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J Alzheimers Dis. 2019 Oct 8. doi: 10.3233/JAD-190898. [Epub ahead of print]

Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products.

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Center for Alzheimer Research, Division of Neurogeriatrics, Department of Neurobiology, Care Sciences, and Society, Karolinska Institutet, Solna, Sweden.
Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany.
Institute of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany.
Laboratory of Chemical Biology & Signal Transduction, The Rockefeller University, New York, NY, USA.
Karolinska University Hospital, Theme Aging, Stockholm, Sweden.
Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet, Stockholm, Sweden.


The amyloid-β protein precursor (AβPP) is critical in the pathophysiology of Alzheimer's disease (AD), since two-step proteolytic processing of AβPP generates the neurotoxic amyloid-β peptide (Aβ). We developed a dual fluorescence labeling system to study the exact subcellular location of γ-secretase cleavage of AβPP. The C-terminal tail of AβPP was fluorescently labeled using a SNAP-tag, while the Aβ region of AβPP was fluorescently tagged with a dye at a genetically-encoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in AβPP using a genetic code expansion strategy and afterwards, the reactive side-chain of the ncAA was coupled to the dye using a bioorthogonal labeling chemistry. In proof-of-concept experiments, HEK293T cells were transfected with plasmids containing engineered AβPP harboring an amber mutation and an amber codon suppression system with an evolved tRNA synthetase/tRNA pair and grown in the presence of a lysine-derived ncAA. Processing of the AβPP variants was validated with ELISA and immunoblotting, and seven AβPP mutants that showed similar cleavage pattern as wild-type AβPP were identified. The AβPP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, AβPP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of Aβ and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label Aβ with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of AβPP.


Alzheimer’s disease; amber codon; amyloid-β precursor protein; cell biology; click chemistry; confocal microscopy; γ-secretase


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