Lysine hydroxylation and O-glycosylation in the globular, C-terminal region of mammalian-expressed, recombinant PrP
Conversion of PrPc, the prion protein, to a conformationally altered isoform, PrPSc, is the major pathogenic event in the transmissible spongiform encephalopathies, a family of neurodegenerative diseases including bovine spongiform encephalopathy, Creutzfeldt-Jakob disease and scrapie. Known post-translational modifications to the protein include disulfide bridge formation, addition of a membrane anchor and N-linked glycosylation. We have previously identified the pro-collagen-like hydroxylation of proline 44 in a murine, recombinant prion protein expressed in Chinese hamster ovary cells and herein report the identification of a second pro-collagen-like modification in this protein. In a proportion of the molecules, Lys193, within the C-terminal, folded domain of the protein, is specifically modified to hydroxylysine with subsequent addition of two hexose units, assumed to be the collagen-like disaccharide modifier Gal-Glu. Proof of the existence of these modifications has been obtained by means of tandem mass spectrometry and Edman degradation. Molecular dynamics simulations show that these modifications lead to a pronounced stabilising effect on the beta(2)-alpha(2) loop, a region of PrP crucial for the disease-associated conversion. If present in vivo, these modifications may have important implications in PrP structure, interactions with ligands or may modulate PrP aggregation.
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Publication
Contributors
Ritchie M A, Hunt L G, Gill A C
Year
2013
Journal
International Journal of Mass Spectrometry
Volume
345
Pages
132-141
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