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Identification of β-secretase-like activity using a mass spectrometry-based assay system

Nature Biotechnology volume 18, pages 66–70 (2000)Cite this article

Abstract

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 β-secretase, the protease that cleaves amyloid precursor protein (APP) at the β-site. Cathepsin D was identified as the enzyme responsible for β-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 β-site but show almost no reactivity with wild-type APP. Treatment of cell lines with pepstatin inhibited the production of amyloid peptide (Aβ) 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 Aβ was increased. This suggests that more than one enzyme is capable of generating Aβ in vivo and that an aspartic protease is involved in the processing of Swedish mutant APP.

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Figure 1: MS assay concept.
Figure 2: Example of β-secretase–like activity detected in crude cell extracts.
Figure 3: Example of an MS profile and its representation by the data system.
Figure 4: Partial purification of in vitro β-secretase.
Figure 5: Differential effect of pepstatin treatment on the amount of Aβ generated from APP-WT or APP-SW transfected HEK cells.

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Acknowledgements

We would like to thank Nicole Soder, Heidi Ortolf, Daniela Hügin, Veronique Horny, Daniel Mona, Peter Jakob, and Daniel Roeder for excellent technical assistance and Manfred Brockhaus, Hansruedi Loetscher, Georg Schmid, Jürgen Schläger, Beat Wipf, and Rommy von Bernhardi for their practical and scientific support. We are also very grateful to Prof. John Kay for generously providing purified cathepsin E and cathepsin E clone.

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Authors and Affiliations

  1. CNS Research, Hoffmann-La Roche Ltd, Grenzacherstrasse 124, Basel, CH-4070, Switzerland

    Fiona Grüninger-Leitch, Peter Nelboeck & Heinz Döbeli

  2. Roche Genetics, Hoffmann-La Roche Ltd, Grenzacherstrasse 124, Basel, CH-4070, Switzerland

    Peter Berndt & Hanno Langen

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  1. Fiona Grüninger-Leitch
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  2. Peter Berndt
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Correspondence to Heinz Döbeli.

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Grüninger-Leitch, F., Berndt, P., Langen, H. et al. Identification of β-secretase-like activity using a mass spectrometry-based assay system. Nat Biotechnol 18, 66–70 (2000). https://doi.org/10.1038/71944

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