Doppel Is an N-Glycosylated, Glycosylphosphatidylinositol-anchored Protein

Abstract

The <i>Prnd</i> gene encodes a homolog of the cellular prion protein (PrP^C) called doppel (Dpl). Up-regulation of <i>Prnd</i> mRNA in two distinct lines of PrP gene ablated (<i>Prnp</i> ^0/0) mice, designated<i>Rcm0</i> and <i>Ngsk,</i> is associated with death of Purkinje cells. Using recombinant Dpl expressed in <i>Escherichia coli</i> and mouse neuroblastoma cells we demonstrate that wild type (wt) Dpl, like PrP^C, adopts a predominantly α-helical conformation, forms intramolecular disulfide bonds, has two<i>N</i>-linked oligosaccharides, and is presented on the cell surface via a glycosylphosphatidylinositol anchor. Dpl protein was detected in testis of wt mice. Using Triton X-114 phase partitioning to enrich for glycosylphosphatidylinositol-anchored proteins, Dpl was detected in brain samples from <i>Rcm0 Prnp</i> ^0/0mice but was absent in equivalent samples from wt mice and <i>ZrchI Prnp</i> ^0/0 mice, indicating that ectopic expression of this protein may cause cerebellar pathology in <i>Rcm0</i> mice. Biochemical and structural similarities between PrP^C and Dpl documented here parallel the observation that ataxic <i>Ngsk Prnp</i> ^0/0 mice can be rescued by overexpression of wild-type PrP transgenes, and suggest that cell surface PrP^C can antagonize the toxic effect of Dpl expressed in the central nervous system.