ADAMTS4 as an enzyme cleaving at APP669 site

Background We have previously identified APP669-711 (a.k.a. Aβ(-3)-40) in human plasma using immunoprecipitation combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IP-MALDI-MS) (Kaneko et al., Proc Jpn Acad Ser B Phys Biol Sci. 2014). Our assay has revealed that the composite biomarker, which is a combination of APP669-711/Aβ1-42 ratio and Aβ1-40/ Aβ1-42 plasma, correlates amyloid PET status (Nakamura Nature 2018). previous research cultured cells suggested A Disintegrin Metalloproteinase Thrombospondin type 1 motif, 4 (ADAMTS4) involved production. Here, we present further evidence on involvement ADAMTS4 APP669 site-cleavage. Method prepared recombinant APP substrate, APP81 corresponding to APP619-699 sequence V5/His tag at C-terminus FLAG N-terminus (FLAG-APP81-V5/His) for vitro cleavage assay. Recombinant protein tagged were incubated either alone or together 37°C. The samples desalted then analyzed by MALDI-TOF MS. In vivo evaluation ADAMTS4, from Adamts4-/- mice WT IP-MALDI-MS. Result assay, N- C-terminal fragments cleaved APP669, Aβ4th, Aβ12th site detected mixture proteins but not sample substrate enzyme alone. Furthermore, peptide derived double digestion site-cleavages (i.e., APP669-Aβ11) was also MS/MS analysis. analysis mouse showed murine level decreased compared mice. Conclusion These results indicated one enzymes can cleave directly contribute generation vivo.