No abstract

Analysis of commercial samples of chicken ovalbumin by reversed-phase high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) showed the presence of several other co-purifying glycoproteins. These were isolated, subjected to tryptic digestion, and two of them were identified as ovomucoid and chicken riboflavin binding-protein following database matching of the peptide masses obtained by MALDI. The N-linked glycans were released from the glycoproteins and their structures were examined by MALDI-MS in combination with exoglycosidase digestion. Ovalbumin was found to be glycosylated mainly with high-mannose and hybrid structures, consistent with profiles obtained on the intact glycoprotein by electrospray. The other glycoproteins contained mainly larger, complex glycans with up to five antennae, many of which had earlier been associated with ovalbumin.

Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potentially of crucial importance in determining many cell interactions. The complexity of cellular organisation of the brain and suggested involvement of N-glycosylation in neural development, make this an ideal system to study the potential role of glycosylation in tissue development, maintenance and function. Neural tissues are known to contain some highly unusual glycan structures but the structures expressed in neural tissue have not as yet been studied systematically. As a first initiative to assess the type of N-glycosylation occurring in neural tissue, we have characterised all of the major neutral N-linked oligosaccharides expressed in adult rat using a combination of matrix-assisted laser-desorption ionisation mass spectrometry, exoglycosidase sequencing combined with normal-phase HPLC, and two-dimensional HPLC mapping. Oligomannosidic glycans, Man (9Ϫ5)GlcNAc2, constituted approximately 15% of the total brain N-glycan pool. The other neutral N-glycan components consisted of a series of diantennary structures (6.5%), (2,6)-branched triantennary glycans (1 %) and hybrid structures (3%). Both the complex and hybrid N-glycans were characterised by the presence of outer-arm A(1,3)-fucosylation (forming the Lewis x determinant), A(1,6)-core fucosylation and a bisecting GlcNAc residue. Some of these are unusual or novel structures not having been reported elsewhere. A large proportion of the diantennary N-glycans either lacked Gal residues entirely or were unsubstituted on one Man residue of the trimannosyl core, notably the Man A(1,3)-arm. This isomeric form is indicative of the action of a novel β-hexosaminidase activity and suggests a modification in the classical biosynthetic pathway for N-linked oligosaccharides. Furthermore, expression of large amounts of oligomannosidic glycans is not usually associated with tissue glycoproteins and suggests a possible involvement of these structures in neural cell interactions.Keywords : neutral N-glycan; central nervous system; two-dimensional HPLC ; Lewis x determinant.Oligosaccharides expressed by glycoconjugates at the cell significant roles in tissue development and maintenance, then surface and in the extracellular matrix are potentially of crucial the brain is the tissue, par excellence, for such studies. For eximportance in determining cell interactions (for reviews on the ample, abrogation of the synthesis of complex and hybrid Nbiological roles of oligosaccharides see [1Ϫ4]). One way this linked oligosaccharides in mice by disruption of N-acetylglucosmay be mediated is via sugarϪlectin recognition requiring an aminyltransferase I activity has its most marked effect in neural array of specific oligosaccharide structures and presentation. No tissue development [5]. other tissue displays the complexity of cellular organisation Much of the detailed structural work on N-linked oligosacshown by the central nervous system. If oligosaccharides play charides has been obtained wit...

The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The large size of the N-linked sugars, together with their dynamic properties, enables them to shield two orthogonal faces of the protein almost completely. Thus, the sugars can protect large regions of the protein surface from proteases and from nonspecific protein-protein interactions. Immunoprecipitation of prion protein with calnexin suggests that in the ER the oligosaccharides may provide a route for protein folding via the calnexin pathway. Major questions relate to the relevance of the glycoform distribution (as defined by glycan site occupancy) to strain type and disease transmission. Glycan analysis has shown that prion protein contains at least 52 different sugars, that these consist of a subset of brain sugars, and that there is site specific glycan processing. PrP(Sc) from the brains of Syrian hamsters contains the same set of glycans as PrP(C), but a higher proportion of tri- and tetra-antennary sugars. This may be attributed to a decrease in the activity of GnTIII. The GPI anchor, which is modified with sialic acid, may allow the prion protein to be mobile in the lipid bilayer. Potentially, this provides a possible means for translocating the prions from one cell to another.