APLP2 is predominantly cleaved by β‐secretase and γ‐secretase in the human brain

Background Amyloid‐β peptide is well‐known as a pathogen of Alzheimer's disease, but its precursor, amyloid‐beta precursor protein (APP), remains unexplained 30 years after its discovery. APP has two homologues called amyloid precursor‐like protein 1 (APLP1) and amyloid precursor‐like protein 2 (APLP2), and shares a similar structural organisation with them and has partially overlapping functions. APP family proteins are essential for survival, shown by the crossbreeding analysis of knockout mice of APP family molecules, including APLP1 and APLP2. APLP2 is known to play the most important role among them, but the molecular metabolism of APLP2 is only partially understood. Here, we analysed ectodomain shedding and γ‐secretase cleavage of APLP2 by molecular biological and biochemical techniques. Method We analysed the culture supernatant of HEK293 cells overexpressing APLP2 and human cerebrospinal fluid. For the analysis of secreted APLP2 fragments, we raised the OA603 antibody that reacts with the juxtamembrane domain of APLP2. Substrate cleavage sites were identified by matrix assisted laser desorption/ionisation mass spectrometry. Results By overexpressing in HEK293 cells, APLP2 undergoes ectodomain shedding at three sites in the extracellular region by α‐ and β‐secretase‐like activity and then is intramembranously cleaved at three sites by γ‐secretase. In particular, in shedding, α‐secretase‐like activity was dominant in HEK cells. Surprisingly, in human cerebrospinal fluid, APLP2‐derived metabolic fragments were mainly cleaved by β‐secretase‐like activity, not by α‐secretase‐like activity. Because APP is also mainly cleaved by beta‐site amyloid precursor protein cleaving enzyme 1 in neurons and APLP1 is expressed exclusively in neurons, these findings suggest that APP family proteins may play a common role via β‐secretase‐like cleavage in the central nerve system. Conclusions Thus, these findings may contribute to a better understanding of the role of APP family proteins in Alzheimer's disease.