title: Structural elucidation of the major Hex4 lipopolysaccharide glycoform from the lgtC mutant of Haemophilus influenzae strain Eagan.
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.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6.
authors.forename: Hussein
authors.initials: H
authors.lastname: Masoud
authors.forename: E Richard
authors.initials: ER
authors.lastname: Moxon
authors.forename: James C
authors.initials: JC
authors.lastname: Richards
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002247:Carbon Isotopes / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D006193:Haemophilus influenzae / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D008070:Lipopolysaccharides; C052253:deacylated lipopolysaccharides; D003903:Deuterium; D005700:Galactosyltransferases