Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase.
We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.
Kakuda
Nobuto
N
Department of Neuropathology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Funamoto
Satoru
S
Yagishita
Sousuke
S
Takami
Mako
M
Osawa
Satoko
S
Dohmae
Naoshi
N
Ihara
Yasuo
Y
D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D010450:Endopeptidases / Q000737:chemistry*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D010446:Peptide Fragments; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases