Identification of β-secretase-like activity using a mass spectrometry-based assay system

We describe an assay system for the identification of site-specific proteases. The assay is based on a protein substrate that is immobilized on ceramic beads. After incubation with cell homogenates, the beads are washed and digested with endoproteinase Lys-C to liberate a defined set of peptides. The peptide fragments are identified by mass spectrometry. The assay was used to screen for β-secretase, the protease that cleaves amyloid precursor protein (APP) at the β-site. Cathepsin D was identified as the enzyme responsible for β-secretase-like activity in two cell lines. Subsequent analysis of the related aspartic protease, cathepsin E, revealed almost identical cleavage specificity. Both enzymes are efficient in cleaving Swedish mutant APP at the β-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Aβ) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wild-type APP, the generation of Aβ was increased. This suggests that more than one enzyme is capable of generating Aβ in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.