Nuclear magnetic resonance and conformational investigations of the pentasaccharide of the Forssman antigen and overlapping di-, tri-, and tetra-saccharide sequences

Grönberg, Gunnar; Nilsson, Ulf J.; Bock, Klaus; Magnusson, Göran

doi:10.1016/0008-6215(94)84106-3

Cited by 49 publications

(31 citation statements)

References 31 publications

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“…Although a different fluorescent tag was used, it only influenced the proton chemical shift of the Glc residue. For compound I, the proton and 13 C chemical shifts were in accord with those reported for Gal-␣1,4-Gal-␤1,4-Glc-␤-1-O-2-(trimethylsilyl)ethanol (25). Compound II was identified as Neu5Ac-␣2,6-Gal-␣1,4-Gal-␤1,4-Glc-␤-1-FEX.…”

Section: Resultssupporting

confidence: 73%

Dependence of the Bi-functional Nature of a Sialyltransferase from Neisseria meningitidis on a Single Amino Acid Substitution

Wakarchuk

Watson

Michael

et al. 2001

Journal of Biological Chemistry

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The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an ␣-linked galactose from a P k epitope is substituted at the O6 position (Wakarchuk, W. W., Gilbert, M., Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards, J. C. (1998) Eur. J. Biochem. 254, 626 -633). Using a synthetic P k -epitope containing acceptor in glycosyltransferase reactions, we were able to show by NMR analysis of the reaction product that the 126E(L1)-derived sialyltransferase can make both ␣-2,3 and ␣-2,6 linkages to the terminal galactose. Gene disruption experiments showed that the lst gene in 126E(L1) was responsible for the in vivo addition of the ␣-2,6-linked N-acetyl-neuraminic acid residue. By site-directed mutagenesis it was possible to change the MC58(L3)-derived enzyme into a bifunctional enzyme with a single amino acid change at position 168, where a glycine was changed to an isoleucine. We performed a gene replacement experiment where the 126E(L1) ␣-2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) ␣-2,3-sialyltransferase and with the mutant MC58(L3) allele G168I. We observed that the level of LOS sialylation with the G168I allele was very similar to that of the wild type 126E(L1), indicating that residue 168 is the critical residue for the ␣-2,6-sialyltransferase activity in vitro as well as in vivo.Mucosal pathogens in the genera Neisseria, Campylobacter, and Haemophilus all possess a major cell surface lipooligosaccharide (LOS) 1 containing N-acetylneuraminic acid (Neu5Ac) (2-4). These carbohydrate structures are not a static feature of the bacterium's outer surface but are produced with significant variation depending on the genetic makeup of the strain and the environment in which it is growing (5-7). This phenomenon is thought to play an important role in the pathogenesis of these organisms (8) (9), relating as it does to the structural similarity of the terminal oligosaccharide portion of the LOS to those found on human glycolipids. In Neisseria a major structure is a terminal ␣-2,3-sialyllacto-N-neotetraose (Fig. 1), in Haemophilus influenzae strain RM118, the LOS structures include ␣-2,3-sialyllactose (7), and in the NTHi 375 strain there is a simple disialic acid structure (perhaps related to the ganglioside GD3) (4). In Campylobacter jejuni there are structures similar to various gangliosides, from GD3 to GT1a (10). Such oligosaccharides provide these pathogens with a means of evading the host immune response through molecular mimicry as well as providing ligands for binding to receptors on human cells (11), and they are therefore potent virulence factors (12).The genetic analysis of the biosynthesis of LOS has shown that variation in the oligosaccharide portion of the LOS can arise in several ways. In H. influenzae, some LOS biosynthesis genes including glycosyltransferases are inactivated through a mechanism involving a variable number of tandem tetranucleotide repeats, whic...

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Section: Resultssupporting

confidence: 73%

Dependence of the Bi-functional Nature of a Sialyltransferase from Neisseria meningitidis on a Single Amino Acid Substitution

Wakarchuk

Watson

Michael

et al. 2001

Journal of Biological Chemistry

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“…This is fortunate because interglycosidic NOEs are usually sparse, and unambiguous determination of solution conformations or conformational families can be a difficult task. First, there are two strong interglycosidic NOEs from H1" to H3d, and from HI" to H4d, and a weak NOE from H1" to H5d (see also Gronberg et al, 1994). By comparison with a corresponding ROESY experiment (data not shown), the latter NOE was shown to result from spin diffusion.…”

Section: Resultsmentioning

confidence: 97%

“…These previous publications discussed the conformational properties of Forssman antigen-related saccharides. One of the more recent studies concluded that the Forssman pentasaccharide has a bent overall shape with some flexibility around the glycosidic linkages (Gronberg et al, 1994), and, unlike the case of BAT (Casset et al, 1996), only one major conformational family was identified. This conformational model of Gronberg et al was used as a basis for our studies, and the discussion of the conformational properties of unliganded FPS will focus on selected effects that have not been addressed before.…”

Section: Resultsmentioning

confidence: 99%

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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

Casset

Pérez²,

Etzler

et al. 1997

European Journal of Biochemistry

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A complex between the Forssman pentasaccharide a-D-GalNAc-( 1+3)-P-~-GalNAc-( 1+3)-a-~-Gal-(1+4)-P-~-Gal-( 1-4)-~-Glc and the seed lectin from Dolichos bz$hrus 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 a-~-GalNAc-(l+3)-P-~-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 a-D-GalNAc-( 1 -3)-p-~-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 a-D-Gal-(1+4)-p-~-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 a-D-GalNAc-(1+3)-p-~-GalNAc and D. bij7oru.s 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.

