Quantifying the Role of Lysine in Prion Replication by Nano-LC Mass Spectrometry and Bioassay

Prions propagate by a template driven process, inducing the normal cellular isoform (PrP^C) to adopt the prion (PrP^Sc) conformation. In PrP^C, the positions of lysines are highly conserved and strongly influence prion propagation. In this study, covalent modification was used to quantitate the role of lysines in the PrP^Sc template that drives prion replication. The ε-amino group of lysines in the PrP^Sc (hamster-adapted scrapie Sc237) template was acetylated by either acetic anhydride (Ac₂O) or the N-hydroxysuccinimide ester of acetic acid (Ac-NHS). The extent of lysine acetylation in PrP^Sc was quantitated by mass spectrometry or Western blot-based analysis. Identical samples were bioassayed to quantitate the loss of infectivity associated with lysine acetylation. The reduction of infectivity at the highest reagent concentration was approximately 90% (∼10-fold). Ten of the eleven prion lysines were acetylated to a greater extent (25-400-fold) than the observed loss of infectivity. Only one lysine, at position 220 (K₂₂₀), had a reactivity that is consistent with the loss of infectivity. Although lysines are highly conserved and play a crucial role in converting PrP^C into the PrP^Sc conformation, once that conformation is adopted, the lysines present in the PrP^Sc template play only a limited role in prion replication. In principle, this approach could be used to clarify the role of other amino acids in the replication of prions and other prion-like protein misfolding diseases.