Molecular dynamics simulations and the conformational mobility of blood group oligosaccharides

Molecular dynamics simulations were carried out without explicit consideration of solvent to explore the conformational mobility of blood group A and H oligosaccharides. The potential energy force field of Rasmussen and co-workers was used with the CHARMM program on a number of disaccharide and trisaccharide models composed of fucose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine chosen to represent various fragments of blood group oligosaccharides. In agreement with results of earlier studies, stable chair conformations were found for each pyranoside from which no transitions were detected in simulations as long as 800 ps. Exocyclic dihedral angles, including that of C5-C6, generally show numerous transitions on a time scale of approximately 5-30 ps. The dihedral angles of some but not all glycosidic linkages of blood group oligosaccharides show transitions on the time scale of 30-50 ps, implying that the extent of internal motion in blood group oligosaccharides depends strongly on linkage stereochemistry. For certain blood group A and H oligosaccharides that show limited internal motion in these simulations, we argue that the calculations are consistent with our previous analysis of 1H nuclear Overhauser enhancement (NOE) data that imply single conformations over a wide range of temperature and solvent conditions. While the trajectories are consistent with 13C T1 values that have been interpreted as indicating rigid conformations, measurements of 13C-NOE and T1 as a function of magnetic field strength are proposed as an improved method for experimental detection of the internal motion that is suggested for certain oligosaccharides in these simulations. The results of these simulations differ substantially from those of peptides of a similar molecular weight in that the oligosaccharides show much less internal motion.     

A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.     

Conformations of type 1 and type 2 oligosaccharides from ovarian cyst glycoprotein by nuclear Overhauser effect spectroscopy and T1 simulations.

To probe differences in conformation of the type 1 and type 2 linkages in blood group oligosaccharides, two-dimensional nuclear Overhauser effect spectroscopy (2D-NOESY) and 1H T1 data were obtained for two blood group A oligosaccharide alditols containing the type 1 and type 2 linkage. The NOE data were interpreted using a complete relaxation matrix approach. Simulations of NOE and T1 values were made using disaccharide and tetrasaccharide model conformations generated by a systemic variation of the glycosidic dihedral angles phi and psi. NOEs from the amide protons of GlcNAc and GalNAc in the type 1 pentasaccharide alditol were obtained, and simulated in a manner similar to those from carbon-bound protons. In addition to providing data for determining the conformation of the type 1 linkage from amide proton NOEs of GlcNAc and GalNAc to neighboring residues, amide proton NOEs also yield information on the orientation of the acetamido side chains. The amide NOE data indicated subtle differences in the orientation of the amide side chain of GlcNAc among the A type 1 pentasaccharide alditol and two previously studied blood group oligosaccharides, lacto-N-difucohexaose 1 and lacto-N-fucopentaose 1. From the NOE and 1H T1 data, and from simple rigid geometry energy calculations, it is concluded that the type 1 and type 2 linkages in the oligosaccharides studied have different conformations and that these conformations are relatively rigid in solution.     

Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity

New neutral oligosaccharides from cow colostrum kappa-casein were identified and characterized by 500-MHz 1H-NMR spectroscopy. Their structures are Gal beta(1----3)GalNAc-ol, Gal beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Fuc alpha(1----3)[Gal beta(1----4)]GlcNAc beta(1----6)]GalNAc-ol. The tetrasaccharide and the cow colostrum kappa-caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow kappa-casein as a function of time after calving is studied.     

Modeling of the structure in aqueous solution of the exopolysaccharide produced by Lactobacillus helveticus 766.

A method is described for constructing a conformational model in water of a heteropolysaccharide built up from repeating units, and is applied to the exopolysaccharide produced by Lactobacillus helveticus 766. The molecular modeling method is based on energy minima, obtained from molecular mechanics calculations of each of the constituting disaccharide fragments of the repeating unit in vacuo, as starting points. Subsequently, adaptive umbrella sampling of the potential of mean force is applied to extract rotamer populations of glycosidic dihedral angles of oligosaccharide fragments in solution. From these analyses, the most probable conformations are constructed for the hexasaccharide-repeating unit of the polysaccharide. After exploring the conformational space of each of the individual structures by molecular dynamics simulations, the different repeating unit conformations are used as building blocks for the generation of oligo- and polysaccharide models, by using a polysaccharide building program. The created models of the exopolysaccharide produced by L. helveticus 766 exhibit a flexible twisted secondary structure and tend to adopt a random coil conformation as tertiary structure.     

