Thimet oligopeptidase cleaves the full-length Alzheimer amyloid precursor protein at a beta-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), an d purified a candidate beta-secretase from bovine brain. N-terminal amino a cid analysis showed the candidate to be thimet. oligopeptidase (TOP), The c DNA 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 norm al substrate (SEVKMDAEFR), We then coexpressed TOP with APP695 in COS cells , collected transfected cells and conditioned media, and analyzed them by i mmunoblotting, The antibody against the specific secreted APP cleaved by be ta-secretase (sAPP beta) detected the secretion of sAPP beta only from APP/ hTOP-overexpressing cells, and not from cells overexpressing of antisense h TOP cDNA, Finally, we analyzed the immunolocalization of overexpressed hTOP in COS cells. Most hTOP was localized in the nuclei, but a small amount wa s localized in the Gels or other organelles around the nuclei. These result s suggest that TOP has a beta-secretase-like activity responsible for the p rocessing of APP.