Brain cathepsin B but not metalloendopeptidases degrade rAPP751 with production of amyloidogenic fragments. Comparison with synthetic peptides emulating beta- and gamma-secretase sites.
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.
Department of Neurochemistry, Nathan Kline Institute for Psychiatric Research, Orangeburg, New York, USA.
N
N
Marks
M J
MJ
Berg
V S
VS
Sapirstein
R
R
Durrie
J
J
Swistok
R C
RC
Makofske
W
W
Danho
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002401:Cathepsin B / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism*; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D006020:Glycopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D008666:Metalloendopeptidases; C008890:phosphoramidon