Abstract
Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrPC isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. LC-electrospray ionization-MS analysis of human PrPC from both biological sources indicated that Gly229 is the ω site in PrPC to which GPI is attached. Gly229 in human PrPC does not correspond to Ser231, the previously reported ω site of Syrian hamster PrPC We found that ∼41% and 28% of GPI anchors in human PrPCs from human and knock-in mouse brains, respectively, have N-acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that N-acetylneuraminic acid in PrPC's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrPC, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.
Highlights
Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC)
Prion diseases are caused by accumulation of the scrapie form of the prion protein (PrPSc), which is converted from the normal cellular prion protein (PrPC) [16]
prion protein (Prnp) knockout mice are resistant to prion disease even after inoculation with PrPSc [17, 18], indicating that the presence of PrPC is critical for the pathogenesis of prion diseases and that prevention of prion conversion can be a therapeutic strategy, the mechanisms of conversion are still unclear
Summary
Lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). We affinity-purified PrPC from knock-in (KI) mouse brains in which Prnp was replaced by human PRNP and from human brains and determined their GPI glycan structure by LC– electrospray ionization (ESI)–MS and the linkage type of Sia using the sialic acid linkage-specific alkylamidation (SALSA) method. To determine the -site amino acid and GPI glycan structures of human PrPC, we first performed LC-ESI-MS analysis.
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