Asparagine-Linked Glycosylation df the Scrapie and Cell'ular Prion Proteins'

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 (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfatepolyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of i%f, 26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPS’ yielded equimolar amounts of two proteins of M, 26K and 28K. The significance of this altered stoichiometry between the 26- and 2%kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 2’7-30, which is produced by limited proteolysis of PrPSc, exhibited a reduced number of charge isomer!3 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 2’7-30 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 iV-acetylglucosamine or ar-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-acetylglucosamine. Whether differences in Asn-linked oligosaccharide structure between PrPC and PrP” exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.