Asparagine-linked glycosylation of the scrapie and cellular 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 sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of Mr26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPSc yielded equimolar amounts of two proteins of Mr26K 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 PrPSc, 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 alpha-methyl-mannoside, 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 PrPC and PrPSc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.
Haraguchi
T
T
Department of Neurology, University of California, San Francisco 94143.
Fisher
S
S
Olofsson
S
S
Endo
T
T
Groth
D
D
Tarentino
A
A
Borchelt
D R
DR
Teplow
D
D
Hood
L
L
Burlingame
A
A
D000818:Animals; D001216:Asparagine; D001921:Brain / Q000378:metabolism / Q000382:microbiology; D003001:Cloning, Molecular; D006224:Cricetinae; D004279:DNA, Viral / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D006031:Glycosylation; D008861:Microsomes / Q000378:metabolism / Q000382:microbiology; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification
D004279:DNA, Viral; D011328:Prions; D014764:Viral Proteins; D001216:Asparagine; D006026:Glycoside Hydrolases