Masoud H, Moxon E R, Martin A, Krajcarski D, Richards J C
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Biochemistry. 1997 Feb 25;36(8):2091-103. doi: 10.1021/bi961989y.
Lipopolysaccharide (LPS) is a major virulence determinant of Haemophilus influenzae. The organism is capable of expressing a heterogeneous population of LPS which exhibits extensive antigenic diversity among multiple oligosaccharide (OS) epitopes. Structural elucidation of variable and conserved OS epitopes of H. influenzae serotype b strain Eagan was determined by the application of high-field NMR techniques and MS-based methods on oligosaccharides obtained from LPS samples by a deacylation strategy. LPS extracted by the hot aqueous phenol method gave complex electrophoretic patterns consisting of at least six low-molecular mass bands. Electrospray ionization-mass spectrometry of O-deacylated LPS revealed a series of related structures differing in the number of hexose residues as well as subpopulations of glycoforms containing additional phosphoethanolamine (PEA) groups. It was demonstrated that the LPS contains a conserved PEA-substituted, heptose-containing trisaccharide inner core moiety attached via a KDO 4-phosphate unit to a lipid A component. Tandem MS experiments unambiguously established the presence of a KDO 4-pyrophosphoethanolamine unit in the subpopulation of LPS containing additional PEA groups. The occurrence of LPS containing this structural feature was found to be dependant on the isolation procedure used. Each heptose of the common inner core element L-alpha-D-Hepp(1-->2)-L-alpha-D-Hepp(1-->3)-L-alpha-D-Hep p(1-->5)-alpha-KDO is substituted by a hexose residue with further chain elongation from the central unit. The structures of the major glycoforms containing four (three Glcs and one Gal), five (three Glcs and two Gals), and six (three Glcs and three Gals) hexoses were determined in detail. The Hex6 glycoform contains the terminal structure, alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glc, providing, for the first time, definitive structural evidence for the expression of the Pk-blood group antigen in H. influenzae LPS. Moreover, an analogue of the Hex4 glycoform was identified in which the third heptose residue carries phosphate at 0-4.
脂多糖（LPS）是流感嗜血杆菌的主要毒力决定因素。该生物体能够表达多种LPS，这些LPS在多个寡糖（OS）表位之间表现出广泛的抗原多样性。通过将高场核磁共振技术和基于质谱的方法应用于通过脱酰策略从LPS样品中获得的寡糖，确定了b型流感嗜血杆菌Eagan菌株可变和保守的OS表位的结构。通过热酚水法提取的LPS给出了由至少六个低分子量条带组成的复杂电泳图谱。O-脱酰化LPS的电喷雾电离质谱显示了一系列相关结构，这些结构在己糖残基数量以及含有额外磷酸乙醇胺（PEA）基团的糖型亚群方面存在差异。结果表明，LPS包含一个保守的、含有PEA取代基的、含庚糖的三糖内核部分，该部分通过一个KDO 4-磷酸单元连接到脂质A成分上。串联质谱实验明确确定了在含有额外PEA基团的LPS亚群中存在KDO 4-焦磷酸乙醇胺单元。发现含有这种结构特征的LPS的出现取决于所使用的分离程序。常见内核元件L-α-D-Hepp(1→2)-L-α-D-Hepp(1→3)-L-α-D-Hep p(1→5)-α-KDO的每个庚糖都被一个己糖残基取代，并从中心单元进一步进行链延长。详细确定了含有四个（三个Glc和一个Gal）、五个（三个Glc和两个Gal）和六个（三个Glc和三个Gal）己糖的主要糖型的结构。Hex6糖型包含末端结构α-D-Galp(1→4)-β-D-Galp(1→4)-β-D-Glc，首次为流感嗜血杆菌LPS中Pk血型抗原的表达提供了明确的结构证据。此外，还鉴定出一种Hex4糖型类似物，其中第三个庚糖残基在0-4位携带磷酸基团。
