A mutant prion protein displays an aberrant membrane association when expressed in cultured cells.
Inherited forms of prion disease have been linked to mutations in the gene encoding PrP, a neuronal and glial protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. One familial form of Creutzfeldt-Jakob disease is associated with a mutant PrP containing six additional octapeptide repeats. We report here our analysis of cultured Chinese hamster ovary cells expressing a murine homologue of this mutant PrP. We find that, like wild-type PrP, the mutant protein is glycosylated, GPI-anchored, and expressed on the cell surface. Surprisingly, however, cleavage of the GPI anchor using phosphatidylinositol-specific phospholipase C fails to release the mutant PrP from the surface of intact cells, suggesting that it has an additional mode of membrane attachment. The phospholipase-treated protein is hydrophobic, since it partitions into the detergent phase of Triton X-114 lysates; and it is tightly membrane-associated, since it is not extractable in carbonate buffer at pH 11.5. Whether membrane attachment of the mutant PrP involves integration of the polypeptide into the lipid bilayer, self-association, or binding to other membrane proteins remains to be determined. Our results suggest that alterations in the membrane association of PrP may be an important feature of prion diseases.
Lehmann
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Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Harris
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DA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D007562:Creutzfeldt-Jakob Syndrome / Q000235:genetics*; D006224:Cricetinae; D017931:DNA Primers; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011328:Prions / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014162:Transfection
D000906:Antibodies; D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D008051:Lipid Bilayers; D010446:Peptide Fragments; D011328:Prions; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase