determinant extends almost perpendicularly to the membrane plane whereas for type 2, 3 and 4 the terminal part of the oligosaccharide chains is more parallel to the membrane. The fucose branch on type 3 and type 4 thereby appears directed towards the environment whereas for type 2 it would face the membrane. Due to restrictions imposed by the membrane layer this core specific orientation is largely preserved even if the flexibility of the saccharide-ceramide linkage is taken into account. Hydrophilic and hydrophobic sites on the surface of the different oligosaccharide chains in their minimum energy conformation were located using the GRID-program. It is suggested that the core-dependent presentation of the A-determinant might explain the chain type specificity observed for different monoclonal anti-A antibodies. The results further suggest that assay systems ensuring a membrane-like presentation of the glycolipid antigen should be used in studies of glycolipid/protein interactions.

Preferred conformation and dynamics of the glycerol backbone in phospholipids. An NMR and X-ray single-crystal analysis.

The conformation of the glycerol group of a number of diacyl and monoacyl (lyso) phospholipids differing in the chemical nature of the head group was studied by 1H high-resolution NMR and X-ray crystallography. The NMR measurements were carried out with solutions or micellar dispersions of the lipids in deuteriated organic solvents or 2H2O. Both solutions, in which the lipid is present as monomers, and lipid micelles give rise to good high-resolution NMR spectra exhibiting spin coupling hyperfine interactions. From 1H spin coupling it is concluded that there are two stable conformations about the glycerol C(2)-C(3) bond of phospholipids. One of these (rotamer A) is characterized by torsion angles theta 3 = antiperiplanar, theta 4 = +synclinal, and the other (rotamer B) by theta 3 = +synclinal, theta 4 = -synclinal. In both rotamers A and B the ester oxygens on the glycerol carbon atoms C(2) and C(3) are synclinal, and hence both types of rotamers readily allow the parallel alignment of the two hydrocarbon chains. By comparison of NMR and single-crystal X-ray data it is obvious that both conformations are minimum free energy conformations. Rotamer A is the conformation prevailing in phospholipid single-crystal structures. The conformation of rotamer B is also found in phospholipid single-crystal structures though to a lesser extent, e.g., in 2,3-dilauroyl-DL-glycero-1-phospho-N,N-dimethylethanolamine and 2,3-dimyristoyl-D-glycero-phospho-DL-glycerol. NMR measurements indicate that in liquid crystals the diacylglycerol part of phospholipids fluctuates between the two stable staggered conformations of rotamers A and B.(ABSTRACT TRUNCATED AT 250 WORDS)

On the dissection of binding epitopes on carbohydrate receptors for microbes using molecular modelling.

The most common receptors for microbes on animal cells seem to be carbohydrates. One characteristic property of microbial protein-carbohydrate interaction is the recognition of sequences placed within an oligosaccharide chain. This leads to a series of isoreceptors defined as saccharides carrying the particular receptor sequence with different neighbouring groups. A microbial ligand may have different binding affinities for such isoreceptors depending upon steric hindrance from neighbouring groups upon access to the binding epitope. By a comparison of binding preferences to a series of isoreceptors with their calculated conformation, the binding epitope on a particular receptor sequence may be approximated by use of molecular modelling. This approach is illustrated for two bacteria recognising lactosylceramide. The potential importance of the procedure for further developments including drug design is briefly discussed.

Identification of a blood group A active hexaglycosylceramide with a type 1 carbohydrate chain in plasma of an A1 Le(a-b-) secretor.

A blood group A active hexaglycosylceramide with a type 1 carbohydrate chain was identified in the plasma of an A1 Le(a-b-) secretor. The analysis was done on the total non-acid glycosphingolipid fraction using mass spectrometry, NMR spectroscopy, and anti-A antibody immunostaining on thin-layer chromatograms.

Conformation and packing properties of membrane lipids: the crystal structure of sodium dimyristoylphosphatidylglycerol.

