Processing of β-amyloid precursor protein by cathepsin D

Abstract

The events leading to the formation of β-amyloid (βA4) from its precursor (βAPP) involve proteolytic cleavages that produce the amino and carboxyl termini of βA4. The enzyme activities responsible for these cleavages have been termed β- and γ-secretase, respectively, although these protease(s) have not been identified. Since βA4 is known to possess heterogeneity at both the amino and carboxyl termini, β- and γ-secretases may actually be a collection of proteolytic activities or perhaps a single proteolytic enzyme with broad amino acid specificity. We investigated the role of cathepsin D in the processing of βAPP since this enzyme has been widely proposed as a γ-secretase candidate. Treatment of a synthetic peptide that spans the γ-secretase site of βAPP with human cathepsin D resulted in the cleavage of this substrate at Ala42-Thr43. A sensitive liquid chromatography/mass spectrometry technique was also developed to further investigate the ability of cathepsin D to process longer recombinant βAPP substrates (156 and 100 amino acids of βAPP carboxyl terminus) in vitro. The precise cathepsin D cleavage sites within these recombinant βAPP substrates were identified using this technique. Both recombinant substrates were cleaved at the following sites: Leu49-Val50, Asp68-Ala69, Phe93- Phe94. No cleavages were observed at putative γ-secretase sites: Val40- Ile41 or Ala42-Thr43, suggesting that cathepsin D is not γ-secretase as defined by these βA4 termini. Under conditions where the βAPP156 substrate was first denatured prior to cathepsin D digestion, two additional cleavage sites near the amino terminus of βA4, Glu-3-Val-2 and Glu3- Phe4, were observed, indicating that cathepsin D cleavage of βAPP is influenced by the structural integrity of the substrate. Taken together, these results indicate that in vitro, cathepsin D is unlikely to function as γ-secretase; however, the ability of this enzyme to efficiently cleave βAPP substrates at nonamyloidogenic sites within the molecule may reflect a role in βAPP catabolism.