Aβ46 Is Processed to Aβ40 and Aβ43, but Not to Aβ42, in the Low Density Membrane Domains*

Abstract

γ-Secretase cleaves the transmembrane domain of β-amyloid precursor protein at multiple sites referred to as γ-, ϵ-, and ζ-cleavage sites. We previously showed that N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a potent dipeptide γ-secretase inhibitor, causes differential accumulation of longer amyloid β-proteins (Aβs) within Chinese hamster ovary cells co-expressing β C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Aβ46 localized exclusively to low density membrane (LDM) domains. Incubating the Aβ46-accumulating LDM domains at 37 °C produced Aβ40, Aβ42, Aβ43, and β-amyloid precursor protein intracellular domain. The addition of L685,458 completely prevented β-amyloid precursor protein intracellular domain generation and resulted in a large decrease in the level of Aβ46 and the concomitant appearance of Aβ40 and Aβ43 but not Aβ42. Further addition of DAPT suppressed the production of Aβ40/43 and abolished the decrease in the amount of Aβ46. These data indicate that preaccumulated Aβ46 is processed by γ-secretase to Aβ40/43 but not to Aβ42 in the LDM domains. The amount of newly produced Aβ40 and Aβ43 was roughly equivalent to the decrease in the amount of Aβ46. Temporal profiles did not show a maximal concentration for Aβ43, suggesting that Aβ46 is processed to Aβ40 and Aβ43 through a nonsuccessive process.