title: Processing of beta-amyloid precursor protein by cathepsin D.
abstract: 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.
authors.affiliation: Scios, Inc., Mountain View, California 94043, USA.
authors.forename: J
authors.initials: J
authors.lastname: Higaki
authors.forename: R
authors.initials: R
authors.lastname: Catalano
authors.forename: A W
authors.initials: AW
authors.lastname: Guzzetta
authors.forename: D
authors.initials: D
authors.lastname: Quon
authors.forename: J F
authors.initials: JF
authors.lastname: Navé
authors.forename: C
authors.initials: C
authors.lastname: Tarnus
authors.forename: H
authors.initials: H
authors.lastname: D'Orchymont
authors.forename: B
authors.initials: B
authors.lastname: Cordell
mesh_terms: D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D015153:Blotting, Western; D002402:Cathepsin D / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008969:Molecular Sequence Data
chemical_list: D016564:Amyloid beta-Protein Precursor; D002402:Cathepsin D