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A Imberty

Publications and source records attributed to A Imberty.

At least 37 records · Page 2Linked to original sources
PubMed1999

The living factory: in vivo production of N-acetyllactosamine containing carbohydrates in E. coli.

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a beta(1,4)GlcNAc-oligosaccharide synthase), and the Neisseria beta(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as betaGal(1,4)[betaGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for betaGal(1,4)GlcNAc alpha(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.

Amino Sugars↗
PubMed1999

Fold recognition study of alpha3-galactosyltransferase and molecular modeling of the nucleotide sugar-binding domain.

The structure and fold of the enzyme responsible for the biosynthesis of the xenotransplantation antigen, namely pig alpha3 galactosyltransferase, has been studied by means of computational methods. Secondary structure predictions indicated that alpha3-galactosyltransferase and related protein family members, including blood group A and B transferases and Forssman synthase, are likely to consist of alternating alpha-helices and beta-strands. Fold recognition studies predicted that alpha3-galactosyltransferase shares the same fold as the T4 phage DNA-modifying enzyme beta-glucosyltransferase. This latter enzyme displays a strong structural resemblance with the core of glycogen phosphorylase b. By using the three-dimensional structure of beta-glucosyltransferase and of several glycogen phosphorylases, the nucleotide binding domain of pig alpha3-galactosyltransferase was built by knowledge-based methods. Both the UDP-galactose ligand and a divalent cation were included in the model during the refinement procedure. The final three-dimensional model is in agreement with our present knowledge of the biochemistry and mechanism of alpha3-galactosyltransferases.

Amino Acid Sequence↗
PubMed1999

Structure and conformation of complex carbohydrates of glycoproteins, glycolipids, and bacterial polysaccharides.

For nuclear magnetic resonance determinations of the conformation of oligosaccharides in solution, simple molecular mechanics calculations and nuclear Overhauser enhancement measurements are adequate for small oligosaccharides that adopt single, relatively rigid conformations. Polysaccharides and larger or more flexible oligosaccharides generally require additional types of data, such as scalar and dipolar coupling constants, which are most conveniently measured in 13C-enriched samples. Nuclear magnetic resonance relaxation data provide information on the dynamics of oligosaccharides, which involves several different types of internal motion. Oligosaccharides complexed with lectins and antibodies have been successfully studied both by X-ray crystallography and by nuclear magnetic resonance spectroscopy. The complexes have been shown to be stabilized by a combination of polar hydrogen bonding interactions and van der Waals attractions. Although theoretical calculations of the conformation and stability of free oligosaccharides and of complexes with proteins can be carried out by molecular mechanics methods, the role of solvent water for these highly polar molecules continues to present computational problems.

Animals↗
PubMed1998

Conserved structural features in eukaryotic and prokaryotic fucosyltransferases.

Fucosyltransferases are the enzymes transferring fucose from GDP-Fuc to Gal in an alpha1,2-linkage and to GlcNAc in alpha1,3-, alpha1,4-, or alpha1,6-linkages. Since all fucosyltransferases utilize the same nucleotide sugar, their specificity will probably reside in the recognition of the acceptor and in the type of linkage formed. A search of nucleotide and protein databases yielded more than 30 sequences of fucosyltransferases originating from mammals, chicken, nematode, and bacteria. On the basis of protein sequence similarities, these enzymes can be classified into four distinct families: (1) the alpha-2-fucosyltransferases, (2) the alpha-3-fucosyltransferases, (3) the mammalian alpha-6-fucosyltransferases, and (4) the bacterial alpha-6-fucosyltransferases. Nevertheless, using the sensitive hydrophobic cluster analysis (HCA) method, conserved structural features as well as a consensus peptide motif have been clearly identified in the catalytic domains of all alpha-2 and alpha-6-fucosyltranferases, from prokaryotic and eukaryotic origin, that allowed the grouping of these enzymes into one superfamily. In addition, a few amino acids were found strictly conserved in this family, and two of these residues have been reported to be essential for enzyme activity for a human alpha-2-fucosyltransferase. The alpha-3-fucosyltransferases constitute a distinct family as they lack the consensus peptide, but some regions display similarities with the alpha-2 and alpha-6-fucosyltranferases. All these observations strongly suggest that the fucosyltransferases share some common structural and catalytic features.

Amino Acid Sequence↗
PubMed1998

Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases.

Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages. During the last few years, several (c)DNA sequences coding for galactosyltransferases became available. We have retrieved these sequences and conducted sequence similarity studies. On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups. Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families. The lengths and locations of the conserved regions as well as the invariant residues are described for each family. In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one.

