Carolina Fontana

Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides. Identification of glycosylation patterns is complicated by the lack of sensitive analytical techniques that can distinguish between epimeric carbohydrates. It has now been shown that ion-mobility tandem mass spectrometry of ions derived from glycopeptides and oligosaccharides enables glycan stereochemistry to be determined, highlighting the potential of this technique for sequencing complex carbohydrates on cell surfaces.

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 Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence

The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManBcore proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β-d-Glcp-(1→4)-α-Kdop-(2→4)[β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5)]-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P), in addition to components lacking one of the terminal β-d-GlcpN and/or the β-d-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of manBcore gives rise to a deep-rough pentasaccharide core (β-d-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β-d-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcore proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the β-d-GlcpN-(1→6)-β-d-GlcpN-(1→4)[β-d-GlcpN-(1→6)]-β-d-GlcpN-(1→3)-α-d-Manp-(1→5) structure in virulence.

Structural studies of the exopolysaccharide from Lactobacillus plantarum C88 using NMR spectroscopy and the program CASPER

Some lactic acid bacteria, such as those of the Lactobacillus genus, have the ability to produce exopolysaccharides (EPSs) that confer favorable physicochemical properties to food and/or beneficial physiological effects on human health. In particular, the EPS of Lactobacillus plantarum C88 has recently demonstrated in vitro antioxidant activity and, herein, its structure has been investigated using NMR spectroscopy and the computer program CASPER (Computer Assisted Spectrum Evaluation of Regular polysaccharides). The pentasaccharide repeating unit of the O-deacetylated EPS consists of a trisaccharide backbone, →4)-α-D-Galp-(1→2)-α-D-Glcp-(1→3)-β-D-Glcp-(1→, with terminal D-Glc and D-Gal residues (1.0 and 0.8 equiv per repeating unit, respectively) extending from O3 and O6, respectively, of the →4)-α-D-Galp-(1→ residue. In the native EPS an O-acetyl group is present, 0.85 equiv per repeating unit, at O2 of the α-linked galactose residue; thus the repeating unit of the EPS has the following structure: →4)[β-D-Glcp-(1→3)][β-D-Galp-(1→6)]α-D-Galp2Ac-(1→2)-α-D-Glcp-(1→3)-β-D-Glcp-(1→. These structural features, and the chain length (∼10(3) repeating units on average, determined in a previous study), are expected to play an important role in defining the physicochemical properties of the polymer.

Glycoengineering of host mimicking type-2 LacNAc polymers and Lewis X antigens on bacterial cell surfaces.

Bacterial carbohydrate structures play a central role in mediating a variety of host–pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram‐negative bacteria, is composed of a lipid A‐core and the O‐antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Galβ1–4(Fucα1–3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram‐negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O‐antigen. A ubiquitous building block in mammalian N‐linked protein glycans is Galβ1‐4GlcNAc, referred to as a type‐2 N‐acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A‐cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly‐LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI‐TOF and NMR analysis. Glycoengineered LOS induced pro‐inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.

Structural Elucidation of the O-Antigen Polysaccharide from Escherichia coli O181

Shiga-toxin-producing Escherichia coli (STEC) is an important pathogen associated to food-borne infection in humans; strains of E. coli O181, isolated from human cases of diarrhea, have been classified as belonging to this pathotype. Herein, the structure of the O-antigen polysaccharide (PS) from E. coli O181 has been investigated. The sugar analysis showed quinovosamine (QuiN), glucosamine (GlcN), galactosamine (GalN), and glucose (Glc) as major components. Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue. The 1H and 13C NMR chemical shift assignments of the OS were carried out using 1 D and 2 D NMR experiments, and the OS was sequenced using a combination of tandem mass spectrometry (MS/MS) data and NMR 13C NMR glycosylation shifts. The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit. Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.

Conformational Preferences of the O‑Antigen Polysaccharides of Escherichia coli O5ac and O5ab Using NMR Spectroscopy and Molecular Modeling

Escherichia coli serogroup O5 comprises two different subgroups (O5ab and O5ac), which are indiscernible from the point of view of standard immunological serotyping. The structural similarities between the O-antigen polysaccharides (PSs) of these two strains are remarkable, with the only difference being the glycosidic linkage connecting the biological tetrasaccharide repeating units. In the present study, a combination of NMR spectroscopy and molecular modeling methods were used to elucidate the conformational preferences of these two PSs. The NMR study was based on the analysis of intra- and inter-residue proton-proton distances using NOE build-up curves. Molecular models of the repeating units and their extension to polysaccharides were obtained, taking into account the conformational flexibility as assessed by the force field applied and a genetic algorithm. The agreements between experimentally measured and calculated distances could only be obtained by considering an averaging of several low energy conformations observed in the molecular models.

