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A new, powerful method is presented for screening the binding in real time and taking place under dynamic conditions of oligosaccharides to lectins. The approach combines an SPR biosensor and HPLC profiling with fluorescence detection, and is applicable to complex mixtures of oligosaccharides in terms of ligand-fishing. Labeling the oligosaccharides with 2-aminobenzamide ensures a detection level in the fmol range. In an explorative study the binding of RNase B-derived oligomannose-type N-glycans to biosensor-immobilized concanavalin A (Con A) was examined, and an affinity ranking could be established for Man(5)GlcNAc(2) to Man(9)GlcNAc(2), as monitored by HPLC. In subsequent experiments and using well-defined labeled as well as nonlabeled oligosaccharides, it was found that the fluorescent tag does not interfere with the binding and that the optimum epitope for the interaction with Con A comprises the tetramannoside unit Manalpha2Manalpha6(Manalpha3)Man[D(3)B(A)4'], rather than the generally accepted trimannoside Manalpha6 (Manalpha3)Man [B(A)4' or 4(4')3]. In a similar experimental setup, the interaction of various fucosylated human milk oligosaccharides with the fucose-binding lectin from Lotus tetragonolobus purpureaus was studied, and it appeared that oligosaccharides containing blood group H could selectively be retained and eluted from the lectin-coated surface. Finally, using the same lectin and a mixture of O-glycans derived from bovine submaxillary gland mucin, minor constituents but containing fucose could selectively be picked from the analyte solution as demonstrated by HPLC profiling.

Glycosylation of Asn-52 of the alpha-subunit (alphaAsn-52) is required for bioactivity of the alphabeta-dimeric human chorionic gonadotropin (hCG), although at a molecular level the effect of the glycan at alphaAsn-52 is not yet understood. To study the role of this glycan for heterodimer stability, the beta-subunit was recombined in solution with either the alpha-subunit or the alpha-subunit enzymically deglycosylated at alphaAsn-52. Enzymic deglycosylation avoids modification of the glycans at alphaAsn-78 and disturbing the protein folding. The efficiency of recombination after 16 h is 80%, independent of whether alphaAsn-52 is glycosylated or not. The dissociation constant of the hCG complex, with or without the glycan at alphaAsn-52, is less than 1 x 10(-5) s(-1), indicating that the glycan at alphaAsn-52 does not contribute significantly to the stability of the dimer. CD and NMR spectra indicate a local conformational difference between both alphabeta-dimeric hCG variants, most probably involving amino acids of the hCG beta-subunit close to the glycan at alphaAsn-52. These data explain the native-like receptor-binding abilities of hCG lacking the glycan at alphaAsn-52. It is proposed that for bioactivity the glycan at alphaAsn-52 is necessary for inducing and stabilizing a conformational change in hCG upon binding to the receptor, resulting in activation of the signal-transduction pathway.

Sponges (Porifera), the simplest and earliest multicellular organisms, are thought to have evolved from their unicellular ancestors about 1 billion years ago by developing cell-recognition and adhesion mechanisms to discriminate against "non-self." Consequently, they are used as models for investigating recognition phenomena. Cellular adhesion of marine sponges is an event involving adherence of extracellular proteoglycan-like molecules, otherwise known as aggregation factors (AFs). In a calcium-independent process the AFs adhere to the cell surface, and in a calcium-dependent process they exhibit AF self-association. A mechanism which has been implied but not definitely proven to play a role in the calcium-dependent event is self-recognition of defined carbohydrate epitopes. For the red beard sponge, Microciona prolifera, two carbohydrate epitopes, a sulfated disaccharide and a pyruvylated trisaccharide, have been implicated in cellular adhesion. To investigate this phenomenon a system has been designed, by using surface plasmon resonance detection, to mimic the role of carbohydrates in cellular adhesion of M. prolifera. The results show self-recognition of the sulfated disaccharide to be a major force behind the calcium-dependent event. The interaction is not simply based on electrostatic interactions, as other sulfated carbohydrates analyzed by using this procedure did not self-associate. Furthermore, the interaction is completely eradicated on substitution of Ca(2+) ions by either Mg(2+) or Mn(2+) ions. This physiologically relevant recognition mechanism confirms the existence of true carbohydrate self-recognition, and may have significant implications for the role of carbohydrates in cellular recognition of higher organisms.

