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 (PrPSc) 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 epsilon-amino group of lysines in the PrPSc(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% (similar to 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 (K-220), 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.