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 prion protein (PrP(Sc)) after conversion of 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 the 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 brain and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. Liquid chromatography-electrospray ionization MS (LC-ESI-MS) analysis of human PrP(C )from both biological sources indicated that Gly-229 is the omega site in PrP(C) to which GPI is attached. Gly-229 in human PrP(C) does not correspond to Ser-231, the previously reported omega site of Syrian hamster PrP(C) We found that approximately 41% and 28% of GPI anchors in human PrP(C)s from human and mouse knock-in brains, respectively, have N-acetylneuraminic acid in the side-chain. Using a sialic acid linkage-specific alkylamidation (SALSA) method to discriminate alpha2,3-linkage from alpha2,6-linkage, we found that N-acetylneuraminic acid in PrP(C)'s GPI side-chain is linked to galactose through an alpha2,3-linkage. In summary, we report the GPI-glycan structure of human PrP(C), including the omega-site amino acid for GPI attachment and the sialic acid linkage type.