Amino Acid Sequence↗
PubMed1997

Transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments reflect the size of the bound segment of the Forssman pentasaccharide in the binding site of Dolichos biflorus lectin.

A complex between the Forssman pentasaccharide alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D- Gal-(1-->4)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNac compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Binding Sites↗
PubMed1997

Oligosaccharide structures: theory versus experiment.

Recently, the interdependency of theoretical and experimental approaches in the structure determination of oligosaccharides has been confirmed. More accurate simulations are possible because of the advances in software and computers. Meanwhile, improvements in NMR techniques permit the measurement of numerous structural and dynamical parameters, either for the free state or for carbohydrate ligands bound to receptors. Several crystal structures of isolated or protein-complexed oligosaccharides give new clues for modeling the intermolecular forces that drive the interactions.

ABO Blood-Group System↗
PubMed1997

Combined NMR and molecular modeling study of an iduronic acid-containing trisaccharide related to antithrombotic heparin fragments.

An iduronic acid-containing trisaccharide, methyl-O-(4-O-methyl-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1-->4)-O- (2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-->4)-O-2,6-di-O-sulfo-alpha-D-glucopyranoside, related to antithrombotic heparin fragments has been subjected to a combined NMR and molecular modeling investigation. The conformational behavior of the two constituting disaccharide segments was investigated using a systematic grid search approach with the MM3 force field along with the proper parameters for the sulfate ester group. The exploration of the potential energy surfaces of the trisaccharide was performed through the use of the CICADA methods interfaced with the MM3 force field. In all cases, the 2-O-sulfo-alpha-L-iduronate moiety was given the three favored ring conformations (1)C4, (4)C1, and (2)S0. Conformations were clustered into families, four of which are likely to exhibit significant occupancy in solution. The different low-energy conformational families display different orientations at the glycosidic linkages and/or different ring shapes for the iduronate ring. The (2)S0 conformation is the major one for the 2-O-sulfo-alpha-L-iduronate but is still in equilibrium with the (1)C4 ring shape. The occurrence of such a conformational equilibrium in solution was probed via high-resolution NMR spectroscopy through measurements of coupling constants and NOE build-up. These results are in keeping with the observation that 2-O-sulfated pentasaccharides display a similar affinity for antithrombin III as their 2-N-sulfated counterparts.

Angiotensin III↗
PubMed1997

Conformational analysis of biantennary glycans and molecular modeling of their complexes with lentil lectin.

Some mannose-binding legume lectins show higher affinity for fucosylated glycans than for glycans without fucose. These lectins possess a secondary binding site. Owing to the possibility of additional fucose binding, oligosaccharides adopt different conformations depending on whether they contain fucose or not. To study these conformational differences, complexes of fucosylated and unfucosylated glycans with Lens culinaris lectin have been modeled. Starting points were X-ray structures of lentil lectin and complexes of the homologous Lathyrus ochrus lectin. The SYBYL molecular modeling package with the TRIPOS force field was used. Two different models were built, displaying in both a network of hydrogen bonds between the saccharide and the binding site. Furthermore, to compare the free and bound ligand, conformational analysis in the free state has been performed. A complete analysis of all possible disaccharide fragments has been performed using the MM3 force field. A CICADA analysis employing the same force field was carried out to study the complete oligosaccharide. Low-energy conformers found by CICADA were clustered in conformational families and analyzed in terms of flexibility and rotational barriers. All values of glycosidic torsion angles are in the range as calculated by MM3 for the disaccharides.

Binding Sites↗
PubMed1997

Fold recognition and molecular modeling of a lectin-like domain in UDP-GalNac:polypeptide N-acetylgalactosaminyltransferases.

By use of threading methods, the C-terminal region of uridine diphospho-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-transferases) was predicted to have the same fold as the lectin-domain of the plant cytotoxins ricin and abrin-a, for which crystal structure are available. The sequence identities are very low. Nevertheless, the amino acids involved in the hydrophobic core essential for the structure stability and the cysteine residues are conserved. In addition, the amino-acids involved in carbohydrate binding are conserved in ppGalNAc-transferases. The extra C-terminal domain of these enzymes is therefore a putative glycan-binding domain. A model of the lectin-like domain of human ppGalNAc-transferase T1 was built using knowledge based methods. Geometry optimization of the complex with galactose allowed prediction that this domain could bind this monosaccharide. However, the interaction seems to be rather weak, and at the moment there is no evidence that ppGalNAc-transferases displays a lectin activity in vivo.

