Identification of ADAMTS4 as an APP‐cleaving enzyme at 669 site in the APP669‐711 production pathway: Biomarkers (non‐neuroimaging)/Plasma/Serum/Urine biomarkers

Background Amyloid‐β peptide (Aβ) is deposited in the brains of Alzheimer disease (AD) patients, and proteolytically derived from its precursor protein, APP. APP669‐711 (a.k.a. Aβ(‐3)‐40) is a novel APP‐derived peptide detected in the plasma using immunoprecipitation‐matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (IP‐MS). We have reported that the composite plasma biomarker, which is combination of APP669‐711/Aβ1‐42 ratio and Aβ1‐40/ Aβ1‐42 ratio, surrogates the accumulation of Aβ in brain (Kaneko et al., Proc Jpn Acad Ser B Phys Biol Sci. 2014; Nakamura et al., Nature 2018). However, the mechanism of APP669‐711 production is largely unclear. Method We analyzed the effects of several inhibitors and genetic knockouts on the production of APP699‐711 in cultured cells. Result Endogenous APP669‐711 was detected in the conditioned medium of BE2‐(C), Neuro2a and A549 cells by IP‐MS. Pharmacological experiments revealed that APP669‐711 is generated by sequential cleavages by GM6001‐sensitive metalloprotease at 669 site and γ‐secretase. Based on the preferences of the substrate sequence, we focused on ADAMTS4, which is the secreted metalloprotease with thrombospondin motif. Overexpression of ADAMTS4 resulted in the overproduction of APP669‐711, which was diminished by the catalytically inactivating mutation (i.e., E362A). Furthermore, endogenous APP669‐711 production was decreased in ADAMTS4 knockout cells. Conclusion These results indicate that ADAMTS4 is involved in the production pathway of APP669‐711, a novel plasma biomarker for Aβ deposition in the brain.