STRUCTURAL ELUCIDATION OF THE BRUCELLA-MELITENSIS M-ANTIGEN BY HIGH-RESOLUTION NMR AT 500-MHZ
STRUCTURAL ELUCIDATION OF THE BRUCELLA-MELITENSIS M-ANTIGEN BY HIGH-RESOLUTION NMR AT 500-MHZ
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DOI：
10.1021/bi00400a034
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发表时间：
1987-12-29
期刊：
影响因子：
2.9
通讯作者：
PERRY, MB
中科院分区：
文献类型：
--
作者：
BUNDLE, DR;CHERWONOGRODZKY, JW;PERRY, MB
来自种型菌株羊种布氏杆菌16 M的布氏杆菌M抗原已被鉴定为细胞壁脂多糖（LPS）的组分。通过温和的酸水解从该LPS释放的O多糖在血清学试验中显示出M活性，并且显示为4-甲酰胺基-4，6-二脱氧-α-葡聚糖的均聚物。通过天然脂多糖的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断，D-吡喃甘露糖残基排列在寡糖重复单元中。通过NMR方法的O多糖的结构分析是困难的，由于明显的微观不均匀性的重复单元，这实际上是由N-甲酰基部分的旋转异构体的存在下引起的。这个问题通过将多糖化学修饰成其氨基和N-乙酰基衍生物来解决，其500-MHz 1H和125-MHz 13 C NMR谱可以通过应用pH依赖性β-NMR分析独特的结构。位移和二维技术，包括COSY，中继COSY，NOESY实验与异相C/H位移相关光谱。基于这些实验并由甲基化和高碘酸盐氧化数据支持，M多糖的结构被确定为由四个1，2-连接的和一个1，3-连接的4，6-二脱氧-4-甲酰胺基-α-葡聚糖组成的无支链五糖重复单元的线性聚合物。D-吡喃甘露糖残基。M抗原和A抗原的显著结构相似性，已知A抗原是4，6-二脱氧-4-甲酰胺基-α-甲酰胺基的1，2-连接的同多糖。D-吡喃甘露糖基单位，解释了两者的交叉血清学反应以及围绕其抗原决定簇性质的长期困惑。结构和血清学方面的考虑，结合十二烷基硫酸钠带型的布鲁氏菌A LPS表明，其生物合成明显不同的M抗原，这似乎是通过预先形成的寡糖重复单元的调节组装合成。温和的溶原性噬菌体可能是造成这种生物合成和结构变异的原因。
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 conjunction 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. Temperate, lysogenic phage may be responsible for such biosynthetic and structural variations.