Identification of beta-secretase-like activity using a mass spectrometry-based assay system.
We describe an assay system for the identification of site-specific proteases. The assay is based on a protein substrate that is immobilized on ceramic beads. After incubation with cell homogenates, the beads are washed and digested with endoproteinase Lys-C to liberate a defined set of peptides. The peptide fragments are identified by mass spectrometry. The assay was used to screen for beta-secretase, the protease that cleaves amyloid precursor protein (APP) at the beta-site. Cathepsin D was identified as the enzyme responsible for beta-secretase-like activity in two cell lines. Subsequent analysis of the related aspartic protease, cathepsin E, revealed almost identical cleavage specificity. Both enzymes are efficient in cleaving Swedish mutant APP at the beta-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Abeta) when they were transfected with a construct bearing the Swedish APP mutant. However, when the cells were transfected with wild-type APP, the generation of Abeta was increased. This suggests that more than one enzyme is capable of generating Abeta in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.
Grüninger-Leitch
F
F
CNS Research, Hoffmann-La Roche Ltd, Grenzacherstrasse 124, CH-4070 Basel, Switzerland.
Berndt
P
P
Langen
H
H
Nelboeck
P
P
Döbeli
H
H
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D002402:Cathepsin D / Q000737:chemistry / Q000378:metabolism*; D020059:Cathepsin E / Q000235:genetics / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002516:Ceramics; D010450:Endopeptidases; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008863:Microspheres; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010436:Pepstatins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity; D013548:Sweden; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D002457:Cell Extracts; D010436:Pepstatins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C014195:Streptomyces pepsin inhibitor; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; D020059:Cathepsin E; C509284:BACE2 protein, human; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse; C509281:Bace1 protein, rat; D002402:Cathepsin D; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; C031375:pepstatin