Thimet oligopeptidase cleaves the full-length Alzheimer amyloid precursor protein at a β-secretase cleavage site in COS cells

We developed an assay method using a novel quenched fluorescent substrate (QFS) flanking the beta-cleavage site of amyloid precursor protein (APP), and purified a candidate beta-secretase from bovine brain. N-terminal amino acid analysis showed the candidate to be thimet. oligopeptidase (TOP), The cDNA for human TOP was cloned from a human brain cDNA library and expressed in COS cells. The enzyme was further purified on a Ni2+-agarose column. TOP cleaved the Swedish Alzheimer's substrate (SEVNLDGEFR) as well as the normal substrate (SEVKMDAEFR), We then coexpressed TOP with APP695 in COS cells, collected transfected cells and conditioned media, and analyzed them by immunoblotting, The antibody against the specific secreted APP cleaved by beta-secretase (sAPP beta) detected the secretion of sAPP beta only from APP/hTOP-overexpressing cells, and not from cells overexpressing of antisense hTOP cDNA, Finally, we analyzed the immunolocalization of overexpressed hTOP in COS cells. Most hTOP was localized in the nuclei, but a small amount was localized in the Gels or other organelles around the nuclei. These results suggest that TOP has a beta-secretase-like activity responsible for the processing of APP.