In Vitro Characterization of the Presenilin-Dependent γ-Secretase Complex Using a Novel Affinity Ligand

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Abstract
γ-Secretase is the enzyme activity releasing the amyloid-β peptide from membrane-bound processing intermediates derived from the β-amyloid precursor protein. Cellular release and subsequent aggregation of the amyloid-β peptide is thought to be causative for the pathogenesis of Alzheimer's disease. γ-Secretase performs an unusual intramembranous cleavage and has been closely linked to a macromolecular complex containing presenilins. To generate a molecular probe for γ-secretase, we have developed a novel biotinylated affinity ligand which is based on a specific inhibitor containing a hydroxyethylene dipeptide isostere, known to serve as a transition state analogue for aspartic proteinases. Using this probe we confirmed the presence of the presenilin heterodimer and mature nicastrin in the active enzyme complex and, furthermore, that substrate binding site(s) and active center(s) are spatially separated. Affinity precipitations suggest that only a discrete fraction of cellular presenilin is present in the active γ-secretase complex and that both γ(40)- and γ(42)-activities are mediated by the same molecular entity. This was also reflected by a co-distribution of both enzyme activities in subcellular fractions enriched for trans-Golgi network membranes.