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), 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 (SEVNLDAEFR) 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 (sAPPbeta) detected the secretion of sAPPbeta 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 Golgi or other organelles around the nuclei. These results suggest that TOP has a beta-secretase-like activity responsible for the processing of APP.
Koike
H
H
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, 153-8902, Japan.
Seki
H
H
Kouchi
Z
Z
Ito
M
M
Kinouchi
T
T
Tomioka
S
S
Sorimachi
H
H
Saido
T C
TC
Maruyama
K
K
Suzuki
K
K
Ishiura
S
S
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001921:Brain / Q000201:enzymology; D019556:COS Cells / Q000378:metabolism; D002417:Cattle; D017077:Culture Media, Conditioned; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D016564:Amyloid beta-Protein Precursor; D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase