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A trisaccharide donor containing a cis-Galpα(1→4)Galp linkage was prepared using a synthetic strategy based on chemoenzymatic oligosaccharide synthesis on a soluble polymeric support. Significantly, only retaining glycosyltransferases gave complete reactions, whereas inverting enzymes showed little or no activity with poly(ethylene glycol) (MPEG)-bound lactose as an acceptor. The MPEG-attached trisaccharide was shown to bind to Verotoxin-1 by transfer NOE studies through the Galpα(1→4)Galp portion of the molecule.

The L1 immunotype strain 126E of Neisseria meningitidis has been shown to have an N-acetyl-neuraminic acid-containing lipooligosaccharide in which an alpha-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 alpha-2,3 and alpha-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 alpha-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) alpha-2,3/6-sialyltransferase was replaced by allelic exchange with the monofunctional MC58(L3) alpha-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 alpha-2,6-sialyltransferase activity in vitro as well as in vivo.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a beta-1, 4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1, 3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked alpha-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOS.

ABSTRACTWe investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidislipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with agalEmutant of N. meningitidisH44/76 (B.15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidisstrains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the β-chain heptose (HepII) of the inner core. In contrast, N. meningitidisstrains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidisLPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused byN. meningitidis.

The type VIII capsular polysaccharide has been isolated and purified from a newly described strain of group B Streptococcus which is a leading cause of sepsis and neonatal meningitis in Japan. The polysaccharide contains D-glucose, D-galactose, L-rhamnose, and sialic acid in the molar ratio 1:1:1:1. By means of high resolution 1H nuclear magnetic resonance (1H NMR), 13C NMR, and homo- and heterocorrelated NMR, the repeating unit structure of the type VIII polysaccharide was delineated as the following, [formula: see text] Enzymatic studies established this polysaccharide as the first from which sialic acid, linked to a branched beta-D-galactopyranosyl residue, is known to be removed by bacterial neuraminidase.

The O-side-chain polysaccharide in the lipopolysaccharide of Klebsiella pneumoniae O1 is based on a backbone structure of repeat units of [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]; this structure is termed D-galactan I. The rfb (O-antigen biosynthesis) gene cluster directs the synthesis of D-galactan I and consists of six genes termed rfbA-FKPO1. In this paper we show that rfbDKPO1 encodes a UDP-galactopyranose mutase (NAD(P)H-requiring) (EC 5.4.99. 9), which forms uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactofuranosyl ester (UDP-Galf), the biosynthetic precursor of galactofuranosyl residues. The deduced amino acid sequence of rfbDKPO1 shows 85% and 37.5% identity to the rfbDKPO8 gene of K. pneumoniae serotype O8 and the glf gene of Escherichia coli, respectively. The molecular mass of the purified RfbDKPO1 enzyme is 45 kDa as determined by SDS-polyacrylamide gel electrophoresis, while gel filtration revealed a molecular mass of 92 kDa, suggesting a dimeric structure for the native protein. The rfbDKPO1 gene product interconverts uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactopyranosyl ester (UDP-Galp) and UDP-Galf. Unlike Glf, RfbDKPO1 showed a requirement for NADH or NADPH, which could not be replaced by NAD or NADP. RfbDKPO1 was used to synthesize milligram quantities of UDP-Galf, allowing this compound to be purified and fully characterized in an intact form for the first time. The structure of UDP-Galf was proven by NMR spectroscopy.

The conformational features of sucrose in the combining site of lentil lectin have been characterized through elucidation of a crystalline complex at 1.9-A resolution, transferred nuclear Overhauser effect experiments performed at 600 Mhz, and molecular modeling. In the crystal, the lentil lectin dimer binds one sucrose molecule per monomer. The locations of 229 water molecules have been identified. NMR experiments have provided 11 transferred NOEs. In parallel, the docking study and conformational analysis of sucrose in the combining site of lentil lectin indicate that three different conformations can be accommodated. Of these, the orientation with lowest energy is identical with the one observed in the crystalline complex and provides good agreement with the observed transferred NOEs. These structural investigations indicate that the bound sucrose has a unique conformation for the glycosidic linkage, close to the one observed in crystalline sucrose, whereas the fructofuranose ring remains relatively flexible and does not exhibit any strong interaction with the protein. Major differences in the hydrogen bonding network of sucrose are found. None of the two inter-residue hydrogen bonds in crystalline sucrose are conserved in the complex with the lectin. Instead, a water molecule bridges hydroxyl groups O2-g and O3-f of sucrose.

A nuclear magnetic resonance technique, the nuclear Overhauser effect, has been used to confirm the presence of Man alpha 1-2, GlcNAc beta 1-2, and Man alpha 1-3 residues and to establish the branching pattern and sequences in four classes of N-linked oligosaccharides. This method offers a rapid and nondestructive approach to the elucidation of sequences in carbohydrate chains, in contrast to enzymatic and chemical methods.