Isolation and structural characterization of the smaller-size oligosaccharides from desialylated human kappa-casein. Establishment of a novel type of core for a mucin-type carbohydrate chain

Alkaline borohydride reductive cleavage (beta-elimination) of desialylated human kappa-caseinoglycopeptide resulted in the release of a series of oligosaccharides. The smaller-size compounds among them were purified to virtual homogeneity by gel filtration followed by high-performance liquid chromatography. The structures of 9 oligosaccharides were determined by 1H-NMR spectroscopy in conjunction with sugar analysis. The tetrasaccharide Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol and various partial structures thereof were characterized. Notably, the disaccharide GlcNAc beta(1----6)GalNAc-ol and the trisaccharide Gal beta(1----4)GlcNAc beta(1----6)GalNAc-ol were identified; they represent a novel type of core structure for mucin-type carbohydrate chains, namely a peptide-linked GalNAc that is mono-substituted at C-6. In addition, some oligosaccharides ending in GlcNAc-ol could be characterized. Their possible origin is discussed.     

MM3 potential energy surfaces of the 2-linked glucosyl trisaccharides alpha-kojitriose and beta-sophorotriose

The adiabatic potential energy surfaces (PES) of two trisaccharides with 2-linkages (alpha-kojitriose and beta-sophorotriose) were obtained using the MM3 force field, and are represented by a single 3D contour map for which the energy is plotted against the two psi glycosidic angles. In spite of the proximity of the positions where the two monosaccharidic units are linked to the central monosaccharide, an almost independent behavior of both linkages was found for the alpha-linked trisaccharide alpha-kojitriose, i.e., the surfaces are those expected from the maps of the disaccharide containing the same linkage. A slight shift of the position of the global minimum is found to occur, due to a hydrogen bond between the third and first monosaccharide units, which also leads to an increase in flexibility. On the other hand, for the beta-linked trisaccharide beta-sophorotriose, the surface is sharply different from that expected by observation of the disaccharide map. Some of the expected minima cannot appear unless a serious deformation of the phi and/or psi angles is produced. Furthermore, the global minimum corresponds to a combination of different conformations for each of the linkages, whereas another minimum with only slightly higher energy has both glycosidic linkages in a conformation less favored for the disaccharide, though close to that predicted in crystal diffraction studies.     

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.     

Efficient modelling protocols for oligosaccharides: from vacuum to solvent

The determination of conformational preferences of oligosaccharides is best approached by describing their preferred conformations on potential energy surfaces as a function of the glycosidic linkage φ, ψ torsional angles. For proper molecular mechanics modelling the flexibility of the rotatable pendant groups must also be considered. The so called adiabatic maps partially mimic the flexibility within the 10 dimensional conformational space of the pendant groups of the given disaccharide. These molecular mechanics maps are considered to be the state-of-the art of the φ, ψ potential energy surface of disaccharides recently calculated. The RAMM (RAndom Molecular Mechanics) method was shown to be able to calculate such profiles automatically. Additionally, based on the continuum solvent approach, RAMM allows the calculation of the effects of solvent on conformational energy profiles. Molecular dynamics simulations are also useful tools to study the influence of solvent on conformational behaviour of oligosaccharides. The capability of the RAMM calculational protocol to locate low-energy conformers on the multidimensional potential energy hypersurfaces of disaccharides is illustrated and compared with molecular dynamics simulations with and without inclusion of the solvent. This revised version was published online in August 2006 with corrections to the Cover Date.     

