Comparison of the level, distribution and form of disease-associated prion protein in variant and sporadic Creutzfeldt-Jakob diseased brain using conformation-dependent immunoassay and Western blot

Abstract

Disease-associated prion protein (PrP Sc ) can be distinguished from the cellular isoform (PrP C ) by conformation-dependent immunoassay (CDI). This technique exploits the presence of an epitope, accessible in PrP C , but only unmasked by denaturation in PrP Sc . In this study, we investigated PrP Sc in different brain regions in variant and sporadic Creutzfeldt-Jakob disease (CJD) by using CDI, and directly compared the results with those obtained using the more commonly employed protease digestion and Western blotting. In general, there was good agreement between the results, although there were certain discrepancies in relative abundance when the regional distribution in variant CJD cases was considered. The results largely confirmed the previously described targeting of different brain regions by variant and sporadic CJD. Additionally, the combination of protease digestion and CDI detection demonstrated, for the first time, the presence of PrP Sc in variant CJD brains that is susceptible to proteolysis under standard conditions.Prion diseases are characterized by the conversion of the host-encoded prion protein (PrP C ) into a disease-associated isoform (PrP Sc ), which is thought to be the main component of the infectious agent or prion (Bolton et al., 1982;Prusiner, 1982Prusiner, , 1998. The conversion of a-helical PrP C into b-sheet-rich PrP Sc accompanies change in biochemical properties, such as protease resistance and detergent solubility (Meyer et al., 1986;Caughey et al., 1991;Pan et al., 1993). The proteolytic treatment of PrP Sc leaves an N-terminally truncated, proteinase K (PK)-resistant core fragment of PrP Sc (termed PrP res ) (Oesch et al., 1985). In human prion diseases, the presence of PrP Sc in a sample of affected tissue (usually grey matter enriched cerebral cortex or cerebellum) has been most commonly demonstrated by detecting PrP res by Western blotting. Variant Creutzfeldt-Jakob disease (vCJD) can be distinguished from sporadic CJD (sCJD) based on the PrP res type as determined by the size of the N-terminally truncated non-glycosylated fragment and the proportion of three possible glycoforms (Collinge et al., 1996;Head et al., 2004). Additionally, the type of PrP res in combination with genotype at PRNP codon 129 has been proposed as the basis of a molecular classification system accounting for the marked clinico-pathological variability of sCJD (Parchi et al., 1999). An alternative approach that also distinguishes PrPSc from PrP C is conformation-dependent immunoassay (CDI), which is based on a conformational transition in the N terminus of PrP during the formation of PrP Sc (Peretz et al., 1997). CDI was initially developed in the form of a direct ELISA using the mAb 3F4 that binds to aa 109-112 of human PrP (Safar et al., 1998). The epitope of mAb 3F4 was found not to be accessible in native PrP Sc , in contrast to its accessibility in PrP C , and was shown to become exposed in PrP Sc after denaturation (Safar et al., 1998). The introduction of sodium phosphotungstic acid (NaPTA) p...

Human prion diseases: Molecular, cellular and population biology

“…We have confirmed these results and extended them to vCJD, which also has more senPrP Sc than PrP res present in the brain (Fig. ) …”

Protease-Sensitive Conformers in Broad Spectrum of Distinct PrPSc Structures in Sporadic Creutzfeldt-Jakob Disease Are Indicator of Progression Rate

“…This method allowed us to compare the conformational features of human PrP Sc independently of proteolytic treatment and in addition provided quantitative data on levels of PrP Sc , sPrP Sc , and rPrP Sc [15], [30]. The CDI techniques represent a major improvement over previously used semi-quantitative WB-based methods, the finding that has been independently confirmed by another group [60], [61]. The dissociation and unfolding of PrPSc in a presence of increasing concentration of Gdn HCl can be described as follows: [PrP Sc ] n →[sPrP Sc ] n →iPrP→uPrP, where [PrP Sc ] n are native aggregates of PrP Sc , [sPrP Sc ] n are soluble protease-sensitive oligomers of PrP Sc , iPrP is an intermedite, and uPrP is completely unfolded (denatured) PrP [7], [43], [62].…”

Variant CJD

“…21 While type 2B PrP res provides a convenient additional diagnostic tool for human BSE identification, 22 it does not provide a complete description of PrP Sc in cases of vCJD, nor does it provide a biochemical definition of the agent. The largest amount of PrP Sc in the vCJD brain is actually protease sensitive 23 and therefore does not figure in conventional PrP res typing. Even within the protease-resistant fraction of vCJD PrP Sc there is evidence of a minority PrP res type.…”