Sialylated N-glycans in adult rat brain tissue

A widespread distribution of disialylated antennae in complex and hybrid structures

Authors


  • Correspondence to D. R. Wing, Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Rd, Oxford OX1 3QU, UK

  • Fax: +44 1865 275216.

  • E-mail:drw@glycob.ox.ac.uk

  • URL:http://www.bioch.ox.ac.uk

  • Abbreviations. AxGyFzB, description of multiantennary complex glycans, A = number of antennae, G = number of galactose residues, F = number of fucose residues, B = bisecting GlcNAc; GnTIII, β-1,4-N-acetylglucosaminyltransferase III; 2D-HPLC, two-dimensional HPLC; IRBP, interphotoreceptor retinol-binding protein; MALDI-MS, matrix-assisted laser desorption/ionisation mass spectrometry; N-CAM, neural-cell-adhesion molecule; NDV, Newcastle disease virus; NGF, nerve growth factor; NP-HPLC, normal-phase HPLC; NPGU, normal-phase (HPLC) glucose unit; PMAA, partially methylated alditol acetate; RP-HPLC, reverse-phase HPLC; WAX-HPLC, weak-anion-exchange HPLC.

  • Enzymes. Newcastle disease virus neuraminidase ( EC3.2.1.18); Arthrobacter ureafaciens neuraminidase ( EC3.2.1.18); bovine testes β-galactosidase ( EC3.2.1.23); Bacteroides fragilis endo-β-galactosidase ( EC3.2.1.103); Diplococcus pneumoniaeβ-galactosidase ( EC3.2.1.23); almond meal α-fucosidase ( EC3.2.1.111); Diplococcus pneumoniaeβ-hexosaminidase (EC 3.3.1.30); Jack bean β-hexosaminidase ( EC3.2.1.30).

  • Core fucosylated biantennary structures (compounds 11 and 12) were recovered mainly in P4 fractions CIII and EIII, and represent the two main peaks shown in Fig. 3.

  • Due to overlap in the P4 separation, small amounts were also recovered in fractions CII and EII (Table 3). Polylactosamine' is used in this paper to denote structures with antennae carrying one or more additional lactosamine repeats

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 α1−6-linked and outer-arm α1−3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewisx and NeuAcα2−3 substituted Galβ1−4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal β1−3-linked galactose residues was observed. The presence of β1−3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAcα2−3Galβ1−3(NeuAcα2−6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one β1−3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing β1−3-linked galactose and ’polylactosamine' N-glycans with one to three terminal β1−3galactose 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 β1−3-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.

Ancillary