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. 2020 May 29;295(22):7789-7798.
doi: 10.1074/jbc.RA120.013444. Epub 2020 Apr 22.

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

Atsushi Kobayashi  1 , Tetsuya Hirata  2   3 , Takashi Nishikaze  4 , Akinori Ninomiya  2 , Yuta Maki  5 , Yoko Takada  3 , Tetsuyuki Kitamoto  6 , Taroh Kinoshita  7   3
Affiliations

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

Atsushi Kobayashi et al. J Biol Chem. .

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.

Keywords: Creutzfeldt–Jakob disease; MS; glycosylation; glycosylphosphatidylinositol (GPI); neurodegeneration; prion; scrapie; sialic acid.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.
Isolation of human PrPC from KI mouse brains. A, schematic of GPI-AP composition. Core and side-chain structures of mammalian GPI-APs are shown. The cleavage site of PI-PLC is indicated by an arrow. Glycan symbols follow the symbol nomenclature for glycans (44). B, PI-PLC treatment of the mouse medulla oblongata. Medulla oblongata homogenates obtained from mice expressing human PrPC were treated with PI-PLC, and then free GPI-GalNAc was detected by Western blotting. GAPDH was used as a loading control. C, isolation of human PrPC from KI mouse brains. The lipid portion of the GPI anchor of human PrPC was removed by PI-PLC, followed by immunoprecipitation with an anti-prion antibody. N-glycans on isolated PrPC were removed by treatment with PNG-F. Bands corresponding to PNG-F and human PrPC are indicated by arrows.
Figure 2.
Figure 2.
Identification of the ω site in human PrPC from KI mouse brains. A, peptide coverage of purified human PrPC from KI mouse brains. Trypsinized peptides were analyzed by LC-ESI-MS, and the obtained data were analyzed using the MASCOT database. B, alignment of PrPC sequences. Sequence alignment was performed using ClustalW and BoxShade. Residues conserved among species are shaded in gray or black. Red boxes indicate the ω sites determined in this and previous studies (28).
Figure 3.
Figure 3.
Determination of GPI glycan structures in human PrPC from KI mice by LC-ESI-MS/MS analysis. A, left panel, the MS/MS spectra of the m/z 788.722+ ion shown in Fig. S1B and the identities of fragment ions. Right panel, the corresponding structure (MW 1576.2) of a C-terminal peptide with GPI containing a HexNAc-Hex side chain. B, left panel, the MS/MS spectra of the m/z 870.252+ ion. Right panel, the corresponding structure (MW 1738.3) of a C-terminal peptide with GPI containing Hex and HexNAc-Hex side chains. C, left panel, the MS/MS spectra of the m/z 934.32+ ion. Right panel, the corresponding structure (MW 1867.4) of a C-terminal peptide with GPI containing a HexNAc-Hex-Neu5Ac side chain. In A–C, all annotated fragment ions are monovalent cations. D, percentages of GPI glycan species in KI mouse brains. Core only, Core + Hex (fourth Man), and Core + HexNAc (GalNAc) were not detected.
Figure 4.
Figure 4.
Linkage analysis of Sia in GPI anchors using the SALSA method. A, structure of a peptide with the GPI glycan displayed in B and C. Numbers indicate m/z values. Boxes show the neutral loss after MS/MS analysis. B, MS spectra of peptides containing the GPI glycan shown in A. A 304-Da difference indicates the presence of α2,3-linked Neu5Ac. C, the MS/MS spectrum of a C-terminal peptide with GPI containing Hex and HexNAc-Hex-Neu5Ac. A 669-Da loss indicates loss of α2,3-linked Neu5Ac, Hex (Gal), and HexNAc (GalNAc).
Figure 5.
Figure 5.
Determination of GPI glycan structures of human PrPC from human brain by LC-ESI-MS/MS analysis. A, left panel, the MS/MS spectra of the m/z 869.752+ ion. All annotated fragment ions are monovalent cations. Right panel, the corresponding structure (MW 1738.3) of a C-terminal peptide with GPI containing Hex and HexNAc-Hex side chains. B, left panel, the MS/MS spectra of the m/z 1015.802+ ion. All annotated fragment ions are monovalent cations. Right panel, the corresponding structure (MW 2029.6) of a C-terminal peptide with GPI containing Hex and HexNAc-Hex-Neu5Ac side chains. C, percentages of GPI glycan species in the human brain. Core only, Core + Hex (fourth Man), and Core + HexNAc (GalNAc) were not detected.
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References

    1. Kinoshita T. (2020) Biosynthesis and biology of mammalian GPI-anchored proteins. Open Biol. 10, 190290 10.1098/rsob.190290 - DOI - PMC - PubMed
    1. Kinoshita T., and Fujita M. (2016) Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling. J. Lipid Res. 57, 6–24 10.1194/jlr.R063313 - DOI - PMC - PubMed
    1. Ferguson M. A. J., Hart G. W., and Kinoshita T. (2015) in Essentials of Glycobiology (Varki A., Cummings R. D., Esko J. D., Stanley P., Hart G. W., Aebi M., Darvill A. G., Kinoshita T., Packer N. H., Prestegard J. H., Schnaar R. L., and Seeberger P. H., eds.), 3rd Ed., pp. 137–150, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY - PubMed
    1. Tanaka S., Maeda Y., Tashima Y., and Kinoshita T. (2004) Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. J. Biol. Chem. 279, 14256–14263 10.1074/jbc.M313755200 - DOI - PubMed
    1. Fujita M., Maeda Y., Ra M., Yamaguchi Y., Taguchi R., and Kinoshita T. (2009) GPI glycan remodeling by PGAP5 regulates transport of GPI-anchored proteins from the ER to the Golgi. Cell 139, 352–365 10.1016/j.cell.2009.08.040 - DOI - PubMed

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