CATHEPSIN-D DISPLAYS IN-VITRO BETA-SECRETASE-LIKE SPECIFICITY

The formation of A beta and A beta-containing fragments is likely a ke y event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Asp-1) of A beta and its C-terminally extended sequences is liberated from the beta-amyloid precursor protein (beta APP) by beta-secretase(s). This activity appears highly increased by t he presence (N-terminally to Asp-1) of a double-mutation (KM --> NL) f ound in several Swedish families affected by early onset Alzheimer's d isease. By means of synthetic peptides encompassing the 'normal' (N pe ptide) and mutated (Delta NL peptide) sequences targeted by beta-secre tase(s), we have detected a human brain protease displaying preferred efficiency for the Delta NL peptide than for the non-mutated analog. T his activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage si tes. Such acidic activity is also detected in rat brain, PC12 cells an d primary cultured astrocytes. The pepstatin sensitivity and pH maximu m of the brain activity that appeared reminiscent of those displayed b y the acidic protease cathepsin D led us to examine this enzyme as a p utative beta-secretase-like candidate. Purified cathepsin D displays h igher catalytic parameters for the Delta NL peptide than for the non-m utated peptide, cleaves these two substrates at the expected M/D or L/ D sites, and is maximally active at acidic pH. However, cathepsin D do es not cleave peptides bearing mutations that were previously shown to drastically lower or fully block A beta secretion by transfected cell s. Furthermore, cathepsin D hydrolyses recombinant baculoviral Delta N LP beta APP(751) at a 6-fold higher rate than beta APP(751) and gives rise to a 12-kDa C-terminal product that is recognized by antibodies f ully specific of the N-terminus of A beta. Altogether, our study indic ates that cathepsin D displays several in vitro beta-secretase-like pr operties that suggests that this protease could fulfill such a role, a t least in the Swedish genetic form of Alzheimer's disease. (C) 1997 E lsevier Science B.V. All rigths reserved.