Prion proteins carrying pathogenic mutations are resistant to phospholipase cleavage of their glycolipid anchors.

Familial prion diseases are linked to mutations in the gene encoding PrP, a protein of unknown function that is attached to the plasma membrane of neurons and several other cell types by a phosphatidylinositol-containing, glycolipid anchor. We have previously found that PrP molecules carrying disease-associated mutations display several biochemical attributes of PrPSc, the pathogenic isoform of PrP, when expressed in cultured Chinese hamster ovary cells. One of the distinctive properties of these mutant PrPs is their abnormal association with cell membranes, as revealed by their retention on the cell surface after treatment with a bacterial phospholipase that normally cleaves the glycolipid anchor. We demonstrate here that mutant PrP molecules, either expressed on intact cells or solubilized in nondenaturing detergents, are partially resistant to phospholipase cleavage. The anchor becomes fully susceptible to the enzyme when the proteins are denatured in SDS. These results suggest that the mutant PrP conformation, state of aggregation, or association with other molecules renders the glycolipid anchor physically inaccessible to cleavage. This conclusion stands in contrast to our previous suggestion that mutant PrP molecules are poorly released from the cell surface because they possess a secondary mechanism of membrane attachment in addition to the glycolipid anchor. Since PrPSc from scrapie-infected brain and cultured cells is also inefficiently released from membranes by phospholipase, resistance to this enzyme may be a molecular marker of the scrapie state.