Abrin↗
PubMed1996

Conformational analysis of blood group A trisaccharide in solution and in the binding site of Dolichos biflorus lectin using transient and transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments.

The present study is concerned with the elucidation of the conformation of the blood group A trisaccharide (alpha-D-GalNAc(1-3)[alpha-L-Fuc(1-->2)] beta-D-Gal-O-R) in the combining site of Dolichos biflorus seed lectin by use of 400-MHz and 600-MHz NMR spectroscopy. D. biflorus lectin displays a unique specificity for GalNAc residues. It occurs in solution as a tetrameric assembly having a molecular mass of 110 kDa, with two carbohydrate-binding sites per molecule. First, NOE build-up curves were obtained for the free blood group A trisaccharide from one-dimensional transient NOE experiments. Simulated NOE build-up curves were constructed from an ensemble of low-energy conformers derived from previous investigations. The comparison of theoretical and experimental data indicates that an equilibrium between two families of low-energy conformers most likely reflects the solution behavior of the trisaccharide in solution. Two-dimensional transferred NOE and rotating-frame enhancements (ROE) were subsequently measured for the trisaccharide complexed with the D. biflorus seed lectin. In addition to the NOEs observed for the free trisaccharide, the transferred NOESY spectrum showed several new NOEs that were identified as spin diffusion using a rotating-frame NOESY (ROESY) experiment. Experimental interglycosidic transferred nuclear Overhauser effect (TRNOE) build-up curves were compared to theoretical curves calculated for both low-energy conformers located in the D. biflorus lectin-binding site. Calculations of theoretical TRNOE were performed using a combination of the full relaxation matrix and the protein-ligand exchange matrix. Comparison between experimental and simulated TRNOE volumes leads to the conclusion that one conformation of blood group A trisaccharide is selected upon binding by D. biflorus lectin.

ABO Blood-Group System↗
PubMed1996

Predicting helical structures of the exopolysaccharide produced by Lactobacillus sake 0- 1.

The viscous exopolysaccharide (EPS) produced by Lactobacillus sake 0- 1 is a high molecular mass polymer (Mm 6 x 10(6) Da) consisting of pentasaccharide repeating units with a composition of D-glucose, L-rhamnose, and sn-glycerol 3-phosphate in molar ratios of 3:2:1. One of the rhamnose residues in the repeating unit is partially 2-O-acetylated. The O-deacetylated, deglycerophosphorylated EPS has been investigated by molecular mechanics calculations. A complete conformational analysis of each of the constituent disaccharide fragments has been performed using the flexible residue approach with the MM3(92) force field. Furthermore, using the same force field, CICADA analyses were accomplished on hexa- and octasaccharide substructure of the polysaccharide. Based on these analyses, insight was obtained into nine conformational minima for the polysaccharide. The low energy conformations found by CICADA were extrapolated to regular polysaccharide structures using a polysaccharide builder program. The generated helices exhibit either 2-fold or 3- or 4-fold right-handed chiralities, and in each case the helices are highly extended.

Carbohydrate Conformation↗
PubMed1996

Solution conformations of pectin polysaccharides: determination of chain characteristics by small angle neutron scattering, viscometry, and molecular modeling.

The solution behavior of pectin polysaccharides has been investigated by small angle neutron scattering (SANS), viscosimetric, and molecular modeling studies. The samples used in the experimental study were obtained from apple and citrus and had degrees of methylation ranging from 28 to 73%, with a rhamnose content lying between 0.6 and 2.2%. Persistence lengths, derived from intrinsic viscosity measurements, ranged from 59 to 126 A, whereas those derived by SANS were between 45 and 75 A. These values correspond to 10-17 monomer units. The modeling simulations were performed for both homogalacturonan itself and homogalacturonan carrying various degrees of rhamnose inserts (rhamnogalacturonan). This required the evaluation of the accessible conformational space for the eight disaccharides that represent the constituent repeating segments of the homogalacturonan and rhamnogalacturonan polysaccharides. For each dimer, complete conformational analysis was accomplished using the flexible residue method of the MM3 molecular mechanics procedure and the results used to access the configurational statistics of representative pectic polysaccharide chains. For homogalacturonan, an extended chain conformation having a persistence length of 135 A (corresponding to 30 monomers) was predicted. The inclusion of varying amounts of rhamnose units (5-25%) in the model in strict alternating sequence with galacturonate residues (equivalent to the rhamnogalacturonan "hairy region" chains) only slightly reduced the calculated persistence length. The extended overall chain conformation remained relatively unchanged as a consequence of the self-cancellation of the kinking effects of successive paired rhamnose units.

