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Structural and Conformational Studies of Oligo- and Polysaccharides
Stockholm University, Faculty of Science, Department of Organic Chemistry. (Göran Widmalm)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The focus of this thesis is to examine the structural properties of polysaccharides produced by bacteria, as well as the dynamic and conformational behavior of a synthetically derived oligosaccharide.

The primary structures of the O-polysaccharide repeating units of four different Escherichia coli (E. coli) strains, namely O175, O177, O103 and TD2158, as well as the first report of a capsular polysaccharide produced by lactic acid bacteria Leuconostoc mesenteroides ssp. cremoris PIA2 are reported in paper I–V. Structural analyses have been performed using a combination of nuclear magnetic resonance spectroscopy and chemical component analysis.

The elucidated structures in paper I–III, as well as paper V, are composed of linear repeating units of varying composition and length. In paper IV, the structure of the O-polysaccharide repeating unit of E. coli TD2158 is determined to be a branched hexasaccharide structure with a heterogeneous substitution pattern, with either a β-GlcpNAc or β-Glcp residue branching to the backbone chain. Incubation with bacteriophage HK620 tailspike protein shows that the polysaccharide is selectively cleaved at the α-GlcpNAc-(1→2)-α-Rhap-linkage of the backbone chain, yielding a 9:1 ratio of β-GlcpNAc/β-Glcp containing hexasaccharides after digestion.

In paper VI the conformational properties of a trisaccharide, which constitutes an internal epitope of the LeaLex hexasaccharide over-expressed on the surface of squamous lung cancer cells, have been analyzed using NMR spectroscopy and molecular dynamics simulations. The β-(1→3)-linkage of the trisaccharide was shown to be highly flexible.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University , 2012. , 54 p.
Keyword [en]
NMR spectroscopy, structural analysis, conformational analysis, carbohydrates, polysaccharides
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-75050ISBN: 978-91-7447-457-2 (print)OAI: oai:DiVA.org:su-75050DiVA: diva2:513867
Public defence
2012-05-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 6: Submitted.

Available from: 2013-04-09 Created: 2012-04-03 Last updated: 2013-11-18Bibliographically approved
List of papers
1. Structural elucidation of the O-antigenic polysaccharide from Escherichia coli O175
Open this publication in new window or tab >>Structural elucidation of the O-antigenic polysaccharide from Escherichia coli O175
2011 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 346, no 3, 449-453 p.Article in journal (Refereed) Published
Abstract [en]

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O175 has been elucidated. Component analysis together with 1H and 13C NMR spectroscopy experiments were used to determine the structure. Inter-residue correlations were determined by 1H,1H-NOESY, and 1H,13C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure:

→2)-α-d-Glcp-(1→4)-α-d-GlcpA-(1→3)-α-d-Manp-(1→2)-α-d-Manp-(1→3)-β-d-GalpNAc-(1→

Cross-peaks of low intensity from an α-linked glucopyranosyl residue were present in the 1H,1H-TOCSY NMR spectra. The α-d-Glcp residue is suggested to originate from the terminal part of the polysaccharide and consequently the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O175 O-antigen is similar to those from E. coli O22 and O83, both of which carry an α-d-Glcp-(1→4)-d-GlcpA structural element, thereby explaining the reported cross-reactivities between the strains.

Keyword
Escherichia coli, lipopolysaccharide, NMR, biological repeating unit
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-59722 (URN)10.1016/j.carres.2010.12.005 (DOI)000293037400010 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2011-07-06 Created: 2011-07-06 Last updated: 2017-12-11Bibliographically approved
2. Structural studies of the O-antigenic polysaccharide from Escherichia coli O177
Open this publication in new window or tab >>Structural studies of the O-antigenic polysaccharide from Escherichia coli O177
2011 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 346, no 14, 2300-2303 p.Article in journal (Refereed) Published
Abstract [en]

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O177 has been determined. Component analysis together with 1H and 13C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by 1H,13C-heteronuclear multiple-bond correlation and 1H,1H-NOESY experiments. PS is composed of tetrasaccharide repeating units with the following structure:

→2)-α-l-Rhap-(1→3)-α-l-FucpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→

An α-l-Rhap residue is suggested to be present at the terminal part of the polysaccharide, which on average is composed of ∼20 repeating units, since the 1H and 13C chemical shifts of an α-linked rhamnopyranosyl group could be assigned by a combination of 2D NMR spectra. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The repeating unit of the E. coli O177 O-antigen shares the →3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→ structural element with the O-antigen from E. coli O15 and this identity may then explain the reported cross-reactivity between the strains.

Keyword
Escherichia coli, Lipopolysaccharide, NMR, Biological repeating unit, Cross-reactivity
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-62339 (URN)10.1016/j.carres.2011.06.022 (DOI)000295754100034 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2011-09-15 Created: 2011-09-15 Last updated: 2017-12-08Bibliographically approved
3. Genetic and structural relationships of Salmonella O55 and Escherichia coli O103 O-antigens and identification of a 3-hydroxybutanoyltransferase gene involved in the synthesis of a Fuc3N derivative
Open this publication in new window or tab >>Genetic and structural relationships of Salmonella O55 and Escherichia coli O103 O-antigens and identification of a 3-hydroxybutanoyltransferase gene involved in the synthesis of a Fuc3N derivative
Show others...
2010 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 6, 679-688 p.Article in journal (Refereed) Published
Abstract [en]

