Structural elucidation of the Brucella melitensis M antigen by high-resolution NMR at 500 MHz.
The Brucella M antigen from the species type strain Brucella melitensis 16M has been identified as a component of the cell wall lipopolysaccharide (LPS). O polysaccharide liberated from this LPS by mild acid hydrolysis exhibited M activity in serological tests and was shown to be a homopolymer of 4-formamido-4,6-dideoxy-alpha-D-mannopyranosyl residues arranged in an oligosaccharide repeating unit as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native lipopolysaccharide. Structural analysis of the O polysaccharide by NMR methods was difficult due to apparent microheterogeneity of the repeating unit, which was in fact caused by the presence of rotational isomers of the N-formyl moiety. This problem was resolved by chemical modification of the polysaccharide to its amino and N-acetyl derivatives, the 500-MHz 1H and 125-MHz 13C NMR spectra of which could be analyzed in terms of a unique structure through application of pH-dependent beta-shifts and two-dimensional techniques that included COSY, relayed COSY, and NOESY experiments together with heteronuclear C/H shift correlation spectroscopy. On the basis of these experiments and supported by methylation and periodate oxidation data, the structure of the M polysaccharide was determined as a linear polymer of unbranched pentasaccharide repeating units consisting of four 1,2-linked and one 1,3-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues. The marked structural similarity of the M antigen and the A antigen, which is known to be a 1,2-linked homopolysaccharide of 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units, accounts for cross-serological reactions of the two and the long-standing confusion surrounding the nature of their antigenic determinants. Structural and serological considerations in conjuction with the sodium dodecyl sulfate banding pattern of Brucella A LPS suggest that its biosynthesis differs appreciably from that of the M antigen, which appears to be synthesized by regulated assembly of preformed oligosaccharide repeating units. Temperature, lysogenic phage may be responsible for such biosynthetic and structural variations.
Division of Biological Science, National Research Council of Canada, Ottawa.
D R
DR
Bundle
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Cherwonogrodzky
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MB
Perry
D000942:Antigens, Bacterial / Q000302:isolation & purification*; D002002:Brucella / Q000276:immunology*; D002236:Carbohydrate Conformation; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D008070:Lipopolysaccharides / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009844:Oligosaccharides / Q000032:analysis; D013045:Species Specificity
D000942:Antigens, Bacterial; D008070:Lipopolysaccharides; D009844:Oligosaccharides; C043431:M antigen, Brucella melitensis