Glycopeptide analysis of different prion strains

Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of infectious neurodegenerative diseases. They are caused by a conformational change of the cellular prion protein (PrPC) to the misfolded form (PrPSc). TSEs differ from one another in incubation time, clinical signs, and biochemical properties. These differences are a result of multiple conformationally different PrPSc states, called strains. One of the main ways to characterize strains is by immunoblotting, since each strain has a specific pattern where different glycoforms can be distinguished. This is due to the fact the prion protein has two N-glycosylation sites, however, the knowledge regarding exact N-glycan structures on different prion strains has not been researched so far. The phenomenon of prion strains still remains to be explained; therefore, the aim of this study was to, for the first time, differentiate individual N-glycan structures on different glycosylation sites. The first part of the project included optimization of the protocol for isolation of large-scale PrPSc. Isolation of the prion protein was shown to be somewhat challenging. Two different approaches were tested, immunopurification and the density medium approach. After performing all of the experiments, the prion protein was successfully isolated from sheep brain tissue, infected with three different prion strains, by using the density medium approach. To analyze the glycan structures, site-specific analysis was performed. The isolated PrPSc was loaded on SDS-PAGE, and after staining the gel with Coomassie, the bands corresponding to PrPSc glycoforms were cut from it and the protein was digested with trypsin. A mixture of peptides and glycopeptides was obtained. To minimize suppression of glycopeptide ionization, an enrichment procedure was performed. Tryptic digests before and after the enrichment procedure were analyzed using reverse phase liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS). Glycan structures detected on sheep PrPSc were identified as “brain-specific” N-glycans, which are usually rich in sialic acid, fucose residues, and have the presence of bisecting N-acetylglucosamine (GlcNAc). The analysis on different strains showed that there are no major differences in the glycan composition, leading to the conclusion that the N-glycan composition does not contribute to prion strain diversities, but rather that the differences come from the protein’s conformation, therefore confirming the “protein-only” hypothesis.