NMR and conformational studies of linear and cyclic oligo-(1→6)-β-d-glucosamines

The conformational behavior of a series of linear and cyclic oligo-(1→6)-β-d-glucosamines and their N-acetylated derivatives, which are related to fragments of natural poly-N-acetylglucosamine, was studied by theoretical molecular modeling and experimental determination of transglycosidic vicinal coupling constants ³J_C,H and ³J_H,H. Molecular dynamics simulations were performed under several types of conditions varying in the consideration of ionization of amino groups, solvent effect, and temperature. Neural network clustering and asphericity calculations were performed on the basis of molecular dynamics data. It was shown that disaccharide fragments in the studied linear oligosaccharides were not rigid, and tended to have several conformers, thus determining the overall twisted shape with helical elements. In addition, it was found that the behavior of C5–C6 bond depended significantly upon the simulation conditions. The cyclic di-, tri-, and tetrasaccharides mostly had symmetrical ring-shaped conformations. The larger cycles tended to adopt more complicated shapes, and the conformational behavior of their disaccharide fragments was close to that in the linear oligosaccharides.     

Disaccharide conformational flexibility. II. Molecular dynamics simulations of sucrose

Molecular dynamics simulations have been used to study the motions in vacuum of the disaccharide sucrose. Ensembles of trajectories were calculated for each of the five local minimum energy conformations identified in the adiabatic conformational energy mapping of this molecule. The model sucrose molecules were found to exhibit a variety of motions, although the global minimum energy conformation was found to be dynamically stable, and no transitions away from this structure were observed to occur spontaneously. In all but one of these vacuum trajectories, the intramolecular hydrogen bond between residues was maintained, in accord with recent nmr studies of this molecule in aqueous solution. Considerable flexibility of the furanoid ring was found in the trajectories. No "flips" to the opposite puckering for this ring were found in the simulations starting from the global minimum, although such a transition was observed for a trajectory initiated with one of the higher local minimum energy conformations. Overall, the observed structural fluctuations were consistent with the experimental picture of sucrose as a relatively rigid molecule.     

Solvation Effects on the Conformational Behaviour of Gellan and Calcium Ion Binding to Gellan Double Helices

The contribution of the presence of solvent to the conformations adopted by disaccharide fragments within the repeat unit of gellan have been studied by molecular modelling techniques. Initial conformational energy searches, using a dielectric continuum to represent the solvent, provided starting geometries for a series of molecular dynamics simulations. The solution behaviour from these simulations was subsequently compared to fibre diffraction data of the potassium gellan salt. The present calculations indicate considerable flexibility of the glycosidic linkages, and this is discussed in relation to its effect on gel formation. One of the fragments was solvated with explicit water molecules. These calculations showed the same conformational behaviour as those simulations conducted in implicit solvent.Finally, a series of molecular dynamics (MD) simulations were performed to study the calcium binding to gellan. The results from this clearly showed a well defined binding site for this ion.Abbreviations MM molecular mechanics - MD molecular dynamics     

Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment

The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.     

Application of restrained minimization, simulated annealing and molecular dynamics simulations for the conformational analysis of oligosaccharides.

The purpose of the present study was to determine the confidence with which the small number of 1H NMR nuclear Overhauser effect (NOE) distance constraints measurable across glycosidic linkages in oligosaccharides could be used for solution conformational analysis. This was assessed by use of these constraints in restrained molecular mechanical minimization of the tetrasaccharide Gal beta 1----4(Fuc alpha 1----3)Glc-NAc beta 1----3Gal, a model compound of the Lewis-X antigenic determinant. This presents a particularly severe test case in view of extreme resonance overlap and a dearth of inter-residue distance constraints. It is concluded that these constraints, when used in conventional restrained minimization, result in the generation of 'virtual conformations' and local minima about glycosidic linkages. However, these restraints are nevertheless found to be useful in the initial stages of a conformational analysis strategy involving restrained minimization combined with dynamical simulated annealing to define more accurately the global minimum energy configuration, together with molecular dynamics simulation to explore conformational mobility about this minimum. Theoretical ROE values calculated over the time course of the MD simulation, using a formalism appropriate for the time scale of the internal motion, are compared with those obtained experimentally in the oligosaccharide.     

