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

We describe an assay system for the identification of site-specific proteas es. The assay is based on a protein substrate that is immobilized on cerami c beads. After incubation with cell homogenates, the beads are washed and d igested with endoproteinase Lys-C to liberate a defined set of peptides. Th e peptide fragments are identified by mass spectrometry. The assay was used to screen for beta-secretase, the protease that cleaves amyloid precursor protein (APP) at the beta-site. Cathepsin D was identified as the enzyme re sponsible for beta-secretase-like activity in two cell lines. Subsequent an alysis of the related aspartic protease, cathepsin E, revealed almost ident ical cleavage specificity, Both enzymes are efficient in cleaving Swedish m utant APP at the beta-site but show almost no reactivity with wild-type APP . Treatment of cell lines with pepstatin inhibited the production of amyloi d peptide (A beta) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wildtype APP, the generation of A beta was increased. This suggests that more than o ne enzyme is capable of generating A beta in vivo and that an aspartic prot ease is involved in the processing of Swedish mutant APP.