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“…6). This effect is probably due to a rigid arrangement of the branched Kdo tetrasaccharide, and it can be compared with the general effect observed in ␣-D-pyranoside residues linked to an axial hydroxyl group that is in close proximity to an equatorial hydroxyl group, as is present for example in galabiose (35). Furthermore, the equatorial H-3 is shifted downfield, but less strongly, which is probably due to an interaction with the lone pair of the glycosidic oxygen possessing a fixed orientation.…”

Section: Structure Of a Baumannii Lipopolysaccharide 28129mentioning

confidence: 99%

Characterization of a Novel Branched Tetrasaccharide of 3-Deoxy-d-manno-oct-2-ulopyranosonic Acid

Vinogradov¹,

Petersen²,

Thomas‐Oates³

et al. 1998

Journal of Biological Chemistry

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For the first time, the tetrasaccharide Kdo␣235Kdo␣235(Kdo␣234)Kdo (Kdo is 3-deoxy-Dmanno-oct-2-ulopyranosonic acid) has been identified in a bacterial lipopolysaccharide (LPS), i.e. in the core region of LPS from Acinetobacter baumannii NCTC 10303. The LPS was analyzed using compositional analysis, mass spectrometry, and NMR spectroscopy. The disaccharide D-GlcpN␤136D-GlcpN, phosphorylated at O-1 and O-4, was identified as the carbohydrate backbone of the lipid A. The Kdo tetrasaccharide is attached to O-6 of this disaccharide and is further substituted by short L-rhamnoglycans of varying length and by the disaccharide D-GlcpNAc␣134D-GlcpNA (GlcpNA, 2-amino-2-deoxy-glucopyranosuronic acid). The core region is not substituted by phosphate residues and represents a novel core type of bacterial LPS. The complete carbohydrate backbone of the LPS is shown in Structure I as follows:where Rha is rhamnose. Except were indicated, monosaccharides possess the D-configuration. Sugars marked with an asterisk are present in non-stoichiometric amounts.Acinetobacter represents a coherent genus within the ␥ subclass of Proteobacteria in which it is placed within the family Moraxellaceae (1). Bacteria of this genus are widespread in Nature, where they occur in soil, water, food, and sewage as free-living saprophytic bacilli (2). However, they are also found in the hospital environment, e.g. on the skin of patients and staff (3), and some species have been recognized as dangerous nosocomial human pathogens that cause different diseases like pneumonia, meningitis, wound infections, septicemia, or endocarditis (4). Hospital strains appear to possess a significant capacity to survive on equipment and human skin for a long time. Moreover, in most cases they are multi-resistant against the major groups of antibiotics (2). On the basis of DNA-DNA hybridization, Acinetobacter is presently classified into 21 DNA groups (also called genomic species), 7 of which have been named to date, together with a number of un-grouped strains (5, 6). Of these 21 DNA groups, Acinetobacter baumannii is the main species associated with nosocomial infections, and most often resistant to antibiotics (2).In common with other Gram-negative bacteria, Acinetobacter possess lipopolysaccharide (LPS) 1 in the outer membrane of their cell wall; these LPSs represent chemotaxonomical and antigenic markers. In early investigations, it had been shown that this LPS is of the rough type, i.e. does not possess an O-specific polysaccharide (7,8). Our further investigations of LPS from several hundred Acinetobacter strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain seem to confirm this; however, when these samples were Western blotted using polyclonal rabbit antisera against LPS, most samples gave a banding pattern characteristic for smooth LPS (9). In the meantime, we and others (Ref. 10 and * Part of this work was presented at the VIIth European Carbohydrate Symposium, August 22-27, 1993, Cracow, Poland. The costs of publication of t...

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Nuclear magnetic resonance and conformational investigations of the pentasaccharide of the Forssman antigen and overlapping di-, tri-, and tetra-saccharide sequences

Cited by 49 publications

References 31 publications

Dependence of the Bi-functional Nature of a Sialyltransferase from Neisseria meningitidis on a Single Amino Acid Substitution

Dependence of the Bi-functional Nature of a Sialyltransferase from Neisseria meningitidis on a Single Amino Acid Substitution

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

Characterization of a Novel Branched Tetrasaccharide of 3-Deoxy-d-manno-oct-2-ulopyranosonic Acid

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