A molecular dynamics study of the effect of glycosidic linkage type in the hemicellulose backbone on the molecular chain flexibility

The macromolecular conformation of the constituent polysaccharides in lignocellulosic biomass influences their supramolecular interactions, and therefore their function in plants and their performance in technical products. The flexibility of glycosidic linkages from the backbone of hemicelluloses was studied by evaluating the conformational freedom of the φ and ψ dihedral angles using molecular dynamic simulations, additionally selected molecules were correlated with experimental data by nuclear magnetic resonance spectroscopy. Three types of β‐(1→4) glycosidic linkages involving the monosaccharides (Glcp, Xylp and Manp) present in the backbone of hemicelluloses were defined. Different di‐ and tetrasaccharides with combinations of such sugar monomers from hemicelluloses were simulated, and free energy maps of the φ – ψ space and hydrogen‐bonding patterns were obtained. The glycosidic linkage between Glc‐Glc or Glc‐Man (C‐type) was the stiffest with mainly one probable conformation; the linkage from Man‐Man or Man‐Glc (M‐type) was similar but with an increased probability for an alternative conformation making it more flexible, and the linkage between two Xyl‐units (X‐type) was the most flexible with two almost equally populated conformations. Glycosidic linkages of the same type showed essentially the same conformational space in both disaccharides and in the central region of tetrasaccharides. Different probabilities of glycosidic linkage conformations in the backbone of hemicelluloses can be directly estimated from the free energy maps, which to a large degree affect the overall macromolecular conformations of these polymers. The information gained contributes to an increased understanding of the function of hemicelluloses both in the cell wall and in technical products.     

Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II

The carbohydrate binding specificity of Allomyrina dichotoma lectin II was investigated by analyzing the behavior of various complex type oligosaccharides and human milk oligosaccharides on an A. dichotoma lectin II-agarose column. Basically, the lectin interacts with the Gal beta 1----4GlcNAc group. Substitution of their terminal galactose residues by Neu5Ac alpha 2----6 will enhance their affinity to the lectin. By contraries, substitution at the C-2 or C-3 position of their terminal galactose with other sugars including sialic acid deprives their affinity to the lectin. With this characteristic, the immobilized lectin column can be used to separate complex type oligosaccharides with the Neu5Ac alpha 2----6Gal beta 1----4GlcNAc group from their isomeric oligosaccharides with the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc group, where Neu5Ac is N-acetylneuraminic acid.     

New strategy for the conformational analysis of carbohydrates based on NOE and 13C NMR coupling constants. Application to the flexible polysaccharide of Streptococcus mitis J22.

For complex oligosaccharides, which are relatively rigid with modest excursions from a single minimum energy conformation, it is straightforward to build conformational models from NOE data. Other oligosaccharides are more flexible with transitions between distinct minima separated by substantial energy barriers. We show that modeling based on scalar coupling data is superior to NOE-based modeling for the latter case. Long range 13C-13C and 13C-1H coupling constants measured for the heptasaccharide repeating subunit of the cell wall polysaccharide from Streptococcus mitis J22 are correlated with individual glycosidic dihedral angles, effectively uncoupling the degrees of freedom of the oligosaccharide and allowing a search for combinations of dihedral angles which are energetically reasonable, i.e., with no bad van der Waals contacts, and which can be combined to satisfy all the measured J values. Allowed values of the individual angles can then be combined to search for overall oligosaccharide conformations which contribute to the ensemble. We show that while the polysaccharide from S. mitis J22 is flexible, requiring multiple conformations, most of the flexibility is localized to a few bonds and only a rather small number of conformations is required to reproduce the experimental NOE and scalar coupling data.     

Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.     

NMR and conformational studies of linear and cyclic oligo-(1→6)-β-D-glucosamines

The conformational behavior of a series of linear and cyclic oligo-(1→6)-β-D-glucosamines and their N-acetylated derivatives, which are related to fragments of natural poly-N-acetylglucosamine, was studied by theoretical molecular modeling and experimental determination of transglycosidic vicinal coupling constants (3)J(C,H) and (3)J(H,H). Molecular dynamics simulations were performed under several types of conditions varying in the consideration of ionization of amino groups, solvent effect, and temperature. Neural network clustering and asphericity calculations were performed on the basis of molecular dynamics data. It was shown that disaccharide fragments in the studied linear oligosaccharides were not rigid, and tended to have several conformers, thus determining the overall twisted shape with helical elements. In addition, it was found that the behavior of C5-C6 bond depended significantly upon the simulation conditions. The cyclic di-, tri-, and tetrasaccharides mostly had symmetrical ring-shaped conformations. The larger cycles tended to adopt more complicated shapes, and the conformational behavior of their disaccharide fragments was close to that in the linear oligosaccharides.