Structural studies of the lipooligosaccharides from Haemophilus influenzae type b strain A2.
The outer membrane lipooligosaccharides (LOS) from Haemophilus influenzae type b strain A2 are a heterogeneous mixture of glycolipids containing a conserved Lipid A structure and a variable oligosaccharide moiety. After O-deacylation by treatment with anhydrous hydrazine, the O-deacylated LOS mixture was analyzed by electrospray mass spectrometry and shown to contain 11 components, ranging in M(r) from 2277.8 to 3416.4. The majority of these structures contained a variable number of hexoses, three L-glycero-D-manno-heptoses, and one 3-deoxy-D-manno-octulosonic acid (KDO) residue attached to a diphosphorylated O-deacylated Lipid A moiety. Additional phosphate and phosphoethanolamine (PEA) groups were also present on the oligosaccharide structures. Two minor high molecular weight components were also observed that contained N-acetylhexosamine and sialic acid. Neuraminidase treatment of the O-deacylated LOS mixture resulted in the loss of sialic acid from these latter two species. After mild acid hydrolysis and separation by size-exclusion chromatography, liquid secondary ion mass spectrometry identified six major and four minor oligosaccharides, ranging in M(r) from 1243.4 to 2215.8. These released oligosaccharides contained a common heptose trisaccharide core structure with anhydro-KDO at the reducing terminus, which arises as an artifact of the hydrolysis procedure by beta-elimination of a phosphate group from the 4-position of KDO. Selected oligosaccharide fractions were subjected to composition and methylation analyses and sequenced by tandem mass spectrometry. Taken together, these data defined the major O-deacylated LOS as follows: [formula: see text] Higher molecular weight structures in the mixture contained galactose, N-acetylglucosamine, and sialic acid as additional branch sugars, suggesting that H. influenzae A2 is capable of forming a sialylated lactosamine structure.
Phillips
N J
NJ
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Apicella
M A
MA
Griffiss
J M
JM
Gibson
B W
BW
D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002648:Child; D006801:Humans; D009980:Influenza A virus / Q000737:chemistry* / Q000302:isolation & purification; D007251:Influenza, Human / Q000134:cerebrospinal fluid / Q000382:microbiology; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D013058:Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data
D002241:Carbohydrates; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides