Cathepsin D displays in vitro β-secretase-like specificity

Abstract

The formation of A β and A β-containing fragments is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Asp-1) of A β and its C-terminally extended sequences is liberated from the β-amyloid precursor protein (βAPP) by β-secretase(s). This activity appears highly increased by the presence (N-terminally to Asp-1) of a double-mutation (KM → NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the 'normal' (N peptide) and mutated (ΔNL peptide) sequences targeted by β-secretase(s), we have detected a human brain protease displaying preferred efficiency for the ΔNL peptide than for the non-mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PC12 cells and primary cultured astrocytes. The pepstatin sensitivity and pH maximum of the brain activity that appeared reminiscent of those displayed by the acidic protease cathepsin D led us to examine this enzyme as a putative β-secretase-like candidate. Purified cathepsin D displays higher catalytic parameters for the ΔNL peptide than for the non-mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. However, cathepsin D does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block A β secretion by transfected cells. Furthermore, cathepsin D hydrolyses recombinant baculoviral ΔNL βAPP751 at a 6-fold higher rate than βAPP751 and gives rise to a 12-kDa C-terminal product that is recognized by antibodies fully specific of the N-terminus of A β. Altogether, our study indicates that cathepsin D displays several in vitro β-secretase-like properties that suggests that this protease could fulfill such a role, at least in the Swedish genetic form of Alzheimer's disease.