Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-beta by cathepsin D.
Amyloid beta (A beta) is a 39-43-residue protein that originates from proteolysis of the beta-protein precursor (beta PP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Mutant beta PP, which incorporates an AD-causing double mutation at positions 687-688, has been shown to enhance A beta production in transfected cells. In this work we investigate the susceptibility of the mutant beta PP sequence to proteolytic cleavage by proteinases from human brain. Internally quenched fluorogenic substrates were used that encompass the NH2-terminal sequence of A beta from wild-type beta PP, the double mutant, and the two single substitutions. Proteinase activity in brain extract cleaved the mutant substrate 100-fold faster than the wild-type substrate and the partial mutants 25-fold faster. The major cleavage site in all substrates was at the amyloidogenic Asp1 site. The brain activity appeared to be cathepsin D (CD), as indicated by similarities to purified CD in 1) the rate and site of substrates cleavage, 2) the pH optima, and 3) the sensitivity to pepstatin A. The increased activity against the mutant substrate was not shared by cathepsins B and C, pepsin, HIV proteinase, and Candida albicans Asp-proteinase. Furthermore, CD cleaved a substrate that incorporates the COOH terminus of A beta at positions equivalent to Thr43 and Ala42, at ratios of 68% and 32%, respectively. CD degraded A beta 1-40 into six fragments but A beta 1-42 was completely resistant to digestion, probably because of its aggregation characteristics. These results indicate that CD is capable of producing the cleavages resulting in A beta production and that it may prove to be a suitable therapeutic target.
Ladror
U S
US
Abbott Laboratories, Abbott Park, Illinois 60064.
Snyder
S W
SW
Wang
G T
GT
Holzman
T F
TF
Krafft
G A
GA
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D001921:Brain / Q000201:enzymology*; D002402:Cathepsin D / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D005625:Frontal Lobe / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D017354:Point Mutation; D013379:Substrate Specificity; D013997:Time Factors
D016229:Amyloid beta-Peptides; D009842:Oligopeptides; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D010450:Endopeptidases; D002402:Cathepsin D; C031375:pepstatin