Hard-sphere Exo-anomeric Calculations Research Articles

Conformational study of asialo-GM1 (GA1) ganglioside.

In order to investigate the significance of preferred conformations of the saccharide for the steric orientation and recognition of glycosphingolipids at the membrane surface, the conformational free energy calculations were carried out on the asialo-GM1 [GA1; beta-D-Gal (1-->3) beta-D-GalNac(1-->4) beta-D-Gal(1-->4) beta-D-Glc-O-ceramide) using a new program CONCARB (CONformational study program for CARBohydrate) in the unhydrated and hydrated states. The overall backbone conformational of GA1 appears to be extended with a little bent at the glycosidic II-III linkage, in which two pyranose rings of Gal(IV)-GalNAc-(III) moiety orient approximately perpendicular to those of Gal(II)-Glc(I) moiety. This is consistent with the structures deduced from high-sensitivity differential scanning calorimetry experiments and the nmr study on GA1. The calculated glycosidic torsion angles of the lowest free energy conformation of GA1 in the hydrated state are in accord with the structures of relevant oligosaccharides deduced from nmr experiments and hard sphere exoanomeric calculations. A comparison of the values of glycosidic torsion angles phi and psi of GA1 and its constituent oligosaccharides indicates that the overall backbone conformation of each oligosaccharide is retained when the oligosaccharide chain becomes longer. This implies that the short-range interactions between the nearest-neighbored saccharides are of significant importance in stabilizing the overall backbone conformation of GA1 in both the unhydrated and hydrated states. The different orientation and hydrogen bonds of hydroxymethyl and hydroxyl groups from one oligosaccharide to another suggest that the medium- and long-range interactions are also of consequence. Hydration seems to affect significantly the confirmation of these groups, but not to perturb remarkably the overall backbone conformation of GA1.

Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.

Novel fucolipids of human adenocarcinoma: monoclonal antibody specific for trifucosyl Ley (III3FucV3FucVI2FucnLc6) and a possible three-dimensional epitope structure.

Immunization of mice with Ley-active trifucosylnonaosylceramide (III3FucV3FucVI2FucnLc6) isolated from human colonic adenocarcinoma (Nudelman, E., Levery, S. B., Kaizu, T., and Hakomori, S. (1986) J. Biol. Chem. 261, 11247-11253) followed by selection of hybridoma by positive reaction with this antigen and negative reaction with two other Ley antigens (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) resulted in successful isolation of the hybridoma producing IgM antibody, termed KH1, specific to Ley-active trifucosylnonaosylceramide, which does not cross-react with Ley-active hexaosyl- or octaosylceramides (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) without internal fucosyl substitution. The three-dimensional structure of the trifucosylnonaosylceramide was simulated based on previously published glycosidic torsion angles for fucosyl type 2 chain (Lex and Ley) and for GlcNAc beta 1----3Gal beta as predicted by hard sphere exo-anomeric calculations (Thøgersen, H., Lemieux, R. U., Bock, K., and Meyer, B. (1982) Can. J. Chem. 60, 44-57). The picture thus constructed showed a broad nonpolar area consisting of the hydrophobic surface of the pyranosyl ring and acetamido group of N-acetylglucosamine and three CH3 groups of L-fucose; this hydrophobic area is adjacent to a hydrophilic area. In analogy to the detailed structure of Leb or Ley involved in their interactions with antibodies and lectins (Spohr, U., Hindsgaul, O., and Lemieux, R. U. (1985) Can. J. Chem. 63, 2644-2652), such a wide hydrophobic area adjacent to a hydrophilic region could be recognized by the antibody KH1, as shown in the model illustrated in the text. Since the axis of ceramide, which is inserted in the lipid bilayer, is perpendicular to the plane of type 2 chain, the epitope recognized by the antibody KH1 is located at the external nonpolar surface of the carbohydrate chain that is overlaid on the lipid bilayer.

Pcilo calculations on 2-acetamido-2-deoxy-α- d-galactose and O-(2-acetamido-2-deoxy-α- d-galactosyl)-(1→3)-2-acetamido-2-deoxy- d-galactose

PCILO semi-empirical quantum-chemical calculations have been conducted on 2-acetamido-2-deoxy-α- d-galactose (GaINAc) and the α- d-(1→3)-linked di-GaINAc disaccharide component of the Forssman antigen, in order to determine the favored orientations of their side groups, and the mutual orientations of the two pyranoid rings in the latter. For the N-acetyl group, three stable positions have been located, in one of which the plane of the amide unit is normal to the mean plane of the pyranoid ring. The orientation of the hydroxyl group attached to the anomeric carbon atom is found to be in accord with the exo-anomeric predictions. Furthermore, all of the side-group orientations of GaINAc found in the present study are in good agreement with the results of X-ray investigations. A grid-search method was used to locate all low-energy conformational regions for the glycosidic dihedral angles of O-(2-acetamido-2-deoxy-α- d-galactosyl)-(1→3)-2-acetamido-2-deoxy- d-galactose. Two allowed regions were found, and energy minimization was then performed in each of them. For this disaccharide, three sets of favored orientations of the (reducing) pyranose ring relative to the nonreducing ring were found. One set of glycosidic torsion angles is very close to that found from our X-ray diffraction results, n.m.r.-spectral observations, and hard-sphere exo-anomeric calculations.

The preferred conformation of oligosaccharides in solution inferred from high resolution NMR data and hard sphere exo-anomeric calculations

Abstract

The preferred conformation of oligosaccharides derived from the complex-type carbohydrate portions of glycoproteins.

The preferred conformation in aqueous solution of oligosaccharides related to the complex‐type carbohydrate portion of glycoproteins has been inferred from high‐resolution nuclear magnetic resonance data (both 1H and 13C) combined with hard‐sphere exo‐anomeric calculations. The results indicate that the N‐acetyl‐lactosamine units are arranged in such a way that they are widely separated in space. Thus, these carbohydrates can cover large surface areas of the attached protein and provide good targets for recognition by other proteins.