Magnus Lundborg

EUROCarbDB: An open-access platform for glycoinformatics.

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.

Complete 1H and 13C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses (1)H and (13)C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the (1)H and (13)C, as well as (31)P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 (1)H and (13)C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D (1)H,(13)C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t(1) incremented (1)H,(13)C-HSQC experiment and a 1D (1)H,(1)H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3Hz apart. The (1)H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental (1)H and (13)C NMR chemical shifts.

Glycosyltransferase functions of E. coli O-antigens

ECODAB (the E. coli O-antigen database) has been expanded to include information about glycosyltransferases (GTs) involved in the assembly of the O-antigen polysaccharide. Similarity searches have been performed to be able to determine GT functions that have not been reported prior to this work. In addition to suggesting the function of 179 GTs, the approach leads to the prediction of part of the O-antigen structures of a number of serogroups. The procedure suggests a novel way of combining genetic information with experimental techniques in structural analysis of oligo- and polysaccharides.

Automatic structure determination of regular polysaccharides based solely on NMR spectroscopy.

The structural analysis of polysaccharides requires that the sugar components and their absolute configurations are determined. We here show that this can be performed based on NMR spectroscopy by utilizing butanolysis with (+)- and (−)-2-butanol that gives the corresponding 2-butyl glycosides with characteristic 1H and 13C NMR chemical shifts. The subsequent computer-assisted structural determination by CASPER can then be based solely on NMR data in a fully automatic way as shown and implemented herein. The method is additionally advantageous in that reference data only have to be prepared once and from a user’s point of view only the unknown sample has to be derivatized for use in CASPER.

Structural studies of the O‐antigen polysaccharide from Escherichia coli TD2158 having O18 serogroup specificity and aspects of its interaction with the tailspike endoglycosidase of the infecting bacteriophage HK620

We have analyzed the O-antigen polysaccharide of the previously uncharacterized Escherichia coli strain TD2158 which is a host of bacteriophage HK620. This bacteriophage recognizes and cleaves the polysaccharide with its tailspike protein (TSP). The polysaccharide preparation as well as oligosaccharides obtained from HK620TSP endoglycosidase digests were analyzed with NMR spectroscopy. Additionally, sugar analysis was performed on the O-antigen polysaccharide and MALDI-TOF MS was used in oligosaccharide analysis. The present study revealed a heterogeneous polysaccharide with a hexasaccharide repeating unit of the following structure: α-D-Glcp-(1→6|) →2)-α-L-Rhap-91→6)-α-D-Glcp-(1→4)-α-D-Ga|lp-(1→3)-α-D-GlcpNAc-(1→ β-D-Glcp/β-D-GlcpNAc-(1→3) A repeating unit with a D-GlcNAc substitution of D-Gal has been described earlier as characteristic for serogroup O18A1. Accordingly, we termed repeating units with D-Glc substitution at D-Gal as O18A2. NMR analyses of the polysaccharide confirmed that O18A1- and O18A2-type repeats were present in a 1:1 ratio. However, HK620TSP preferentially bound the D-GlcNAc-substituted O18A1-type repeating units in its high affinity binding pocket with a dissociation constant of 140 μM and disfavored the O18A2-type having a β-D-Glcp-(1→3)-linked group. As a result, in hexasaccharide preparations, O18A1 and O18A2 repeats were present in a 9:1 ratio stressing the clear preference of O18A1-type repeats to be cleaved by HK620TSP.

CarbBuilder: An Adjustable Tool for Building 3D Molecular Structures of Carbohydrates for Molecular Simulation

CarbBuilder is a software tool for building 3D structures of carbohydrates, which are the most structurally varied of all molecular classes. CarbBuilder was designed with the dual aims of portability and adaptability, using an iterative software development approach. CarbBuilder employs a simple algorithm, using heuristics based upon experimental data to convert a primary structure description of a carbohydrate molecule into a three-dimensional structure file. This straightforward approach means that CarbBuilder can be easily adapted: users can add additional monosaccharide building blocks or alter the conformational defaults to suit specific requirements. The output carbohydrate structure can be used for subsequent molecular simulation investigations. CarbBuilder is freely available and portable: it is a text-based stand-alone program that can run on Windows, Linux and MacOS X systems without installation.

Small molecules containing hetero-bicyclic ring systems compete with UDP-Glc for binding to WaaG glycosyltransferase

The α-1,3-glucosyltransferase WaaG is involved in the synthesis of the core region of lipopolysaccharides in E. coli. A fragment-based screening for inhibitors of the WaaG glycosyltrasferase donor site has been performed using NMR spectroscopy. Docking simulations were performed for three of the compounds of the fragment library that had shown binding activity towards WaaG and yielded 3D models for the respective complexes. The three ligands share a hetero-bicyclic ring system as a common structural motif and they compete with UDP-Glc for binding. Interestingly, one of the compounds promoted binding of uridine to WaaG, as seen from STD NMR titrations, suggesting a different binding mode for this ligand. We propose these compounds as scaffolds for the design of selective high-affinity inhibitors of WaaG. Binding of natural substrates, enzymatic activity and donor substrate selectivity were also investigated by NMR spectroscopy. Molecular dynamics simulations of WaaG were carried out with and without bound UDP and revealed structural changes compared to the crystal structure and also variations in flexibility for some amino acid residues between the two WaaG systems studied.

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and (1)H, (13)C, and (31)P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by (1)H, (13)C-heteronuclear multiple-bond correlation, and (1)H, (1)H-NOESY experiments. D-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the alpha-D-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure: -->4)-alpha-D-Glcp6-(P-2-D-GroA)-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->3)-beta-D-GlcpNAc-(1--> Cross-peaks of low intensity from an alpha-D-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-D-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.

NMR Chemical Shift Prediction of Glycans: Application of the Computer Program CASPER in Structural Analysis

Carbohydrate molecules have highly complex structures and the constituent monosaccharides and substituents are linked to each other in a large number of ways. NMR spectroscopy can be used to unravel these structures, but the process may be tedious and time-consuming. The computerized approach based on the CASPER program can facilitate rapid structural determination of glycans with little user intervention, which results in the most probable primary structure of the investigated carbohydrate material. Additionally, (1)H and (13)C NMR chemical shifts of a user-defined structure can be predicted, and this tool may thus be employed in many aspects where NMR spectroscopy plays an important part of a study.

An in silico virtual screening study for the design of norovirus inhibitors: fragment-based molecular docking and binding free energy calculations.

Gastrointestinal infections caused by noroviruses may be prevented by the inhibition of their binding to histo-blood group carbohydrate antigens. A fragment-based virtual screening approach was used, employing docking followed by molecular dynamics simulations in order to enable binding free energy calculations using the linear interaction energy method. The resulting structures, composed of high-affinity fragments, can be a good starting point for lead optimizations and four molecules that pass both REOS and SYLVIA filters, which can remove known toxic features and assess the synthetic accessibility, respectively, are proposed as inhibitors.