Glycosylation differences between the normal and pathogenic prion protein isoforms

Abstract

Prion protein consists of an ensemble of glycosylated variants or glycoforms. The enzymes that direct oligosaccharide processing, and hence control the glycan profile for any given glycoprotein, are often exquisitely sensitive to other events taking place within the cell in which the glycoprotein is expressed. Alterations in the populations of sugars attached to proteins can reflect changes caused, for example, by developmental processes or by disease. Here we report that normal (PrP C ) and pathogenic (PrP Sc ) prion proteins (PrP) from Syrian hamsters contain the same set of at least 52 bi-, tri-, and tetraantennary N-linked oligosaccharides, although the relative proportions of individual glycans differ. This conservation of structure suggests that the conversion of PrP C into PrP Sc is not confined to a subset of PrPs that contain specific sugars. Compared with PrP C , PrP Sc contains decreased levels of glycans with bisecting GlcNAc residues and increased levels of tri-and tetraantennary sugars. This change is consistent with a decrease in the activity of N-acetylglucosaminyltransferase III (GnTIII) toward PrP C in cells where PrP Sc is formed and argues that, in at least some cells forming PrP Sc , the glycosylation machinery has been perturbed. The reduction in GnTIII activity is intriguing both with respect to the pathogenesis of the prion disease and the replication pathway for prions. N-acetylglucosaminyltransferase III scrapie

Misfolding of the prion protein: linking biophysical and biological approaches

“…When PrP is purified from natural sources, it was in some instance observed as a mixture of nonglycosylated, monoglycosylated, and biglycosylated species. Furthermore, the number of glycosylated variants (glycoforms) was estimated up to 50 different bi-, tri-, and tetra-antennary Nlinked oligosaccharides [103]. PrP inserts into the cellular plasma membrane through the GPI anchor attached to the C-terminus [116].…”

Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures

“…Post translational modifications include two Asn-linked glycosylation sites [14,15], a GPI anchor [21], a single disulphide bond [22], and partial proline hydroxylation near the N-terminus [23]. The glycosylation of the prion protein has been suggested to play a fundamental role in the transmissible spongiform encephalopathies (TSEs) [24 -26].…”

“…Crucially occupancy of the 2 N-linked glycosylation sites varies depending on the strain of TSE disease [27] and blockade of glycosylation of PrP can promote scrapie-like pathogenesis in cultured cells [24]. Differences in the structures of individual carbohydrates have also been observed between the normal, PrP C , and diseased PrP Sc isoforms of the protein [15]. Although there were no gross differences in carbohydrate structures, PrP Sc had fewer saccharides containing bisecting GlcNAc moieties, consistent with down regulation of the saccharide transferase enzyme GnT III during disease pathogenesis.…”

“…The method also allows carbohydrates to be characterized site specifically, even if a mixture of glycosylation sites is present in the same mixture. Our strategy has been developed using the standard glycopeptide fetuin, and has then been applied to the analysis of the N-linked glycans of hamster PrP Sc , the scrapie-associated prion protein, which carries up to 60 different glycoforms attached to two different sites [14,15].…”

“…Characterization of the carbohydrates attached to PrP Sc is therefore essential to understand fully the role that this protein plays in pathogenesis of these fatal, neurodegenerative diseases. Previous work in this area has focused on the characterization of carbohydrates attached to murine PrP Sc from a single TSE strain [14] or on the differences in carbohydrate structures between PrP Sc and PrP C , the normal, cellular isoform [15]. Our novel methodology has been developed to provide a rapid method of mapping carbohydrate structure profiles without lengthy labeling or digestion steps and should enable the identification of any structures specific to individual TSE strains.…”