A presenilin-independent aspartyl protease prefers the γ-42 site cleavage

β-Amyloid peptides (Aβ40 and Aβ42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimer's disease (AD). The Aβ is derived from sequential cleavages of amyloid precursor protein (APP) by β- and γ-secretases. γ-Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the γ-site cleavage of APP. Secretion of both Aβ40 and Aβ42 is significantly reduced in PS1 knock-out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Aβ from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Aβ42 accumulation; thus the intracellular Aβ42 appears to be generated in a PS-independent manner. Here we present the first biochemical evidence indicating that Aβ, especially Aβ42, can be generated in the absence of PS based on an in vitroγ-secretase assay employing membranes prepared from PS-deficient Blastocyst-derived (BD) cells. This PS-independent γ-secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Aβ40 and Aβ42 generation, respectively. These results indicate that these PS-independent γ-site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42-site cleavage over the 40-site cleavage, thereby generating Aβ42 as the predominant product.