Effects of Different Experimental Conditions on the PrPSc Core Generated by Protease Digestion

Abstract

The discovery of molecular subtypes of the pathological prion protein PrP Sc has provided the basis for a novel classification of human transmissible spongiform encephalopathies (TSEs) and a potentially powerful method for strain typing. However, there is still a significant disparity regarding the understanding and nomenclature of PrP Sc types. In addition, it is still unknown whether a specific PrP Sc type is associated with each TSE phenotypic variant. In sporadic CreutzfeldtJakob disease (sCJD), five disease phenotypes are known, but only two major types of PrP Sc , types 1 and 2, have been consistently reproduced. We further analyzed PrP Sc properties in sCJD and variant CJD using a high resolution gel electrophoresis system and varying experimental conditions. We found that pH varies among CJD brain homogenates in standard buffers, thereby influencing the characteristics of protease-treated PrP Sc . We also show that PrP Sc type 1 and type 2 are heterogeneous species which can be further distinguished into five molecular subtypes that fit the current histopathological classification of sCJD variants. Our results shed light on previous disparities in PrP Sc typing, provide a refined classification of human PrP Sc types, and support the notion that the pathological TSE phenotype is related to PrP Sc structure.

A refined method for molecular typing reveals that co-occurrence of PrPSc types in Creutzfeldt–Jakob disease is not the rule

“…The western blot profile of PrP27-30 in these cases appears almost invariably characterized by the association of two PrP Sc core fragments, which include a classic 19 kDa type 2 band and a slower migrating band of about 20 kDa. As previously reported by us, 29,30 the two bands are readily detected by 3F4. Furthermore, their relative amount varies significantly according to the brain region analyzed, although the type 2 band is usually dominant over the 20 kDa band (P Parchi et al, unpublished data).…”

“…In particular, much attention was given to performing PK digestion of tissue homogenate at a given pH value (using a high buffer capacity solution) and at a given total protein concentration. This is because PrP Sc digestion by PK is significantly affected by both the actual pH 30 and the total protein concentration in the homogenate, which can also significantly differ among different areas (data not shown). In these conditions, the amount ratio between the type 1-like band and type 2 signal was not significantly influenced by the brain structure analyzed (Figure 7a).…”

“…34, 35 We also detected a type 1-like band in all CJD type 2 subtypes but came up to a different conclusion concerning the origin and biological significance of this band. In a previous study, 30 where we originally applied a highresolution electrophoretic system, which better discriminates between distinct PrP isoforms, we showed that PK digestion of both PrP Sc types 1 and 2 produces additional fragments migrating slightly slower than the resistant core. Since these fragments were significantly more sensitive to protease digestion than the PrP Sc core, we thought they might represent the effect of a relatively inefficient hydrolysis of PrP Sc .…”

“…29 More recently, the study of PrP Sc under stringent pH conditions using a gel electrophoresis technique with increased sensitivity demonstrated that PrP Sc types 1 and 2 are heterogeneous species, which can be further distinguished into molecular subtypes that better fit the current histopathological classification of sporadic CJD (sCJD) into six subtypes. 30 Despite these significant advances, much remains to be performed to explore the molecular basis of CJD heterogeneity and strain diversity in full. One intriguing aspect of CJD molecular pathology relates to the co-occurrence of PrP Sc types 1 and 2 in the same brain.…”

Recent Developments in Prion Disease Research: Diagnostic Tools and In Vitro Cell Culture Models

“…Therefore, electrophoretic mobility is influenced by host genotype and prion strain [75,129]. In other words, the mobility index is often used for discriminating the CJD subtype [iatrogenic CJD (iCJD), sCJD, and vCJD], BSE, and scrapie strains [20,36,72,77,111,136].…”