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.
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Division of Biological Science, National Research Council of Canada, Ottawa.
Cherwonogrodzky
J W
JW
Perry
M B
MB
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