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

Abstract

The amyloid-␤ protein precursor (A␤PP) is critical in the pathophysiology of Alzheimer's disease (AD), since two-step proteolytic processing of A␤PP generates the neurotoxic amyloid-␤ peptide (A␤). We developed a dual fluorescence labeling system to study the exact subcellular location of ␥-secretase cleavage of A␤PP. The C-terminal tail of A␤PP was fluorescently labeled using a SNAP-tag, while the A␤ region of A␤PP was fluorescently tagged with a dye at a geneticallyencoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in A␤PP 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 A␤PP 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 A␤PP variants was validated with ELISA and immunoblotting, and seven A␤PP mutants that showed similar cleavage pattern as wild-type A␤PP were identified. The A␤PP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, A␤PP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of A␤ and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label A␤ with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of A␤PP.

Minimal genetically encoded tags for fluorescent protein labeling in living neurons

“…As a proof-of-principle, they showed incorporation of O-methyltyrosine in a reporter GFP TAG protein in neurons. In the meantime, other types of UAAs were applied in further studies of neuronal proteins, such as irreversible or reversible light-induced control of NMDA-and AMPA-type glutamate receptors with photoresponsive UAAs [34][35][36][37] , fluorescent labeling of NMDA receptors 38 and the Shaker B voltage-dependent potassium channel 39 , as well as the development of reporters for amyloid precursor protein trafficking and processing 40,41 . However, all of these studies were performed using standard cell lines, and the number of studies showing UAA incorporation in neurons has remained limited.…”

Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint

“…Dual-color labeling of different proteins in the same cell using GCE is currently a challenging task, as it requires exploiting two codons from the codon table and introducing two mutually orthogonal tRNA/ tRNA synthetase pairs to the cells, which is extremely difficult in mammalian cells [46]. Although unAAbased labeling can be readily combined with conventional protein tags for a dual-color labeling [35,47], several laboratories proposed strategies to overcome the challenge of GCE-based dual-color labeling [3,46].…”

“…The ratio between the specific labeling of the POI and the unspecific labeling of other proteins in the cell can be quantified by using in-gel fluorescence [22,23,30,36,47]. In addition, the overall background level in cells can be evaluated by measuring the signal intensity after adding all the GCE and click reaction components to the cells-except the TAG-modified POI [22,23,30,35,36,38,47]. Despite these potential background-related limitations, GCEbased labeling demonstrates relatively high signal-tonoise ratios (SNRs), which enable quantitative imaging in either fixed or live cells [24,35,36].…”

“…Such cases may result in the unspecific labeling of endogenous proteins, which will increase background levels. The ratio between the specific labeling of the POI and the unspecific labeling of other proteins in the cell can be quantified by using in‐gel fluorescence [22,23,30,36,47]. In addition, the overall background level in cells can be evaluated by measuring the signal intensity after adding all the GCE and click reaction components to the cells—except the TAG‐modified POI [22,23,30,35,36,38,47].…”

A Genetic Code Expansion‐Derived Molecular Beacon for the Detection of Intracellular Amyloid‐β Peptide Generation

“…The cy5 labeling pattern also indicated we captured post‐endocytic APP and its derivatives, as the APP(H609BCNK)‐EGFP subpopulation residing at the ER envelope that has not yet trafficked to the cell surface is not labeled with cy5. The ability to precisely track post‐endocytic APP and its processing differentiated our reporter from other dual‐labeled APP reporter designs [17] …”