Cofactor and glycosylation preferences for in vitro prion conversion are predominantly determined by strain conformation

Abstract

Prion diseases are caused by the misfolding of a host-encoded glycoprotein, PrP C , into a pathogenic conformer, PrP Sc. Infectious prions can exist as different strains, composed of unique conformations of PrP Sc that generate strain-specific biological traits, including distinctive patterns of PrP Sc accumulation throughout the brain. Prion strains from different animal species display different cofactor and PrP C glycoform preferences to propagate efficiently in vitro, but it is unknown whether these molecular preferences are specified by the amino acid sequence of PrP C substrate or by the conformation of PrP Sc seed. To distinguish between these two possibilities, we used bank vole PrP C to propagate both hamster or mouse prions (which have distinct cofactor and glycosylation preferences) with a single, common substrate. We performed reconstituted sPMCA reactions using either (1) phospholipid or RNA cofactor molecules, or (2) di-or un-glycosylated bank vole PrP C substrate. We found that prion strains from either species are capable of propagating efficiently using bank vole PrP C substrates when reactions contained the same PrP C glycoform or cofactor molecule preferred by the PrP Sc seed in its host species. Thus, we conclude that it is the conformation of the input PrP Sc seed, not the amino acid sequence of the PrP C substrate, that primarily determines species-specific cofactor and glycosylation preferences. These results support the hypothesis that strain-specific patterns of prion neurotropism are generated by selection of differentially distributed cofactors molecules and/or PrP C glycoforms during prion replication.

Identification of a homology-independent linchpin domain controlling mouse and bank vole prion protein conversion

“…All HEK-expressed constructs were partially deglycosylated using PNGase F as previously described ( S1 Fig ) to improve the conversion efficiency of Mo PrP C [ 28 ] ( S2 Fig ) . This effect is likely due to the previously observed inhibitory effects of PrPC glycosylation on the propagation of mouse prion strains[ 28 , 29 ], which may be due to negatively charged sialic acid groups within the glycan structure[ 30 ], and which may be further aggravated by hyper-glycosylation in HEK 293 cells. To ensure our system faithfully reproduces similar seed specificity as brain homogenate substrates in sPMCA, we first tested the susceptibility of cell-expressed BV PrP and Mo PrP in sPMCA ( Fig 2 ) .…”

Posttranslational modifications define course of prion strain adaptation and disease phenotype

“…Replication of transmissible, yet clinically silent PrP Sc states would not result in a clinical disease (76,77). Fourth, a spread of prions between brain regions is likely to be affected by region-specific differences in PrP C , such as differences in their sialylation status, and expression of strain-specific cofactors (59,78).…”

Site-specific analysis of N-glycans from different sheep prion strains

“…Another study, using desialylated PrP C , led to a proposal that PrP Sc amplification is increased because of the removal of electrostatic repulsion between N-glycans [ 68 ]. Although prion conversion is possible without glycans, the importance of investigating glycosylation in strains has been emphasized in a recent study, in which it was shown that strain conformation determines both cofactor and glycosylation preferences, supporting the strain-specific neurotropism hypothesis [ 69 ]. The same group of researchers has also shown in vitro that the second glycosylation site affects prion conversion, suggesting this site has a role in controlling the conversion of PrP Sc to PrP C [ 70 ].…”