Structural studies and biosynthetic aspects of the O-antigen polysaccharide from Escherichia coli O42.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O42 has been investigated by NMR spectroscopy as the main method, which was complemented with sugar analysis, mass spectrometry, and analysis of biosynthetic information. The O-specific chain of the O-deacylated lipopolysaccharide (LPS-OH) consists of branched tetrasaccharide-glycerol repeating units joined by phosphodiester linkages. The lipid-free polysaccharide contains 0.8equiv of O-acetyl groups per repeating unit and has the following teichoic acid-like structure: Based on biosynthetic aspects, this should also be the biological repeating unit. This O-antigen structure is remarkably similar to that of E. coli O28ac, differing only in the presence or absence, respectively, of a glucose residue at the branching point. The structural similarity explains the serological cross-reactivity observed between strains of these two serogroups, and also their almost identical O-antigen gene cluster sequences.

Structural studies of the O-antigen polysaccharide from Escherichia coli O115 and biosynthetic aspects thereof

The structure of the O-antigen polysaccharide (PS) of Escherichia coli O115 has been investigated using a combination of component analysis and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy experiments. The repeating unit of the O-antigen was elucidated using the O-deacetylated PS and has the following branched pentasaccharide structure: →3)[β-L-Rhap-(1 → 4)]-β-D-GlcpNAc-(1 → 4)-α-D-GalpA-(1 → 3)-α-D-Manp-(1 → 3)-β-D-GlcpNAc-(1→. Cross-peaks of low intensity, corresponding to a β-L-Rhap-(1 → 4)-β-D-GlcpNAc-(1→ structural element, were present in the NMR spectra and attributed to the terminal part of the PS; this information defines the biological repeating unit of the O-antigen by having a 3-substituted N-acetyl-D-glucosamine (GlcNAc) residue at its reducing end. Analysis of the NMR spectra of the native PS revealed O-acetyl groups distributed over different positions of the l-Rhap residue (∼0.70 per repeating unit) as well as at O-2 and O-3 of the D-GalpA residue (∼0.03 and ∼0.25 per repeating unit, respectively), which is in agreement with the presence of two acetyltransferases previously identified in the O-antigen gene cluster (Wang Q, Ruan X, Wei D, Hu Z, Wu L, Yu T, Feng L, Wang L. 2010. Mol Cell Probes. 24:286-290.). In addition, the four glycosyltransferases initially identified in the O-antigen gene cluster of E. coli O115 were analyzed using BLAST, and the function of two of them predicted on the basis of similarities with glycosyltransferases from Shigella dysenteriae type 5 and 12, as well as E. coli O58 and O152.

Rapid structural elucidation of polysaccharides employing predicted functions of glycosyltransferases and NMR data: Application to the O-antigen of Escherichia coli O59

A computerized method that uses predicted functions of glycosyltransferases (GTs) in conjunction with unassigned NMR data has been developed for the structural elucidation of bacterial polysaccharides (PSs). In this approach, information about the action of GTs (consisting of possible sugar residues used as donors and/or acceptors, as well as the anomeric configuration and/or substitution position in the respective glycosidic linkages) is extracted from the Escherichia coli O-antigen database and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, which alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen PS of E. coli O59. The repeating unit of the O-specific chain was determined using the O-deacylated PS and has a branched structure, namely, →6)[α-d-GalpA3Ac/4Ac-(1 → 3)]-α-d-Manp-(1 → 3)-α-d-Manp-(1 → 3)-β-d-Manp-(1 → 3)-α-d-GlcpNAc-(1→. The identification of the O-acetylation positions was efficiently performed by comparison of the (1)H,(13)C HSQC NMR spectra of the O-deacylated lipopolysaccharide and the lipid-free PS in conjunction with chemical shift predictions made by the CASPER program. The side-chain d-GalpA residue carries one equivalent of O-acetyl groups at the O-3 and O-4 positions distributed in the LPS in a 3:7 ratio, respectively. The presence of O-acetyl groups in the repeating unit of the E. coli O59 PS is consistent with the previously proposed acetyltransferase WclD in the O-antigen gene cluster.