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Updated: Sep 11, 2025
Protein Misfolding Cyclic Amplification of Prions
Published on: November 7, 2012
Variably protease-sensitive prionopathy: mass spectrometry analysis of the pathogenic prion protein provides a new
S K Nemani1,2, X Xiao3, S Notari4
1Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada.
Abstract:
Variably protease-sensitive prionopathy (VPSPr) is a rare and complex prion disease that differs from sporadic Creutzfeldt-Jakob disease (sCJD) in its clinical and histopathological phenotypes. VPSPr also features a variety of fragments generated by the disease-causing prion protein (PrPD). However, accurately determining the number and sequence of these fragments has been challenging when relying solely on epitope mapping with existing antibodies. To address these challenges, we performed mass spectrometry analyses and designed epitope mapping experiments to determine the primary structure and verify the presence or absence of the anchor in the VPSPr proteinase K-resistant and deglycosylated PrPD fragments. All three N-terminus fragments, with reported molecular weights of 20, 17, and 7 kDa, likely share Ser97 as the N-terminal amino acid. The C-terminus of the internal 7 kDa fragment is ragged, extending from Phe141 to Met154, while the 20 kDa and 17 kDa fragments differ only in the absence of the anchor in the latter. The three fragments belonging to the C-terminus group have previously been reported to have electrophoretic mobilities of 18, 12/13, and 8-9 kDa. After deglycosylation, the 18 kDa fragment was not detected. The 12 kDa component of the 12/13 kDa fragment was found to have a ragged N-terminus between Tyr162 and Asp181 and the anchor, while the 8 kDa fragment represented the anchorless version of the 12 kDa fragment. Unexpectedly, a second approximately 8 kDa fragment was identified that bore the anchor but had a shorter, ragged N-terminus ranging from Gly195 to Phe198. Calculation based on sequencing data revealed that the actual molecular masses of the 20, 17, and 12 kDa fragments are 1-2 kDa lighter. Moreover, the primary structures of the 20, 17, and 12 kDa fragments match those of the 19, 17, and 12 kDa fragments associated with sCJD type 2. Our findings provide new insights into the characteristics of the deglycosylated, PK-resistant fragments in VPSPr, which will likely assist in interpreting future high-resolution studies of amyloid fibrils in this disease.
Insights
Variably protease-sensitive prionopathy (VPSPr) involves unique prion protein fragments. Mass spectrometry and epitope mapping identified specific N- and C-terminal fragments, clarifying their structures and relation to sporadic CJD.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Variably protease-sensitive prionopathy (VPSPr) is a rare prion disease with distinct clinical and pathological features compared to sporadic Creutzfeldt-Jakob disease (sCJD).
- The prion protein (PrPD) in VPSPr generates diverse fragments, but their precise number and sequence have been difficult to determine using traditional epitope mapping.
Purpose of the Study:
- To characterize the primary structure of proteinase K-resistant and deglycosylated PrPD fragments in VPSPr.
- To verify the presence or absence of the anchor in these fragments using mass spectrometry and epitope mapping.
Main Methods:
- Mass spectrometry analysis of VPSPr PrPD.
- Epitope mapping experiments to determine fragment sequences and anchor presence.
- Analysis of N-terminal and C-terminal fragments, including molecular weight determination and deglycosylation.
Main Results:
- Identified three N-terminal fragments (20, 17, 7 kDa) likely sharing Ser97 as the N-terminus, with the 7 kDa fragment showing ragged C-termini.
- Characterized three C-terminal fragments (previously 18, 12/13, 8-9 kDa), with the 18 kDa fragment undetected after deglycosylation. The 12 kDa fragment contains the anchor and has ragged N-termini, while the 8 kDa fragment is anchorless. A second ~8 kDa fragment with the anchor but a shorter N-terminus was also found.
- Calculated actual molecular masses for 20, 17, and 12 kDa fragments are 1-2 kDa lighter than reported. Their primary structures match those of sCJD type 2 fragments.
Conclusions:
- The study provides detailed structural insights into deglycosylated, PK-resistant PrPD fragments in VPSPr.
- The findings clarify fragment heterogeneity and reveal structural similarities between VPSPr and sCJD type 2 fragments.
- This research will aid in interpreting future high-resolution studies of VPSPr amyloid fibrils.
