Structural elucidation of the major Hex4 lipopolysaccharide glycoform from the lgtC mutant of Haemophilus influenzae strain Eagan |
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Authors: | Masoud Hussein Moxon E Richard Richards James C |
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Affiliation: | Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6. |
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Abstract: | Lipopolysaccharide (LPS) oligosaccharide epitopes are major virulence factors of Haemophilus influenzae. The structure of LPS glycoforms of H. influenzae type b strain Eagan containing a mutation in the gene lgtC is investigated. LgtC is involved in the biosynthesis of globoside trisaccharide [alpha-D-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-D-Glcp-(1-->], an LPS epitope implicated in the virulence of this organism. Glycose and methylation analyses provided information on the composition while electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS (LPS-OH) indicated the major glycoform to contain 4 hexoses attached to the common H. influenzae triheptosyl inner-core unit. The structure of the Hex4 glycoform in LPS-OH and core oligosaccharide samples was determined by NMR. It consists of an l-alpha-D-HepIIIp-(1-->2)-[PEtn-->6]-l-alpha-D-HepIIp-(1-->3)-l-alpha-D-HepIp-(1-->5)-[P-->4]-alpha-D-Kdop-(2--> to which a beta-D-Glcp-(1-->4)-alpha-D-Glcp disaccharide unit is extended from HepII at the C-3 position, while HepI and HepIII are substituted at the C-4 and C-2 positions with beta-D-Glcp and beta-D-Galp, respectively. This structure corresponds to that expressed as a subpopulation in the parent strain. 31P NMR studies permitted the identification of subpopulations of LPS containing Kdo substituted at the C-4 position with monophosphate or pyrophosphoethanolamine (PPEtn). HepIII was found to be substituted with either phosphate at the C-4 position or acetate at the C-3 position, but not both of them together in the same subpopulation. The subpopulations containing phosphate and acetate at HepIII and their location have not previously been reported. |
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Keywords: | DOC-PAGE, deoxycholate-polyacrylamide gel electrophoresis GLC-MS, gas liquid chromatography-mass spectrometry ESI-MS, electrospray ionization-mass spectrometry COSY, correlated spectroscopy TOCSY, total correlated spectroscopy HMQC, heteronuclear multiple quantum coherence NOE, nuclear Overhauser effect NOESY, two-dimensional NOE spectroscopy Kdo, 3-deoxy- smallcaps" >d-manno-octulosonic acid LPS, lipopolysaccharide LPS-OH, O-deacylated LPS OS, oligosaccharide Etn, ethanolamine PEtn, phosphoethanolamine PPEtn, pyrophosphoethanolamine: and PCho: phosphocholine. |
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