Characterization of Truncated Forms of Abnormal Prion Protein in Creutzfeldt-Jakob Disease

Abstract

In prion disease, the abnormal conformer of the cellular prion protein, PrP Sc , deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP Sc to proteolytic digestion in vitro generates a core fragment of 19 -21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5-or 17-kDa fragments and the previously described 13-kDa PrP Sc C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes.

Gerstmann-Sträussler-Scheinker Disease and “Anchorless Prion Protein” Mice Share Prion Conformational Properties Diverging from Sporadic Creutzfeldt-Jakob Disease

“…The contribution of the anchor to the molecular mass of PrP has been previously calculated to account for 2-3 kDa and 4 kDa, under experimental conditions using, respectively, hydrofluoric acid and proaerolysin (16,26). These data are in keeping with our findings showing a 2.5 kDa difference between "anchored PrP" C57BL and "anchorless PrP" Tg mice in the orthogonal migration of either native or PK-resistant PrP Sc isoforms.…”

“…3, C and D), in MV2 cases the reduced number of spots, and in particular the absence of basic 17.5-kDa isoforms, is consistent with mAb 12B2 (epitope 89 -93) missing the capture of G92, S97, and W99 N-terminal species that are highly expressed in this sCJD subtype (21). Therefore, using an extensive panel of anti-PrP mAbs and a high sensitive separation technique, we were unable to confirm the presence of anchorless PrP species, as suggested by Notari et al using a conventional SDS-PAGE technique and an antiPrP rabbit antiserum (16 6D11, and ICSM-35 (Fig. 5A).…”

“…Recently, Notari et al reported on the presence of anchorless PrP Sc in different sCJD subtypes, hence widening the spectrum of molecular PrP quasispecies detected in this human disorder (16). Anchorless PrP forms were tentatively identified based on the immunodecoration of PK-resistant species migrating 2 kDa faster than the unglycosylated PrP27-30, under SDS-PAGE separation.…”

“…Recently, the generation of anchorless PrP forms has been also claimed in sCJD, hence suggesting that in addition to anchored PrP Sc conformers, anchorless molecules could contribute to the phenotypic heterogeneity of this disorder (16). This issue raises additional concerns regarding the neuroinvasive properties of these quasispecies and the potential infectivity of human body fluids and peripheral tissues of sCJD patients.…”

“…Sc species in all sCJD subtypes, and their enrichment after PK digestion (16). Anchorless PrP quasispecies were tentatively identified based on their migration, ϳ2 kDa faster than the core fragment, and their detection with mAbs directed to epitopes along PrP residues 89 -221, including 12B2 and 3F4, but not with a rabbit antiserum directed to residues 220 -231.…”