A POSSIBLE ROLE FOR CATHEPSIN-D, CATHEPSIN-E, AND CATHEPSIN-B IN THE PROCESSING OF BETA-AMYLOID PRECURSOR PROTEIN IN ALZHEIMERS-DISEASE

Formation of the 4-kDa peptides, which are essential constituents of t he extracellular plaques in Alzheimer's disease, involves the sequenti al cleavage of the amyloid precursor protein (APP) by beta- and gamma- secretases. The carboxy-terminal 99-amino-acid peptide which is libera ted from APP by beta-secretase was used as a potential native substrat e of the gamma-secretase(s). With the addition of an initiator Met and a FLAG sequence at the C-terminus (beta APP100-FLAG), it was expresse d in Escherichia coli under the control of the T7 promotor. The prefer red site(s) of cleavage in the N-terminal 30-amino-acid beta-amyloid p eptide and beta APP100-FLAG by potential gamma-secretase(s) were rapid ly identified using matrix-assisted laser-desorption/ionization time-o f-flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since gamma-secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. S tudies using different detergents indicated that the cleavage preferen ce of cathepsin D for the beta APP100-FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins wer e found to be capable of catabolizing both beta-amyloid peptides and t he beta APP100-FLAG. As cathepsin D was found to cleave the beta APP10 0-FLAG in the vicinity of the C-terminus of the beta-amyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the po ssibility cannot be excluded that cathepsins D and B are involved in t he amyloidogenic processing of APP.