The conformation and molecular packing of sodium 1,2-dimyristoyl-sn-glycero-phospho-rac-glycerol (DMPG) have been determined by single crystal analysis (R = 0.098). The lipid crystallizes in the monoclinic spacegroup P2(1) with the unit cell dimensions a = 10.4, b = 8.5, c = 45.5 A and beta = 95.2 degrees. There are two independent molecules (A and B) in the asymmetric unit which with respect to configuration and conformation of their glycerol headgroup are mirror images. The molecules pack tail to tail in a bilayer structure. The phosphoglycerol headgroups have a layer-parallel orientation giving the molecules an L-shape. At the bilayer surface the (-) phosphoglycerol groups are arranged in rows which are separated by rows of (+) sodium ions. Laterally the polar groups interact by an extensive network of hydrogen, ionic and coordination bonds. The packing cross-section per molecule is 44.0 A2. The hydrocarbon chains are tilted (29 degrees) and have opposite inclination in the two bilayer halves. In the chain matrix the chain planes are arranged according to a so far unknown hybride packing mode which combines the features of T parallel and O perpendicular subcells. The two fatty acid substituted glycerol oxygens have mutually a - synclinal rather than the more common + synclinal conformation. The conformation of the diacylglycerol part of molecule A and B is distinguished by an axial displacement of the two hydrocarbon chains by four methylene units. This results in a reorientation of the glycerol back bone and a change in the conformation and stacking of the hydrocarbon chains. In molecule A the beta-chain is straight and the gamma-chain is bent while in molecule B the chain conformation is reversed.

Interactions and space requirements of the phosphate head group in membrane lipids. The crystal structure of disodium lysophosphatidate dihydrate.

The packing arrangement and molecular conformation of lysophosphatidate (disodium 3-lauroyl-DL-glycero-1-phosphate dihydrate (LPA] was determined by single crystal analysis. The lipid crystallizes in the triclinic space group P1 with unit cell dimensions of a = 7.74, b = 5.54, c = 32.87 A and alpha = 92.6, beta = 99.2, gamma = 128.3 degrees. The molecules are arranged tail to tail in a bilayer structure. Thereby the D- and L-enantiomers pack separately in the opposite halves of the bilayer and are conformationally related by mirror symmetry. The hydrocarbon chains adopt the triclinic (T parallel) chain packing mode and are tilted by 55 degrees with respect to the layer normal. The phosphate groups are linked by one of the sodium ions to tightly packed rows. Between these rows the second sodium ion and the two water molecules of hydration are accommodated forming an extensive network of hydrogen, ionic and coordination bonds. The two sodium ions are positioned 1 A below and above the plane defined by the centres of gravity of the phosphate charges. The phosphate group and its 4 ligands thus pack effectively with a small cross-sectional area in the layer plane of 33.6 A2.

New gangliosides from human erythrocytes.

We have identified a number of gangliosides from human erythrocytes that have not previously been detected in these cells, including two new compounds. The gangliosides were separated into monosialo- and disialoganglioside fractions by DEAE-column chromatography. Two monosialogangliosides that have not been previously detected in these cells are GM2 and GM1. Two other monosialogangliosides have the same carbohydrate structure, NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4) Glc-Cer, but they contain different fatty acids. The compound with higher chromatographic mobility (MG-5) contains a predominance of C22 and C24 fatty acids, whereas the principal fatty acid of the slower compound (MG-6) is C16. Both gangliosides are receptors for human anti-p and anti-Gd cold agglutinins. Six disialogangliosides not identifed previously in human red cells include GD3, GD1a, GD1b, DG-3, (formula: see text). The latter two are newly identified compounds and DG-4 contains a sugar sequence that has not been described previously, sialic acid residues linked to different hydroxyl groups of the same galactose.

Further characterization of the structure of GM1b ganglioside from rat ascites hepatoma.

A ganglioside named GM1b (Hirabayashi, Y., Taki, T., and Matsumoto, M. (1979) FEBS Let. 100, 253-257) with a sugar composition identical with that of GM1, II3 alpha NeuAc-GgOse4Cer (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 leads from 2 alpha NeuAc) beta 1 leads to 4G1c beta 1 leads to 1'ceramide), isolated from rat ascites hepatoma was further characterized. In contrast to GM1, the sialic acid in this ganglioside was susceptible to clostridial sialidase in the absence of bile salts. Based on the sequential enzymic hydrolysis, permethylation analysis and direct probe mass spectrometric analysis, the structure of this ganglioside is determined to be: NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc leads to ceramide. The structure of this ganglioside is identical with that biosynthesized in vitro (Stoffyn, A., Stoffyn, P., and Yip, M. C. M. (1975) Biochim. Biophys. Acta 409, 97-103).

Chemical structures of three fucogangliosides isolated from nervous tissue of mini-pig.

In the nervous tissue of miniature pig, type Göttingen, three fucosyl-containing gangliosides are major gangliosides. All three were found in dorsal-root ganglia and spinal cord, but only two of them in the forebrain. The concentrations of these gangliosides were increased in chloroquine intoxication. Their chemical structures were determined by component analysis, partial acid and enzymatic degradation and assay of the partially methylated sugars after hydrolysis, reduction and acetylation. Mass spectrometric analysis of the intact gangliosides as permethylated, permethylated-reduced and permethylated-reduced-silylated derivatives confirmed the following structures of the three gangliosides. (See Formula in text).