Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-β by cathepsin D

Amyloid β (Aβ) is a 39-43-residue protein that originates from proteolysis of the β-protein precursor (βPP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Mutant βPP, which incorporates an AD-causing double mutation at positions 687-688, has been shown to enhance Aβ production in transfected cells. In this work we investigate the susceptibility of the mutant βPP sequence to proteolytic cleavage by proteinases from human brain. Internally quenched fluorogenic substrates were used that encompass the NH₂-terminal sequence of Aβ from wild-type βPP, 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 Aβ at positions equivalent to Thr⁴³ and Ala⁴², at ratios of 68% and 32%, respectively. CD degraded Aβ 1-40 into six fragments but Aβ 1-42 was completely resistant to digestion, probably because of its aggregation characteristics. These results indicate that CD is capable of producing the cleavage resulting in Aβ production and that it may prove to be a suitable therapeutic target.