In a previous study [Pantophlet, R., Brade, L., Dijkshoorn, L., and Brade, H. (1998) J. Clin. Microbiol. 36, 1245-1250] the O-polysaccharide of the lipopolysaccharides (LPS) from Acinetobacter haemolyticus strains 57 and 61 exhibited indistinguishable banding-patterns following Western blot and immunostaining with homologous or heterologous rabbit antiserum. In this report, the molecular basis for the observed cross-reactivity was elucidated, by determining the chemical structure of the polysaccharides by compositional analysis and NMR spectroscopy. The structures are: [sequence: see text] for strain 61 [GulpNAcA, 2-acetamido-2-deoxy-gulopyranosyluronic acid; ManpNAcA, 2-acetamido-2-deoxy-mannopyranosyluronic acid; QuipN4N, 2,4-diamino-2,4,6-trideoxy-glucopyranose; acyl (S)-3-hydroxybutyryl], thus, differing only in the anomeric configuration of the QuipN4N residue. The antigenic structures were determined by generating murine monoclonal antibodies, which were characterized by Western blot using LPS as antigen, by ELISA using LPS and de-O-acylated LPS as solid-phase antigens, and by ELISA inhibition studies using LPS, polysaccharide, and de-O-acylated LPS as inhibitors. Of the four antibodies selected, two were specific for the respective LPS moieties and two were cross-reactive. All antibodies were found to require the presence of the O-acetyl group for reactivity.

A polysaccharide containing D-GalNAc, D-Glc and 4-acetamido-4,6-dideoxy-D-glucose (Qui4NAc) was isolated from the phenol-soluble lipopolysaccharide originating from Acinetobacter baumannii strain 9. The structure of the repeating unit was shown by means of monosaccharide analyses, Smith-degradation, partial acid hydrolysis, mass spectrometry, and NMR spectroscopy to be a branched pentasaccharide, in which the tetrasaccharide backbone is built from amino sugars only. [structure: see text] The polysaccharide was identified by serological and western blot analyses as the O-antigen of the lipopolysaccharide.

A polysaccharide containing D-GlcN, 2-amino-2,6-dideoxy-L-galactose (L-FucN), and 7-acetamido-5-acylamino-3,5,7,9-tetradeoxy-L-glycero-D-galacto-nonulo sonic acid (LegAX), in which the acyl group (X) is either S-3-hydroxybutyryl (50%) or acetyl (50%), was isolated by mild acid hydrolysis treatment, followed by gel-permeation chromatography, of the water-soluble lipopolysaccharide from Acinetobacter baumannii serogroup O24. The polysaccharide, characterised by means of monosaccharide analyses, partial acid hydrolysis, methylation analysis and NMR studies, was shown to have a linear tetrasaccharide repeating unit, as depicted below. Serological tests indicated that the polymer corresponded to the O24 antigen. -->6)-alpha-D-GlcpNAc-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-Glcp NAc-(1-->4)-beta-LegpAX-(1-->.

A polysaccharide containing D-Gal, D-GalNAc, 3-(L-2-acetoxypropionamido)-3,6-dideoxy-D-galactose (approximately 80%) and 3-(L-2-hydroxypropionamido)-3,6-dideoxy-D-galactose (approximately 20%) was isolated by mild acid hydrolysis, followed by gel-permeation chromatography, from the phenol-soluble lipopolysaccharide (phenol/water extracted) derived from Acinetobacter strain 94. The polysaccharide, characterised by means of monosaccharide analyses, partial acid hydrolysis, and NMR studies, consisted of a branched tetrasaccharide repeating unit, as depicted below, in which Fucp3Nacyl represents 3-(L-2-hydroxypropionamido)-3,6-dideoxy-D-galactose, in which approximately 80% of the acyl residues are O-acetylated. These Fucp3N derivatives and an O-acetylated acyl group are therefore constituents of bacterial LPS, but to our knowledge are not present in any other natural carbohydrates. [sturcture: see text]

A polysaccharide containing rhamnose (Rha) and Gal was isolated by acetic acid hydrolysis, followed by gel-permeation chromatography, from the water-soluble lipopolysaccharide (phenol/water extracted) from Acinetobacter junii strain 65. The polysaccharide was characterised by means of monosaccharide analyses, Smith degradation, and NMR studies, and was shown to have a linear pentasaccharide repeating unit, as depicted below. This structure was specifically recognised in western blots and enzyme immunoassays by polyclonal rabbit antisera. [structure in text]

Water-soluble lipopolysaccharide (phenol/water extraction) isolated from Acinetobacter strain 90, which belongs to DNA group 10, was hydrolysed with 1% acetic acid, ultracentrifuged, and water-soluble products finally eluted from a Sephadex G-50 column. The major fraction, a polysaccharide, contained D-Gal, D-GlcNAc, D-GalNAc, and 4,6-dideoxy-4-[(R)-3-hydroxybutyramido]-D-galactose (Fuc4NBuOH). The polysaccharide was characterised by means of monosaccharide analyses, Smith-degradation, N-deacetylation/deamination, and NMR studies, and was shown to have a branched pentasaccharide repeating unit. [structure in text] This structure was specifically recognised in western blots and enzyme immunoassays by polyclonal rabbit antisera.

