Application of N‑Terminal Labeling Methods Provide Novel Insights into Endoproteolysis of the Prion Protein in Vivo

Alternative α- and β-cleavage events in the cellular prion protein (PrP^C) central region generate fragments with distinct biochemical features that affect prion disease pathogenesis, but the assignment of precise cleavage positions has proven challenging. Exploiting mouse transgenic models expressing wild-type (WT) PrP^C and an octarepeat region mutant allele (S3) with increased β-fragmentation, cleavage sites were defined using LC–MS/MS in conjunction with N-terminal enzymatic labeling and chemical in-gel acetylation. Our studies profile the net proteolytic repertoire of the adult brain, as deduced from defining hundreds of proteolytic events in other proteins, and position individual cleavage events in PrP^C α- and β-target areas imputed from earlier, lower resolution methods; these latter analyses established site heterogeneity, with six cleavage sites positioned in the β-cleavage region of WT PrP^C and nine positions for S3 PrP^C. Regarding α-cleavage, aside from reported N-termini at His110 and Val111, we identified a total of five shorter fragments in the brain of both mice lines. We infer that aminopeptidase activity in the brain could contribute to the ragged N-termini observed around PrP^C’s α- and β-cleavage sites, with this work providing a point of departure for further in vivo studies of brain proteases.