Gerstmann-Sträussler-Scheinker disease revisited: accumulation of covalently-linked multimers of internal prion protein fragments

Abstract

Despite their phenotypic heterogeneity, most human prion diseases belong to two broadly defined groups: Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). While the structural characteristics of the disease-related proteinase K-resistant prion protein (resPrP^D) associated with the CJD group are fairly well established, many features of GSS-associated resPrP^D are unclear. Electrophoretic profiles of resPrP^D associated with GSS variants typically show 6-8 kDa bands corresponding to the internal PrP fragments as well as a variable number of higher molecular weight bands, the molecular nature of which has not been investigated. Here we have performed systematic studies of purified resPrP^D species extracted from GSS cases with the A117V (GSS^A117V) and F198S (GSS^F198S) PrP gene mutations. The combined analysis based on epitope mapping, deglycosylation treatment and direct amino acid sequencing by mass spectrometry provided a conclusive evidence that high molecular weight resPrP^D species seen in electrophoretic profiles represent covalently-linked multimers of the internal ~ 7 and ~ 8 kDa fragments. This finding reveals a mechanism of resPrP^D aggregate formation that has not been previously established in prion diseases.

Keywords: Aggregate formation; Creutzfeldt-Jakob disease; Epitope mapping; Mass spectrometry; Multimers; Prion protein.

Fig. 1

Epitope mapping combined with deglycosylation of purified resPrP^D associated with GSS^F198S, GSS^A117V and sCJDMV1 used as control. PNGase F-treated or untreated, PK-resistant PrP^D (resPrP^D) purified (see Materials and Methods) from GSS^F198S, GSS^A117V and sCJDMV1 control were blotted and probed with the Abs to PrP denoted, with their epitopes, at the top of each panel. Panel a: Only full-length (PK-untreated) recombinant PrP (23–231) was detected by this Ab to the proximal N-terminal region confirming the absence of resPrP^D with complete N-terminus. Panels b to e: Both GSS^F198S and GSS^A117V resPrP^D conformers were selectively detected by the same Abs to PrP N- and C-terminal regions; both also showed no major variation of the resPrP^D banding pattern following deglycosylation indicating that most of the resPrP^D is unglycosylated in both conditions. Although overall similar, the two resPrP^D profiles differed especially in the higher molecular weight region suggesting a distinct repertoire, or relative proportions, of polymers in the two GSS variants. The GSS banding patterns clearly differed from the three-band pattern of sCJDMV1, two of which are glycosylated (See Results for detailed description). Panel c includes the bands of both 23–231 and 90–231 recombinant PrP which have been used as molecular weight markers. In b the portion of the panel with the GSS^A117V samples required longer exposure. In panel f, * indicates the 13 kDa component of the glycosylated and anchor bearing PrP 12/13 C-terminus fragments with N-termini at residues 162–167 and 154–156, respectively [24, 41]

Fig. 2

Mass spectrometry (MS)-based sequencing of PrP fragments in the material extracted from individual gel bands of two GSS variants. GSS^A117V resPrP^D (a) and GSS^F198S resPrP^D (b). The material present in these bands was subjected to trypsin digestion, followed by MS analysis of tryptic fragments. The fragments identified by MS for species extracted from individual gel bands are shown as blue, red and green lines. Amino acid sequence within the 70-152 region is shown above the lines. Residues marked in red represent potential cleavage sites in the relevant region of PrP; 129M/V polymorphic residues are marked in blue

Fig. 3

Relative abundance of 129M and 129V PrP variants in resPrP^D associated with GSS^A117V and GSS^F198S. a: GSS^A117V; b: GSS^F198S. The relative abundance of resPrP^D with M or V at residue 129 reflects the representation of the PrP mutation, which is coupled with the 129V in both GSS variants. Approximately 10–25% of resPrP^D could be identified as non-mutated (wild type) in GSS^F198S while only mutated resPrP^D could be detected in GSS^A117V. The relative populations were determined by mass spectrometry using the spectral counting method