A polysaccharide containing 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 2.4-diacetamido-2,4,6-trideoxy-D-glucose (QuiNAc4NAc), and D-alanine (Ala) was isolated from the water-soluble lipopolysaccharide (LPS) originating from the reference strain for Acinetobacter haemolyticus (DNA group 4) strain ATCC 17906. The polysaccharide, characterised by means of monosaccharide analyses and NMR studies, was shown to be based on a linear trisaccharide repeating unit, as shown below, with the alanine group amide-bound to position 6 of one GalNAcA residue. It was specifically recognised in western blots by polyclonal rabbit antisera. [structure: see text]

A polysaccharide containing D-galactose (Gal), 2-acetamido-2-deoxy-D-galactose (GalNAc), 2-acetamido-2-deoxy-D-glucose (GlcNAc), and 3-deoxy-3-(D-3-hydroxybutyramido)-D-quinovose (Qui3NR) was isolated from lipopolysaccharide (LPS) obtained from cells walls of the reference strain for Acinetobacter baumannii O23. By means of NMR studies, methylation analysis, and chemical degradations, the repeating unit of the polymer was identified as a branched pentasaccharide with the structure 1. The same polymer was apparently also present in LPS of the reference strain for serogroup O12, together with a second polymer based on a branched tetrasaccharide with the structure 2. This second polymer has previously been isolated as the O16 antigen of A. baumannii [Haseley, S.R., Diggle, H.J. & Wilkinson, S. G. (1996) Carbohydr. Res. 293, 259-265] and is probably present as a minor component of the LPS of A. baumannii O11 [Haseley, S.R. & Wilkinson, S.G. (1996) Eur. J. Biochem. 237, 266-271]. [Sequence: see text]

S-form lipopolysaccharide was isolated by phenol/water extraction from a strain of Acinetobacter calcoaceticus (DNA group 1 ). The structure of the O-antigenic polysaccharide was determined by compositional analysis and NMR spectroscopy of the de-O-acylated lipopolysaccharide. The isolated polysaccharide obtained after hydrolysis of lipopolysaccharide in 0.01 M trifluoroacetic acid has the following structure: [STRUCTURE IN TEXT] in which Pyr is pyruvate. The O-acetyl substitution of D-Gal was non-stoichiometric. The O-antigen was specifically recognised in western blots by polyclonal rabbit antisera.

A major polysaccharide containing D-galactose, D-glucose and 2-acetamido-2-deoxy-D-galactose was obtained after mild acid hydrolysis of the water-soluble material released by treatment of cell walls from Acinetobacter baumannii strain O11 with hot, aqueous phenol. By means of NMR studies, Smith degradation and N-deacetylation/deamination, the repeating unit of the polymer was identified as a branched pentasaccharide of the structure shown. Also present was a minor polymer containing glucose, 2-acetamido-2-deoxyglucose-and 2-acetamido-2-deoxygalactose, the structure of which was not elucidated. On serological testing, the polymeric material was shown to correspond to the O-antigenic moiety of the parent extract (assumed to be lipopolysaccharide) and circumstantial evidence indicated that O11 specificity was conferred by the major polymer. [formula: see text]

A polysaccharide containing 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-fucose (FucNAc), and 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA) was isolated from an aqueous phenol extract of lipid-free, isolated cell walls of the reference strain for Acinetobacter baumannii serogroup O5, by mild acid hydrolysis of the extract and chromatography of the water-soluble products on Sephadex G-50. By means of NMR studies, methylation analysis, carboxyl reduction and chemical degradations, the repeating unit of the polymer was identified as a branched tetrasaccharide of the structure shown. The serologically active polymer is believed to correspond to the side chain of the O5 lipopolysaccharide: [table: see text]

A polysaccharide containing D-galactose, 2-deoxy-2-N-acetylamino-D-galactose and 3,6-dideoxy-3-N-(D-3-hydroxybutyryl)amino-D-galactose, probably corresponding to the lipopolysaccharide side chain, was obtained from an aqueous phenol extract of isolated cell walls from Acinetobacter baumannii strain O2. By means of NMR studies and chemical degradations, the repeating unit of the polymer was identified as a branched hexasaccharide of the structure shown, where Fuc3N represents 3-amino-3,6-dideoxygalactose and R represents D-3-hydroxybutyryl. Serological tests indicated that the polymer corresponded to the O2 antigen.