Intracellular accumulation of the cellular prion protein after mutagenesis of its Asn-linked glycosylation sites.
The cellular isoform of the prion protein (PrPC) is a sialoglycoprotein bound almost exclusively on the external surface of the plasma membrane by a glycosyl phosphatidylinositol anchor. The deduced amino acid sequence of Syrian hamster PrPC identifies two potential sites for the addition of Asn-linked carbohydrates at amino acids 181-183 (Asn-Ile-Thr) and 197-199 (Asn-Phe-Thr). We have altered these sites by replacing the threonine residues with alanine and expressed the mutant proteins transiently in CV1 cells utilizing a mutagenesis vector with the T7 promoter located upstream from the PrP gene. The T7 RNA polymerase was supplied by infection with a recombinant vaccinia virus. The 3 mutant proteins (PrPAla183, PrPAla199 and PrPAla183/199) have a reduced relative molecular weight compared to wild-type (wt) PrP. Deglycosylation as well as synthesis in the presence of tunicamycin reduced the relative molecular weight of all the PrP species to that of the double mutant PrPAla183/199. Our results indicate that both single-site mutant prion proteins are glycosylated at non-mutated sites and they suggest that both potential sites for Asn-linked glycosylation are utilized in wt PrPC. Immunofluorescence studies demonstrate that while wt PrPC localizes to the cell surface, all the mutant PrP molecules accumulate intracellularly. The site of accumulation of PrPAla183 is probably prior to the mid-Golgi stack since this protein does not acquire resistance to endoglycosidase H. Whether the intracellular locations of the mutant PrPC species are the same as those identified for the scrapie isoform of the prion protein (PrPSc) remains to be established.
Rogers
M
M
Department of Neurology, University of California, San Francisco 94143.
Taraboulos
A
A
Scott
M
M
Groth
D
D
Prusiner
S B
SB
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D015151:Immunoblotting; D007700:Kinetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D018620:PrPSc Proteins; D011328:Prions / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D018620:PrPSc Proteins; D011328:Prions; D011994:Recombinant Proteins; D001216:Asparagine