Positive–negative epitope-tagging of β amyloid precursor protein to identify inhibitors of Aβ processing

In this report, a novel positive–negative epitope tagging approach was developed to study the cellular processing of β amyloid precursor protein (βAPP). Amino acids centered around the α-secretase cleavage site within the Aβ sequence were replaced with residues comprising an epitope for which high-affinity monoclonal antibodies are commercially available. The resulting mutant βAPP cDNAs were expressed in human embryonic kidney cells (HEK 293). Cleavage of labeled βAPP by β- and γ-secretase(s) results in the release of an epitope-tagged Aβ peptide, whereas cleavage by α-secretase results in destruction of the epitope. Highly sensitive and specific immunoassays were developed to study processing of this labeled βAPP via the amyloidogenic pathway. Secretion of epitope-tagged Aβ was prevented by MDL 28170, a previously described γ-secretase inhibitor. Confocal microscopic studies revealed that processing and cellular trafficking of epitope-tagged βAPP was not different from wild-type βAPP. These results suggest that positive–negative epitope-tagged βAPP is normally processed within the cell and may be used to identify secretase inhibitors as therapeutics for Alzheimer’s disease.