ADAMTS4 as an enzyme cleaving at APP669 site

Abstract 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 ratio in human plasma, correlates with amyloid PET status (Nakamura et al., Nature 2018). Our previous research using cultured cells has suggested that A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) is involved in APP669‐711 production. Here, we present further evidence on the involvement of ADAMTS4 in the APP669 site‐cleavage. Method We prepared a recombinant APP substrate, which is APP81 corresponding to APP619‐699 sequence with a V5/His tag at the C‐terminus and a FLAG tag at the N‐terminus (FLAG‐APP81‐V5/His) for in vitro cleavage assay. Recombinant human ADAMTS4 protein and the tagged APP81 were incubated either alone or together at 37°C. The incubated samples were desalted and then analyzed by MALDI‐TOF MS. In vivo evaluation of ADAMTS4, we analyzed plasma samples from Adamts4‐/‐ mice and WT mice using IP‐MALDI‐MS. Result In vitro cleavage assay, N‐ and C‐terminal fragments cleaved at APP669, Aβ4 th , Aβ12 th site were detected in the mixture of the recombinant APP81 and ADAMTS4 proteins but not in the sample of substrate or enzyme alone. Furthermore, a peptide derived from double digestion by APP669 and Aβ12 th site‐cleavages (i.e., APP669‐Aβ11) was also identified by MS/MS analysis. The analysis of mouse plasma showed that murine APP669‐711 level was decreased in Adamts4‐/‐ mice compared to WT mice. Conclusion These results indicated that ADAMTS4 is one of the enzymes that can cleave the APP669 site directly and contribute to the generation of APP669‐711 in vivo.