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]