Brain cathepsin B but not metalloendopeptidases degrade rAPP751 with production of amyloidogenic fragments

Lysosomal cathepsin B but not L degraded rAPP751 to yield C-terminal 19-25 kDa fragments containing beta A4, reinforcing the view that acidic proteases participate in endosomal-lysosomal processing to yield amyloidogenic fragments in situ. This mechanism is consistent with fragmentation of endogenous APPs within clathrin-coated vesicles (CVs) by vesicular hydrolases, with the appearance of C-terminal amyloidogenic fragments following incubation at pH 6.5. A neutral endopeptidase resembling NEP 24.11 (PS-NEP) purified from detergent extracts of human brain degraded rAPP751; however, breakdown was not blocked robustly by metal chelators or phosphoramidon, suggesting the presence of an alternative processing enzyme. Effects of other inhibitors showed that breakdown was mediated by serine-protease-like component(s). A phosphoramidon-insensitive metalloendopeptidase (PI-NEP) partially purified from rat brain P2 using detergents, and resembling NEP 24.15, showed no activity towards rAPP751. Peptides containing putative beta- or gamma-secretase sites were synthesized for purposes of examining their metabolism by the brain enzymes. Those containing beta-secretase sites were hydrolysed at one or more sites by the four enzymes, but only PI- and PS-NEP acted at the Met-Asp site of Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg.NH2. In the case of substrates containing the gamma-site, these two categories of enzymes were the only ones degrading N-Ac-Ile-Ala.NH2. These data imply that the brain metalloendopeptidases, while inactive towards intact precursors, may be involved in turnover of intermediates containing beta- or gamma-sites.