Prion Propagation in Cells Expressing PrP Glycosylation Mutants

Abstract

Infection by prions involves conversion of a host-encoded cell surface protein (PrP C ) to a disease-related isoform (PrP Sc ). PrP C carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP C glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP Sc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP Sc , while PrP C with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP C , were able to form infectious PrP Sc . Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Mammalian prions

“…Their removal from an otherwise well tolerated insert indeed impaired prion replication ( Table 1, compare lanes 1 and 8 or lanes 5 and 9). Accordingly, substitution in wild-type PrP of the F or the T residues of the NFT glycosylation site was not always compatible with prion conversion 36 (see also Table 1). Another salient finding was that prion conversion also accommodated insertion of as much as 8 additional amino acids in the C-terminal region of H2 (Fig.…”

Generating Bona Fide Mammalian Prions with Internal Deletions

“…Rov cells are epithelial RK13 cells that stably express either wild-type or mutant ovine PrP using a tetracycline-inducible system (38,39). They were obtained by transfection and puromycin selection, grown in Opti-MEM medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) and antibiotics, and split at 1:4 after trypsin dissociation once a week.…”

“…For PrP res , otherwise-indicated samples corresponding to the PK-resistant PrP present in either 25 or 50 g of protein of cellular lysate were loaded on the gel. The transfer of proteins, their detection, and their revelation were described previously (39,47).…”

“…Cells were infected with the 127S prion strain using 1% (wt/vol) brain pool homogenates of terminally ill tg338 mice, as previously described (38,39). For infections with sCJDderived T1…”

Mechanisms of Cell Death in the Transmissible Spongiform Encephalopathies

“…Initial reports from studies in cell lines suggested that removing prion glycosylation produced spontaneously misfolded protein (Lehmann & Harris, 1997), however, this may have been a result of overexpression, since more recent studies have shown that blocking glycosylation of endogenously expressed PrP C does not produce this phenotype (Cancellotti, et al, 2005). In some cases, studies have been hampered by the folding and trafficking abnormalities that can occur when PrP C is expressed without glycosylation (Cancellotti, et al, 2005 depending on the specific mutations used to prevent glycosylation (Capellari et al, 2000, Ikeda et al, 2008, Salamat et al, 2011, Wong et al, 2000b. Neuendorf et al selected deglycosylating mutations that retained authentic PrP C cellular trafficking and mice in which these proteins were over-expressed were susceptible to both scrapie and BSE (Neuendorf et al, 2004).…”