The precise molecular structure of the cell-surface lipopolysaccharide (LPS) of the gram-negative bacterium Yersinia ruckerii (Serovar 11) has been investigated. This bacterium is the causative agent of enteric redmouth disease in fish, a disease which constitutes an important economic problem in commercial rainbow trout hatcheries. We present in this letter positive ion fast atom bombardment mass spectral studies carried out on the 9-specific antigen, one of the distinct segments of the LPS and the portion containing the epitopes. chemical-ionization fragmentation) of the derived alditol acetates and of the acetylated derivatives of the methyl and the (-)-2-butyl glycosides, the 0-specific antigen was found to be composed of 2-acetamido-2,6-~ideoxy-~-glucose (N-acetyl-gquinovosamine), 2-acetamido-2-deoxy-~-glucose (N-acetyl-gglucosamine) and N-acetyl-q-muramic acid in the molar ratio of -1:l:l. The chemical structure of this 0-specific antigen was determined using sugar and methylation analyses, specific degradations, chromium trioxide oxidation and both 'H and 13C-nuclear magnetic resonance'. We have established that this -0-specific antigen was an unbranched linear heteropolymer, formed of a repeating trisaccharide unit and having the following structure:On the basis of combined GC-MS (electron-impact and OAc J. 6 [ 4 ) -a-g-MurpNAc--( 1-13) -a-D-QuipNAc--( 14)-BD-GlcpNAc---( 1-11It is well documented that mass spectrometry using surface ionization modes is a powerful technique for the structural investigation of complex carbohydrates2 and we have determined that positive ion FAB was the most suitable method for the analysis of this 0-specific polysaccharide.In effect, tLe fully permethylated 0-specific antigen gave an excellent positive ion FAB-MS that was easy to interpret and that contained complete sequence information. The major ions formed were derived from the single pathway A cleavage2, which occurred predominantly at each hexosaminal residue yielding a series of characteristic sequence-ions. determination is shown in the following Fig. 1 permethylated 0-specific antigen, we could recognize the major characteristic-sequence-ions originating from the complete native heteropolymer backbone (main series), in addition to two minor series (series I and 11) affording low abundance sequence-ions. These two minor sequences starting with QuiNAc as well as GlcNAc may well explain the phenomenon of microheterogeneity always associated with the incomplete biosynthesis of bacterial smooth lipopoly~accharide~.
Conjugation of simple ketoses (such as 3-deoxy-~-manno-2-octu~osonic acid and N-acetylneuraminic acid) and of various 0-specific polysaccharides (from Aerornonas hydrophila and Aeromonas salmonicida) to the bifunctional spacer 2,6-hexanediamine, was achieved by reductive amination. The saccharide -1-(ti-amino)-hexane alkyamines obtained were converted into the corresponding isothiocyanate derivatives and coupled to the free c-amino group of lysine residues of the protein carrier bovine serum albumin. In similar manner, the aldehyde group introduced by selective periodate oxidation into the partially 0-deacylated lipopolysaccharide of Vibrio anguillarum was conjugated to 1,6-hexanediamine, converted into the corresponding isothiocyanate and covalently attached to bovine serum albumin.During the past decade, artificial glycoconjugates or neoglycoproteins, obtaincd by covalent attachment of polysaccharide to protein, have been extensively used for antibody -polysaccharide interactions [l] and found to be of great utility as vaccines against encapsulated bacteria [2].Numerous methods, each with their own merits and purposes, have been developed for the synthesis of glycoconjugates [3]. Most of these studies have generally resorted to the use of naturally occurring bacterial oligo-and polysaccharides or chemically synthesized carbohydrate haptens which mimic the natural saccharide sequences of the carbohydrate portion of the conjugate. One method that is both simple and effective is the direct covalent attachment of reducing carbohydrates to the amino groups of proteins by reductive amination using sodium cyanoborohydride [4, 51. A major disadvantage of this method is the opening of the ring structure of the terminal reducing sugar to generate an acyclic amine which, in certain cases, could be detrimental to the biological specificities of the glycoconjugate [6]. However, this disadvantage is of no importance if the haptenic saccharide is large, as in the case of bacterial polysaccharide [7]. Another drawback to this method is that it is not possible to conjugate oligosaccharides having reducing ketose residues, such as the terminal 3-deoxy-~-manno-2-octulosonk acid (dOclA), to proteins [8]. This is consistent with the fact that a successful reductive amination attachment of oligosaccharides containing terminal dOclA residues to proteins can be achieved only if a functionalized spacer molecule is introduced at the ketonic carbonyl group of dOclA [8, 91. This paper presents a new method for coupling different 0-specific polysaccharides and a partially 0-deacylated oxidized lipopolysaccharide of representative species of the Vibrionaceae family to the lysine residues of the carrier protein bovine serum albumin. The method presented here is based on the attachment of the new spacer (or bridging arm) 1,6-hexanediamine, by reductive amination, to the ketonic carbonyl group of the only dOclA residue of the coreoligosaccharide portion [lo, 111 of the native 0-specific polysaccharides of Aeromonas hydrophila and Aeromonas s...
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