Analysis of the interaction between lectins and tetra- and tri-saccharide mimics of the Sd(a) determinant by surface plasmon resonance detection.

The binding properties of a spacer-linked synthetic Sd(a) tetrasaccharide beta-D-GalpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (1), two tetrasaccharide mimics beta-D-Galp-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (2) and beta-D-GlcpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (3), and two trisaccharide mimics beta-D-GalpNAc-(1-->4)-3-O-(SO(3)H)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (4) and beta-D-GalpNAc-(1-->4)-3-O-(CH(2)COOH)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (5) with lectins from Dolichos biflorus (DBL), Maackia amurensis (MAL), Phaseolus limensis (PLL), Ptilota plumosa (PPL), Ricinus communis 120 (RCL120) and Triticum vulgaris (wheat germ agglutinin, WGA) have been investigated by surface plasmon resonance (SPR) detection. MAL, PPL, RCL120 and WGA did not display any binding activity with compounds 1-5. However, DBL and PLL, both exhibiting GalNAc-specificity, showed strong binding activity with compounds 1, 4 and 5, and 1, 3, 4 and 5, respectively. The results demonstrate that SPR is a very useful analysis system for identifying biologically relevant oligosaccharide mimics of the Sd(a) determinant.

Carbohydrate self-recognition mediates marine sponge cellular adhesion.

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.

Studies on the structure of a lithium-treated soybean pectin: characteristics of the fragments and determination of the carbohydrate substituents of galacturonic acid.

Two galacturonic-acid-containing polysaccharide fractions (ChSS and P) were isolated from soybean meal and subjected to lithium treatment. The fragments obtained were analyzed by using monosaccharide and methylation analyses, and NMR spectroscopy. Lithium degradation of ChSS, followed by sodium borodeuteride reduction, hydrolysis, sodium borohydride reduction, and acetylation afforded alditol acetates, of which the labeled ones reflected residues linked to GalA. As followed from quantifications of the labeled and non-labeled alditols from each constituent monosaccharide by GLC-EIMS, 6 mol% of Ara, 22 mol% of Fuc, 13 mol% of Gal, 53 mol% of Rha, and 57 mol% of Xyl are glycosidically linked to GalA. Analysis of the lithium-treated polymer revealed that it contains arabinogalactan side chains linked to Rha O-4, which consist of a beta-(1 --> 4)-linked galactan substituted with highly branched arabinan chains. On average, an arabinogalactan chain contains up to 29 Gal and 25 Ara residues. Surface plasmon resonance was used to determine conditions for affinity chromatography. Furthermore, this technique confirmed the presence of terminal alpha-Fuc residues in ChSS. Polysaccharide P turned out to be relatively resistant to lithium degradation.

Characterization of the carbohydrate binding specificity and kinetic parameters of lectins by using surface plasmon resonance.

An accurate, rapid, and sensitive method for characterizing the carbohydrate binding properties of lectins using a BIAcore apparatus and the detection method of surface plasmon resonance is described. As a model study, the sialic acid binding lectins from Sambucus nigra and Maackia amurensis, which are specific for the epitopes Neu5Ac(alpha2-6)Gal and Neu5Ac(alpha2-3)Gal, respectively, were chosen as suitable candidates. Two systems, one for the analysis of oligosaccharides and the other for glycoproteins, were developed after a rigorous analysis and evaluation of such parameters as binding conditions, buffers, and regeneration conditions. The systems take into account nonspecific binding, using the respective denatured lectin as negative blank, and avoid loss of activity: regeneration of the surface using either 10 mM NaOAc (pH 4.3) buffer (oligosaccharide system) or 20 mM HCl (glycoprotein system). The specificity of the lectins is well illustrated, while the kinetics parameters are shown to be sensitive to subtle changes in the recognized epitopes, and to be affected by steric hindrance. Surface plasmon resonance is a suitable technique for the analysis and characterization of lectins.

Chemical and antigenic structure of the O-polysaccharide of the lipopolysaccharides from two Acinetobacter haemolyticus strains differing only in the anomeric configuration of one glycosyl residue in their O-antigens.

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.

Structure of the O-7 antigen from Acinetobacter baumannii.

The polymeric O-antigen was isolated from the lipopolysaccharide of the reference of the reference strain for Acinetobacter baumannii serogroup O-7. Both the lipopolysaccharide and the isolated polymer reacted with the homologous antiserum. Monosaccharide analyses and NMR spectra showed that the polymer had a hexasaccharide repeating unit constructed from residues of L-rhamnose (4) and N-acetyl-D-glucosamine (2). The following structure for the repeating unit was established by means of detailed interpretation of the NMR spectra, methylation analysis, and chemical degradations. The tetrasaccharide backbone is identical to that for the O-10 antigen of A. baumannii, which has alpha-D-ManpNAc as the lateral substituent in place of the disaccharide present in the O-7 antigen. [formula: see text]

Structural studies of the O-antigen isolated from the phenol-soluble lipopolysaccharide of Acinetobacter baumannii (DNA group 2) strain 9.

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.

Structural studies of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter (DNA group 11) strain 94 containing 3-amino-3,6-dideoxy-D-galactose substituted by the previously unknown amide-linked L-2-acetoxypropionic acid or L-2-hydroxypropionic acid.

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]

Structure of the O18 antigen from Acinetobacter baumannii.

The polymeric O antigen was obtained from lipopolysaccharide extracted from isolated, defatted cell walls of the reference strain for Acinetobacter baumannii serogroup O18. Monosaccharide analyses and NMR spectra established that the polymer had a regular structure with a repeating unit based on residues of D-galactose (2), N-acetyl-D-galactosamine (1), and N-acetyl-D-mannosamine (1). Further interpretation of the NMR spectra, combined with the results of methylation analysis and a Smith degradation, showed that the repeating unit had the following structure. beta-D-ManpNAc-(1-->4)-alpha-D-Galp 1 decreases 4 -->3)-beta-D-GalpNAc-(1-->3)-beta-D-Galp-(1-->.

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter strain 90 belonging to DNA group 10.

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.

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter junii strain 65.

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]

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter haemolyticus strain ATCC 17906.

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]

Structures of polymeric products isolated from the lipopolysaccharides of reference strains for Acinetobacter baumannii O23 and O12.

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]

Structural and serological characterisation of the O-specific polysaccharide from lipopolysaccharide of Acinetobacter calcoaceticus strain 7 (DNA group 1).

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.

Structural studies of the putative O-specific polysaccharide of Acinetobacter baumannii O24 containing 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-D-galacto-nonulosonic acid.

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-->.

Structure of the O-specific polysaccharide of Acinetobacter baumannii O5 containing 2-acetamido-2-deoxy-D-galacturonic acid.

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]

Structural studies of the putative O-specific polysaccharide of Acinetobacter baumannii O11.

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]

Structural studies of the putative O-specific polysaccharide of Acinetobacter baumannii O2 containing 3,6-dideoxy-3-N-(D-3-hydroxybutyryl)amino-D-galactose.

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.

Structure of the putative O10 antigen from Acinetobacter baumannii.

A polysaccharide containing L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-mannose was obtained from an aqueous phenol extract of isolated cell walls from the reference strain for Acinetobacter baumannii serogroup O10. By means of NMR studies and chemical degradations, the repeating unit of the polymer (the putative O10 antigen) was identified as a branched pentasaccharide of the structure shown.