title: Sialylated N-glycans in adult rat brain tissue--a widespread distribution of disialylated antennae in complex and hybrid structures.
abstract: This paper extends our earlier work on the analysis of neutral N-glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40% of the total (acidic and neutral) N-glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion-exchange chromatography, normal and reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption/ionisation-mass spectrometry and combined gas chromatography/mass spectrometry. Mono-, di- and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7%, respectively, relative to the total N-glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N-glycans substituted with short chains of polysialic acid. Sialylated bi-, tri- [mainly the (2,4)-branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for 'brain-type' N-glycosylation, these sialylated structures were variously modified by the presence of core alpha1-6-linked and outer-arm alpha1-3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewis(x) and NeuAc alpha2-3 substituted Galbeta1-4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal beta1-3-linked galactose residues was observed. The presence of beta1-3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAc alpha2-3Galbeta1-3(NeuAc alpha2-6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one beta1-3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing beta1-3-linked galactose and 'polylactosamine' N-glycans with one to three terminal beta1-3galactose residues were additional novel features. The N-glycans modified by polysialylation lacked outer-arm fucose and bisecting GlcNAc residues but all contained one or more terminal beta1-3-linked galactose residues. These may be representative, therefore, of the polysialylated N-glycans expressed mainly on neural cell-adhesion molecules and known to be present in adult rat brain. The diversity of presentation of terminal sialylated groups in rat brain implies potential specificity for possible charge or lectin-mediated interactions. The distinguishing sets of sialylated structures described here are indicative of differences in the natural glycosylation processing pathways in different cell types within the central nervous system, a specificity that may be further magnified on the individual glycoproteins.
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, UK.
authors.forename: S
authors.initials: S
authors.lastname: Zamze
authors.forename: D J
authors.initials: DJ
authors.lastname: Harvey
authors.forename: Y J
authors.initials: YJ
authors.lastname: Chen
authors.forename: G R
authors.initials: GR
authors.lastname: Guile
authors.forename: R A
authors.initials: RA
authors.lastname: Dwek
authors.forename: D R
authors.initials: DR
authors.lastname: Wing
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005643:Fucose / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011134:Polysaccharides; D005643:Fucose; D019158:N-Acetylneuraminic Acid; D005690:Galactose