Carbohydrate Conformation↗
PubMed1996

Recognition of the blood group H type 2 trisaccharide epitope by 28 monoclonal antibodies and three lectins.

The patterns of cross-reaction of 30 monoclonal antibodies and three lectins were determined by ELISA with 21 ABH, Ii or Lewis related synthetic oligosaccharides coupled to bovine serum albumin. At least seven main groups of cross-reactive patterns were identified among the antibodies, plus several isolated antibodies which had intermediate patterns between two of the main antibody groups. The three lectins had different cross-reaction patterns, Galactia tenuiflora was different from all the antibodies, Ulex europaeus lectin 1 and Lotus tetragonolobus were similar, but not identical to groups III and V of antibodies respectively. The anti-H antibodies cross-reacting with A type 2 gave similar agglutination scores with all the normal ABO erythrocytes, while the anti-H antibodies not cross-reacting with A type 2 reacted with different scores: O > A2 > A2B > B > A1 > A1B > O(h), suggesting that these antibodies react better with the free H epitopes and do not recognize the H in A or B epitopes. Based on the ELISA and agglutination results and the lowest energy conformations of each oligosaccharide obtained by computer modelling, the most probable oligosaccharide surface areas recognized by each antibody main group are illustrated.

ABO Blood-Group System↗
PubMed1996

Knowledge-based modeling of a legume lectin and docking of the carbohydrate ligand: the Ulex europaeus lectin I and its interaction with fucose.

Ulex europaeus isolectin I is specific for fucose-containing oligosaccharide such as H type 2 trisaccharide alpha-L-Fuc (1-->2) beta-D-Gal (1-->4) beta-D-GlcNAc. Several legume lectins have been crystallized and modeled, but no structural data are available concerning such fucose-binding lectin. The three-dimensional structure of Ulex europaeus isolectin I has been constructed using seven legume lectins for which high-resolution crystal structures were available. Some conserved water molecules, as well as the structural cations, were taken into account for building the model. In the predicted binding site, the most probable locations of the secondary hydroxyl groups were determined using the GRID method. Several possible orientations could be determined for a fucose residue. All of the four possible conformations compatible with energy calculations display several hydrogen bonds with Asp-87 and Ser-132 and a stacking interaction with Tyr-220 and Phe-136. In two orientations, the O-3 and O-4 hydroxyl groups of fucose are the most buried ones, whereas two other, the O-2 and O-3 hydroxyl groups are at the bottom of the site. Possible docking modes are also studied by analysis of the hydrophobic and hydrophilic surfaces for both the ligand and the protein. The SCORE method allows for a quantitative evaluation of the complementarity of these surfaces, on the basis of molecular lipophilicity calculations. The predictions presented here are compared with known biochemical data.

Artificial Intelligence↗
PubMed1996

How do antibodies and lectins recognize histo-blood group antigens? A 3D-QSAR study by comparative molecular field analysis (CoMFA).

The cross-reaction patterns of nine antibodies and three lectins against 12 H type 2 related oligosaccharides have been analysed by means of 3D-QSAR study. Three-dimensional descriptors of the molecular properties have been used in comparative molecular field analysis (CoMFA). Three different alignments were considered for the oligosaccharides. One, based on the superimposition of the oligosaccharide core, could be correlated to most of the antibody activities. A second alignment, based on a superimposition of the fucose residue, had to be taken into account for explaining the binding properties of Ulex europaeus isolectin I. Analysis of the QSAR data gives indications on the carbohydrate epitopes essential for antibody recognition and yields some insights about the nature of the molecular recognition. This study complements previous biochemical estimates of the H type 2 related oligosaccharide binding areas (Mollicone, R.; Cailleau, A.; Imberty, A.; Gane, P.; Pérez, S.; Oriol, R. Glycoconj. J. 1996, 13, 263-271).

Antibodies↗
PubMed1996

Crystal and molecular structure of a histo-blood group antigen involved in cell adhesion: the Lewis x trisaccharide.

This work describes the first crystal structure ever reported of a histo-blood group carbohydrate antigen: Le(x). This study provides a detailed description of the conformation of two crystallographic independent molecules in a highly hydrated environment along with their hydrogen bonding properties and packing features. Some interactions observed between adjacent trisaccharides can provide the basis for involvement of Le(x)-Le(x) interactions in cell-cell adhesion.

Carbohydrate Conformation↗