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The events leading to the formation of beta-amyloid (betaA4) from its precursor (betaAPP) involve proteolytic cleavages that produce the amino and carboxyl termini of betaA4. The enzyme activities responsible for these cleavages have been termed beta- and gamma-secretase, respectively, although these protease(s) have not been identified. Since betaA4 is known to possess heterogeneity at both the amino and carboxyl termini, beta- and gamma-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 betaAPP since this enzyme has been widely proposed as a gamma-secretase candidate. Treatment of a synthetic peptide that spans the gamma-secretase site of betaAPP 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 betaAPP substrates (156 and 100 amino acids of betaAPP carboxyl terminus) in vitro. The precise cathepsin D cleavage sites within these recombinant betaAPP 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 gamma-secretase sites: Val40-Ile41 or Ala42-Thr43, suggesting that cathepsin D is not gamma-secretase as defined by these betaA4 termini. Under conditions where the betaAPP156 substrate was first denatured prior to cathepsin D digestion, two additional cleavage sites near the amino terminus of betaA4, Glu-3-Val-2 and Glu3-Phe4, were observed, indicating that cathepsin D cleavage of betaAPP is influenced by the structural integrity of the substrate. Taken together, these results indicate that in vitro, cathepsin D is unlikely to function as gamma-secretase; however, the ability of this enzyme to efficiently cleave betaAPP substrates at nonamyloidogenic sites within the molecule may reflect a role in betaAPP catabolism.
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