Production pathway of APP669‐x peptides in the cultured cells

Abstract

AbstractBackgroundWe have 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), and revealed that the composite biomarker using APP669‐711/Aβ1‐42 ratio correlates with amyloid PET status (Kaneko et al., 2014; Nakamura et al., 2018). We also recently found that A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) is responsible for the APP669‐site cleavage of APP (Matsuzaki et al., Mol Psychiatry 2023). Here, we analyzed the production pathway APP669‐x peptides from the C‐terminal stub c102 of APP, which is c99 with 3 extra N‐terminal amino acid residues (i.e., the cleaved APP fragment at APP669‐site).MethodWe established the endo‐specific antibody against the N terminus of APP669‐x peptide, an anti‐c102 antibody. We analyzed the effect of ADAMTS4 on the production of Aβ‐related peptides from the APP‐expressing HEK293 cells using IP‐MALDI‐MS.ResultWe successfully detected the endogenous C‐terminal fragment of APP specifically reacted with anti‐c102 antibody in A549 cells. We found that the overexpression of the recombinant c102 resulted in the production of a series of APP669‐x peptides by the γ‐secretase. No Aβ starting at the 1st nor 4th position was generated from c102‐expressing cells irrespective of ADAMTS4 expression. Co‐expression of ADAMTS4 with APP increased the production of both APP669‐711 and Aβ4‐40. However, the addition of extracellular ADAMTS4 to HEK293 cells expressing APP resulted in the increase in the Aβ4‐40, but not APP669‐711 nor Aβ1‐40, suggesting that no further N‐terminal trimming of c102 is occurred within the cells after ADAMTS4‐mediated APP669 cleavage.ConclusionThese results indicated that ADAMTS4 cleaves APP in the secretory pathway to generate c102, which is a direct substrate for the γ‐secretase.