CLEAVAGE AT THE AMINO AND CARBOXYL TERMINI OF ALZHEIMERS AMYLOID-BETABY 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 position s 687-688, has been shown to enhance A beta production in transfected cells. In this work we investigate the susceptibility of the mutant be ta 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 brai n extract cleaved the mutant substrate 100-fold faster than the wild-t ype substrate and the partial mutants 25-fold faster. The major cleava ge site in all substrates was at the amyloidogenic Asp(1) site. The br ain activity appeared to be cathepsin D (CD), as indicated by similari ties 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 a ctivity against the mutant substrate was not shared by cathepsins B an d C, pepsin, HIV proteinase, and Candida albicans Asp-proteinase. Furt hermore, CD cleaved a substrate that incorporates the COOH terminus of A beta at positions equivalent to Thr(43) and Ala(42), at ratios of 6 8% and 32%, respectively. CD degraded A beta 1-40 into six fragments b ut 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.