O-antigen (O-polysaccharide), a part of the outer membrane of Gram-negative bacteria, is one of the most variable cell constituents and is related to bacterial virulence. O-antigen diversity is almost entirely due to genetic variations in O-antigen gene clusters. In this study, the O-polysaccharide structures of Salmonella O55 and Escherichia coli O103 were elucidated by chemical analysis and nuclear magnetic resonance spectroscopy. It was found that the O-polysaccharides have similar pentasaccharide O-units, which differ only in one sugar (glucose versus N-acetylglucosamine) and in the N-acyl group (acetyl versus 3-hydroxybutanoyl) on 3-amino-3,6-dideoxy-d-galactose (d-Fuc3N). The Salmonella O55 antigen gene cluster was sequenced and compared with the E. coli O103 antigen gene cluster reported previously. The two gene clusters were found to share high-level similarity (DNA identity ranges from 53% to 76%), except for two putative acyl transferase genes (fdtC in Salmonella O55 and fdhC in E. coli O103) which show no similarity. Replacement of the fdtC gene in Salmonella O55 with the fdhC gene from E. coli O103 resulted in production of a modified O-antigen, which contains a 3-hydroxybutanoyl derivative of Fuc3N in place of 3-acetamido-3,6-dideoxygalactose. This finding strongly suggests that fdhC is a 3-hydroxybutanoyltransferase gene. The sequence similarity level suggested that the O-antigen gene clusters of Salmonella O55 and E. coli O103 originate from a common ancestor, and this evolutionary relationship is discussed.

Place, publisher, year, edition, pages
Oxford University Press, 2010
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-39137 (URN)10.1093/glycob/cwq015 (DOI)000277448400004 ()
Available from: 2010-05-10 Created: 2010-05-10 Last updated: 2017-12-12Bibliographically approved
4. 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
Open this publication in new window or tab >>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
Show others...
2012 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 357, 118-125 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2012
Keyword
Escherichia coli, Tailspike, Endoglycosidase, Lipopolysaccharide, NMR, Mass spectrometry
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-75048 (URN)10.1016/j.carres.2012.05.022 (DOI)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2012-04-03 Created: 2012-04-03 Last updated: 2017-12-07Bibliographically approved
5. Structural studies of the capsular polysaccharide produced by Leuconostoc mesenteroides ssp. cremoris PIA2
Open this publication in new window or tab >>Structural studies of the capsular polysaccharide produced by Leuconostoc mesenteroides ssp. cremoris PIA2
2011 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, no 7, 2496-2501 p.Article in journal (Refereed) Published
Abstract [en]

The structure of the capsular polysaccharide (CPS) produced by Leuconostoc mesenteroides ssp. cremoris PIA2 has been determined using component analysis and NMR spectroscopy. 1H and 13C resonances were assigned using 2D NMR experiments, and sequential information was obtained by 1H,1H-NOESY and 1H,13C-HMBC experiments. The CPS consists of linear pentasaccharide repeating units with the following structure: →3)-β-d-Galf-(1→6)-β-d-Galf-(1→2)-β-d-Galf-(1→6)-β-d-Galf-(1→3)-β-d-Galp-(1→, in which four out of the five sugar residues have the furanoid ring form, a structural entity found in bacteria but not in mammals. The analysis of the magnitude of the homonuclear three-bond coupling constants of the anomeric protons for the five-membered sugar rings indicates that the sugar residues substituted at a primary carbon atom show one kind of conformational preferences, whereas those substituted at a secondary carbon atom show another kind of conformational preferences.

National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-59717 (URN)10.1021/bm200177z (DOI)000292617700009 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2011-07-06 Created: 2011-07-06 Last updated: 2017-12-11Bibliographically approved
6. Conformational Dynamics of a Central Trisaccharide Fragment of the LeaLex Tumor Associated Antigen Studied by NMR Spectroscopy and Molecular Dynamics Simulations
Open this publication in new window or tab >>Conformational Dynamics of a Central Trisaccharide Fragment of the LeaLex Tumor Associated Antigen Studied by NMR Spectroscopy and Molecular Dynamics Simulations
Show others...
2012 (English)In: European Journal of Organic Chemistry, ISSN 1434-193X, E-ISSN 1099-0690, no 25, 4705-4715 p.Article in journal (Refereed) Published
Abstract [en]

Certain carbohydrate structures are recognized as cancer antigens, and identification of these and relevant epitopes are essential in fighting the disease. The trisaccharide beta-D-GlcpNAc-(1?3)-beta-D-Galp-(1?4)-beta-D-GlcpNAc-OMe represents a model for the central region of the LeaLex hexasaccharide and it has herein been investigated by 1D 1H,1H-NOESY experiments to obtain effective interresidue protonproton distances as well as by 2D J-HMBC experiments to determine transglycosidic 3JCH coupling constants. Molecular dynamics (MD) simulations using explicit water as solvent and three different carbohydrate force fields, namely, GLYCAM06, PARM22/SU01, and CHARMM2011, were employed for the interpretation of experimental data. Overall, the force field based MD simulations are able to reproduce the experimental data and the ? torsion angle at the beta-(1?3)-linkage is concluded to be flexible. In addition, different minor states were present for the three force fields with either anti-? or non-exo-anomeric conformations. Transitions between the exo-anomeric and the non-exo-anomeric conformations for the f torsion angle at the beta-(1?4)-linkage in one of the MD simulations were analyzed in detail. It was found that hydrogen-bonding water molecules, interresidue hydrogen bonds and the transitions between antiperiplanar and synperiplanar conformations for the tH torsion angle of an N-acetyl group were all essential in the description of the glycosidic transition process. In particular, the transition of tH may be a general way of regulating other transitions into less populated but biologically important conformational regions.

Keyword
Carbohydrates, Conformation analysis, NMR spectroscopy, Molecular dynamics
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-81235 (URN)10.1002/ejoc.201200569 (DOI)000307937800008 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:6;

Available from: 2012-10-18 Created: 2012-10-15 Last updated: 2017-12-07Bibliographically approved

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