Abstract
The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.
Highlights
The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and -elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrixassisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and 1H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides
The polysaccharide is characterized by a number of rather novel structural features for an O-chain, including a relatively low molecular weight (3330 Da), uniform size, numerous endogenously Omethylated components (3-O-methyl-6-deoxytalose, 2,3-di-Omethylfucose, 2-O-methylfucose, and 2,3,4-tri-O-methylfucose), and O-acetylation
The latter features appear to confer a fairly high level of hydrophobicity to the glycan, a property that may be important during infection thread development and the establishment of symbiosis4 [6, 47]
Summary
Growth of Bacteria—R. etli CE3, the streptomycin-resistant derivative of wild-type strain CFN42, was grown in tryptone/yeast extract supplemented with Ca2ϩ as described previously [10]. Acidic glycosyl residues were analyzed as the alditol acetates by converting the carboxyl groups to methyl esters (1 M methanolic HCl, 80 °C for 2 h), followed by carboxyl reduction with NaBD4 in water, trifluoroacetic acid hydrolysis, and conversion to the alditol acetates [33]. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry was performed on an LDI 1700XP time-of-flight (TOF) spectrometer (Linear Scientific, Reno, NV) in the positive and negative modes, using a matrix of 100 mM 2,5-dihydroxybenzoic acid in 90% methanol. High Pressure Liquid Chromatography Fractionation of Oligosaccharides—Oligosaccharides derived from the O-chain by -elimination were separated by SEC as described above and fractionated using a Vydac 238TP510 5-m monomeric C18 semi-preparative reverse phase column (10 ϫ 250 mm) using a linear gradient (0 –30%) of methanol in water, containing 0.05% trifluoroacetic acid with a flow rate of 3 ml/min. The Kdo was assumed to be in the D-configuration
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