Mechanism of the Cleavage Specificity of Alzheimer's Disease γ -secretase Identified by Phenylalanine-Scanning Mutagenesis of the Transmembrane Domain of the Amyloid Precursor Protein

Proteolytic processing of the amyloid precursor protein by β -secretase yields A4CT (C99), which is cleaved further by the as yet unknown γ -secretase, yielding the β -amyloid (Aβ) peptide with 40 (Aβ40) or 42 residues (Aβ42). Because the position of γ -secretase cleavage is crucial for the pathogenesis of Alzheimer's disease, we individually replaced all membrane-domain residues of A4CT outside the Aβ domain with phenylalanine, stably transfected the construct in COS7 cells, and determined the effect of these mutations on the cleavage specificity of γ -secretase (Aβ42/Aβ40ratio). Compared with wild-type A4CT, mutations at Val-44, Ile-47, and Val-50 led to decreased Aβ42/Aβ40ratios, whereas mutations at Thr-43, Ile-45, Val-46, Leu-49, and Met-51 led to increased Aβ42/Aβ40ratios. A massive effect was observed for 145F (34-fold increase) making this construct important for the generation of animal models for Alzheimer's disease. Unlike the other mutations, A4CT-V44F was processed mainly to Aβ38, as determined by mass spectrometry. Our data provide a detailed model for the active site of γ -secretase:γ -secretase interacts with A4CT by binding to one side of the α -helical transmembrane domain of A4CT. Mutations in the transmembrane domain of A4CT interface with the interaction between γ -secretase and A4CT and, thus, alter the cleavage specificity of γ -secretase.