A Possible Role for Cathepsins D, E, and B in the Processing of β‐amyloid Precursor Protein in Alzheimer's Disease

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Abstract
Formation of the 4‐kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by β‐ and γ‐secretases. The carboxy‐terminal 99‐amino‐acid peptide which is liberated from APP by β‐secretase was used as a potential native substrate of the γ‐secretase(s). With the addition of an initiator Met and a FLAG sequence at the C‐terminus (β5APP100‐FLAG), it was expressed in Escherichia coli under the control of the T7 promoter. The preferred site(s) of cleavage in the N‐terminal 40‐amino‐acid β‐amyloid peptide and βAPP100‐FLAG by potential γ‐secretase(s) were rapidly identified using matrix‐assisted laser‐desorption/ionization time‐of‐flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since γ‐secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. Studies using different detergents indicated that the cleavage preference of cathepsin D for the βAPP100‐FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both β‐amyloid peptides and the βAPP100‐FLAG. As cathepsin D was found to cleave the βAPP100‐FLAG in the vicinity of the C‐terminus of the β‐amyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.