Carolina Fontana

Stockholm University, Stockholm, Stockholm, Sweden

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Publications (10)71.26 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: 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. In this approach information about the action of glycosyltransferases (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 E. coli O-antigen database (ECODAB) and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, that alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen polysaccharide (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 were efficiently performed by comparison of the (1)H,(13)C-HSQC NMR spectra of the O-deacylated lipopolysaccharide (LPS-OH) and the lipid-free polysaccharide 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.
    Glycobiology 02/2014; · 3.54 Impact Factor
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    ABSTRACT: 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.
    Nature Chemistry 01/2014; 6(1):65-74. · 21.76 Impact Factor
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    ABSTRACT: 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.
    Biomacromolecules 05/2013; · 5.37 Impact Factor
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    ABSTRACT: Extensive analysis by NMR spectroscopy of the delipidated lipopolysaccharide of Shigella flexneri serotype 6 strain MDC 2924-71 confirmed the most recently reported structure of the O-antigen repeating unit as {→4)-β-D-GalpA-(1→3)-β-D-GalpNAc-(1→2)-α-L-Rhap3Ac/4Ac-(1→2)-α-L-Rhap-(1→}, and revealed the non-stoichiometric acetylation at O-3C/4C. Input from the CASPER program helped to ascertain the fine distribution of the three possible patterns of O-acetylation. The non-O-acetylated repeating unit (ABCD) corresponded to about 2/3 of the population, while 1/4 was acetylated at O-3C (3AcCDAB), and 1/10 at O-4C (4AcCDAB). Di- to tetrasaccharides with a GalpA residue (A) at their reducing end were synthesized as their propyl glycosides following a multistep linear strategy relying on late-stage acetylation at O-3C. Thus, the 3C-O-acetylated and non-O-acetylated targets were synthesized from common protected intermediates. Rhamnosylation was most efficiently achieved by using imidate donors, including at O-4 of a benzyl galacturonate acceptor. In contrast, a thiophenyl 2-deoxy-2-trichloroacetamido-D-galactopyranoside precursor was preferred for chain elongation involving residue B. Final Pd/C-mediated deprotection ensured O-acetyl stability. All of the target molecules represent parts of the O-antigen of S. flexneri 6, a prevalent serotype. Non-O-acetylated oligosaccharides are also fragments of the Escherichia coli O147 O-antigen.
    European Journal of Organic Chemistry 05/2013; 2013(19):4085-4106. · 3.34 Impact Factor
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    ABSTRACT: The structure of the O-antigen polysaccharide (PS) of Escherichia coli O115 has been investigated using a combination of component analysis together with 1D and 2D 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 polysaccharide; this information defines the biological repeating unit of the O-antigen by having a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Analysis of the NMR spectra of the native polysaccharide 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 et al. 2010). 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 based on similarities with glycosyltransferases from Shigella dysenteriae type 5 and 12, as well as Escherichia coli O58 and O152.
    Glycobiology 11/2012; · 3.54 Impact Factor
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    ABSTRACT: 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 analyzed 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 analyze the role of defined carbohydrate structures in different biological processes.
    Molecular Microbiology 11/2012; · 4.96 Impact Factor
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    ABSTRACT: O-Mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galβ1-4GlcNAcβ1-2Manα, which is reported to be the most abundant O-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a “one-pot” enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described.
    Journal of the American Chemical Society 04/2012; · 10.68 Impact Factor
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    ABSTRACT: The structure of the repeating unit of the O-antigenic polysaccharide (PS) from Escherichia coli O174 has been determined. Component analysis together with (1)H and (13)C 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. The PS is composed of tetrasaccharide repeating units with the following structure: [formula see text] Cross-peaks of low intensity were present in the NMR spectra consistent with a β-D-GlcpNAc-(1→2)-β-D-GlcpA(1→ structural element at the terminal part of the polysaccharide, which on average is composed of ∼15 repeating units. Consequently the biological repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.
    Carbohydrate research 03/2012; 354:102-5. · 2.03 Impact Factor
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    ABSTRACT: O-mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galβ1-4GlcNAcβ1-2Manα, which is reported to be the most abundant O-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a "one-pot" enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described.
    Journal of the American Chemical Society 03/2012; 134(10):4521-4. · 10.68 Impact Factor
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    Magnus Lundborg, Carolina Fontana, Göran Widmalm
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    ABSTRACT: 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 (1)H and (13)C 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.
    Biomacromolecules 09/2011; 12(11):3851-5. · 5.37 Impact Factor