Equimolar production of amyloid β-protein and amyloid precursor protein intracellular domain from β-carboxyl-terminal fragment by β-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 ( A beta) 49 in cells resulted in predominant production of A beta 40 and that of A beta 48 leads to preferential production of A beta 42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between A beta andAICD, 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 A beta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various A beta species, including A beta 40, A beta 42, A beta 43, A beta 45, A beta 48, and A beta 49, 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, A beta 48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced A beta 48 andA beta 49 rapidly undergo gamma-cleavage, resulting in generation of various A beta species.