A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein

The cellular form of the prion protein (PrPC) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrPC proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109 down arrow His-110 (mouse sequence), termed alpha-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. beta-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the alpha-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu2+ redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu2+ redox-generated reactive oxygen species do produce fragmentation corresponding to beta-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu2+- and Zn2+-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of alpha-cleavage, Lys-109 down arrow His-110 (MoPrP sequencing); however, upon addition of Cu2+, the location of alpha-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in alpha-cleavage at Lys109 down arrow His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119 down arrow Val-120, with additional cleavage by ADAM10 at Gly-227 down arrow Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrPC fragmentation responds to Cu2+ and Zn2+.