Asparagine-linked glycosylation of the scrapie and cellular prion proteins

Abstract

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrP Sc) and cellular (PrP C) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrP C yielded two proteins of M r 26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrP Sc yielded equimolar amounts of two proteins of M r 26K and 28K. The significance of this altered stoichiometry between the 26- and 28-kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 27–30, which is produced by limited proteolysis of PrP Sc, exhibited a reduced number of charge isomers after PNGase F digestion. The molecular weight of PrP 27–30 was reduced from 27K–30K by PNGase F digestion to 20K–22K while anhydrous hydrogen fluoride or trifluoromethanesulfonic acid treatment reduced the molecular weight to 19K–21K and 20K–22K, respectively. Denatured PrP 27–30 was radioiodinated and then assessed for its binding to lectin columns. PrP 27–30 was bound to wheat germ agglutinin (WGA) or lentil lectins and eluted with N-acetylglucosamine or α-melthylmannoside, respectively. Digestion of PrP 27–30 with sialidase prevented its binding to WGA but enhanced its binding to Ricinus communis lectin. These findings argue that PrP 27–30 probably possesses Asn-linked, complex oligosaccharides with terminal sialic acids, penultimate galactoses, and fucose residues attached to the innermost N-acetyl-glucosamine. Whether differences in Asn-linked oligosaccharide structure between PrP C and PrP Sc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.