α2,3 linkage of sialic acid to a GPI anchor and an unpredicted GPI attachment site in human prion protein

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 (PrP(C)). The glycosylphosphatidylinositol (GPI) anchor of PrP(C) is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrP(C) conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrP(C). 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 PrP(C) 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 PrP(C) from both biological sources indicated that Gly(229) is the ? site in PrP(C) to which GPI is attached. Gly(229) in human PrP(C) does not correspond to Ser(231), the previously reported ? site of Syrian hamster PrP(C). We found that ?41% and 28% of GPI anchors in human PrP(C)s 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 ?2,3 linkage from ?2,6 linkage, we found that N-acetylneuraminic acid in PrP(C)'s GPI side chain is linked to galactose through an ?2,3 linkage. In summary, we report the GPI glycan structure of human PrP(C), including the ?-site amino acid for GPI attachment and the sialic acid linkage type.