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.
γ-Secretase cleaves the transmembrane domain of β-amyloid precursor protein at multiple sites. These are referred to as γ-, ζ-, and ε-cleavages. We showed previously that DAPT, a potent dipeptide γ-secretase inhibitor, caused differential accumulations of longer amyloid β-proteins (Aβs) (Aβ43 and Aβ46) in CHO cells that are induced to express the β C-terminal fragment (CTF). To learn more about the cleavage mechanism by γ-secretase, CHO cell lines coexpressing βCTF and wild-type or mutant presenilin (PS) 1/2 were generated and treated with DAPT. In all cell lines treated with DAPT, as the levels of Aβ40 decreased, Aβ46 accumulated to varying extents. In wild-type PS1 or M146L mutant PS1 cells, substantial amounts of Aβ43 and Aβ46 accumulated. In contrast, this was not the case with wild-type PS2 cells. In M233T mutant PS1 cells, significant amounts of Aβ46 and Aβ48 accumulated differentially, whereas in N141I mutant PS2 cells, large amounts of Aβ45 accumulated concomitantly with a large decrease in Aβ42 levels. Most interestingly, in G384A mutant PS1 cells, there were no significant accumulations of longer Aβs except for Aβ46. Aβ40 was very susceptible to DAPT, but other Aβs were variably resistant. Complicated suppression and accumulation patterns by DAPT may be explained by stepwise processing of βCTF from a ζ- or ε-cleavage site to a γ-cleavage site and its preferential suppression of γ-cleavage over ζ- or ε-cleavage.
We previously showed that cells expressing wild–type (wt) β–amyloid precursor protein (APP) or coexpressing wtAPP and wt presenilin (PS) 1/2 produced APP intracellular domain (AICD) 49–99 and AICD50–99, 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 AICD49–99 than AICD50–99. Furthermore, the expression of amyloid β–protein (Aβ) 49 in cells results in predominant production of Aβ40 and that of Aβ48 leads to preferential production of Aβ42. These observations suggest that ϵ–cleavage and γ–cleavage are interrelated. To determine the stoichiometry between Aβ and AICD produced by γ–secretase. We have established a CHAPSO–solubilized γ–secretase assay system that exhibits high specific activity. Microsomal fractions of CHO cells were solubilized in 1% CHAPSO and incubated with C99–FLAG purified from Sf9 cells to produce Aβ and AICD. Reaction mixtures were subjected to western blotting for quantification of Aβ and AICD with authentic peptides. Aβ and AICD species were identified by 8 M Urea containing gel and mass spectrometry, respectively. Equal amounts of Aβ and AICD are produced from C99 by γ–secretase, irrespective of wt or mtAPP and PS1/2. Although various Aβ species, including Aβ40, Aβ42, Aβ43, Aβ45, Aβ48 and Aβ49, are generated, only two species of AICD, AICD49–99 and AICD50–99, are detected. In addition, M233T mtPS1 produces only one species of AICD, AICD49–99, and only one of its counterpart, Aβ48, in contrast to wt and other mtPS1s. These strongly suggest that ϵ–cleavage is the primary event, and produced Aβ48 and Aβ49 rapidly undergo γ–cleavage, resulting in generation of various Aβ species.
