Expression and characterization of human beta-secretase candidates metalloendopeptidase MP78 and cathepsin D in beta APP-overexpressing cells.
Human beta-secretase candidates, MP78 (h-MP78, EC 3.4.24.15) and cathepsin D (Cat D, EC 3.4.23.5), were evaluated for their ability to enhance amyloid-beta-protein (A beta) secretion when overexpressed in beta APP-containing cells. HEK-293 cells stably co-expressing h-MP78 or Cat D and h-beta APP695 were metabolically labeled with [35S]methionine and A beta secretion was quantified in the conditioned media by immunoprecipitation and ELISA without showing any significant increase in A beta production. Because Cat D is known to have a higher affinity for APP-substrate containing the Swedish familial Alzheimer's disease double mutation (SFAD, K595N and M596L substitutions in beta APP695) than for the wild type substrate [Dreyer et al., Eur. J. Biochem., 224 (1994) 265-271], the effect of Cat D overexpression was tested in a HEK293/beta APPSFAD stable cell line. ELISA analysis of the conditioned media from these cells did also not reveal any increase in A beta generation. In addition, recombinant h-MP78 purified from E. coli cleaved an APP-derived substrate spanning the beta-secretase site (ISEVKMD1AEFRHDS) at multiple sites, but the beta-site cleavage was only a minor one; cleavage occurred predominantly at K-M and E-F bonds. Human liver Cat D also cleaved the same substrate at multiple sites, yet the major cleavage at pH 4.0 occurred at the amyloidogenic D1 site. These findings indicate that h-MP78 does not have the cleavage specificity required for a beta-secretase protease and although Cat D fulfilled the amyloidogenic cleavage specificity, the results of the co-expression experiments make both enzymes less likely candidates as relevant beta-secretases.
Thompson
A
A
Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Grueninger-Leitch
F
F
Huber
G
G
Malherbe
P
P
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis*; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D / Q000032:analysis / Q000096:biosynthesis*; D002460:Cell Line; D010450:Endopeptidases / Q000032:analysis / Q000096:biosynthesis*; D006801:Humans; D008666:Metalloendopeptidases / Q000032:analysis / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis
D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D002402:Cathepsin D; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase