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

Abstract Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often‐small size and intricate post‐translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue‐specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid‐β peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.


Reference
. APP expression via genetic code expansion. (a) Criteria for amino acid position selection to be mutated to an amber stop codon for genetic code expansion. (b,c) Confirming BCNK-dependent expression of APP. HEK293 cells were transfected with transgenes encoding various APP(TAG)-myc mutants plasmids, incubated 45 hours in the presence or absence of 60 µM BCNK and analysed on western blots using anti-α-myc antibody. TAG mutation is represented by *. APP m and APP im indicate bands corresponding to mature-APP and immature-APP, respectively. (d) Quantification of intensity ratio between mature-APP and immature-APP of various APP(TAG) mutants.    Figure S5. Detection of amyloid-β species via antibody and amyloid plaque-specific dye. (a) High background upon performing Western blotting with anti-amyloid antibody. HEK293 expressing APP(H609BCNK)-myc were lysed with RIPA buffer either immediately (0h) or 2-hour to allow APP processing (2h), before analysis by Western Blotting with anti-Aβ antibody (Merck AB5078P). Controls were shown with untransfected (Un) cells, and HEK293 cells expressing exogenous Aβ40 and Aβ42. (b) Specific labeling could be seen upon immunofluorescence staining and imaging with anti-amyloid antibody. HEK293 expressing APP(H609BCNK)-myc were labeled live with 1.2 µM tetrazine-cy5 at 4 °C for 30 minutes and further grown for additional 2 hours at 37 °C to permit APP internalization and processing before fixation. Cells were counterstained with anti-myc for APP expression, and anti-Aβ for detection of amyloid-β. Yellow arrows indicate intact APP (coincident mycstaining and cy5 label) that was stained with anti-Aβ. White arrows indicate liberated Aβ (standalone cy5 signal) that was stained with anti-Aβ. (c) Plaque-specific Amylo-Glo failed to specifically label intracellular APP and Aβ. Amylo-glo staining was performed per manufacturer's instruction.

Plasmid assembly
The APP mutant constructs were cloned via site-directed mutagenesis using pEGFP-n1-APP695 plasmid (Addgene #69924) as a template. The site-directed mutagenesis was carried out by PCR amplification using mutagenic primers to place the amber stop codon (TAG (*)) at a desired position within the amyloid-β (Aβ) segment of the APP including H602*, D603*, H609*, H610* and Q611*.
Subsequently, all APP mutants as well as the wild-type APP gene were subcloned into a previously reported plasmid (pPB) containing 4 copies of amber-suppressor Methanosarcina mazei pyrrolysyl tRNA for genetic code expansion in mammalian cells [1] using restriction cloning at the NheI and NotI restriction sites. To construct the APP-EGFP reporter plasmid, pEGFP-n1-APP(H609TAG) was digested with NheI and NotI restriction enzymes and sub-cloned into the pPB vector to generate pPB-APP(H609TAG)-EGFP_4xPylT. To construct the APP-HaloTag molecular beacon reporter plasmid, HaloTag was amplified from YIP-aga1P-HaloTag and subcloned into pPB-APP(H609TAG)-EGFP_4xPylT via restriction enzyme digest (with AgeI and NotI sites to remove EGFP and replace with HaloTag) to obtain pPB_APP(H609TAG)-Halotag_4xPylT. pPB-MmPylRS-AF-4xPylT was a gift from Jason Chin.

Live cell labeling of amyloid precursor protein and Aβ
HEK293 cells were seeded into 6-well plates (Corning) for western blot analysis or on glass coverslips placed in 24-well plates for imaging studies. The coverslips were incubated for 1 hour with 0.1 mg/mL poly-D-lysine (Sigma) prior to cell plating. Upon reaching ~ 80% confluence, the media was exchanged for Optimem (Thermo Fisher), transfection mixture of DNA (pPB_APPs : MmPylRS-AF = 9 : 1), plus optional 20 ng of organelle marker plasmids including mApple-Rab7a-7 (Addgene #54945) or mApple-TGNP-N-10 (Addgene #54954)), lipofectamine-3000 and its accessory reagent (Invitrogen). After incubation for 6 hours at 37 °C, the media was exchanged for DMEM (Thermo Fisher) supplemented with 10% FBS (Thermo Fisher), 1% Pen-strep (Thermo Fisher) and 60 uM BCNK (Sichem, catalogue no. SC-8016). Subsequently, cells were grown in presence of BCNK for 45 hours. To label the amyloidβ (Aβ) segment of APP, cells were washed twice with PBS to remove excess BCNK. Cells were labeled membrane was washed several times with TBS-T and developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer's instructions. The signal on the membrane was photographed using ImageQuant LAS500 (GE Healthcare).

Fixed cell imaging
After labeling, the cells were fixed using 4% paraformaldehyde in PBS for 10 mins at room temperature.

In-gel cy5 fluorescence visualization of APP and Aβ
HEK293 cells expressing APP(H609BCNK)-α-myc, APP(H609BCNK)-EGFP or APP(H609BCNK)-HaloTag were labeled for 30 minutes with 1.2 µM tetrazine-cy5 in cold DMEM/FBS/Pen-strep and subsequently incubated at 37 °C for 2 hours post-labeling in order to allow proteolytic processing of APP as described in the previous section. Cells were lysed on ice for 20 minutes and the lysates were cleared by centrifugation at 20,000 g at 4 °C. The lysates were collected and protein concentration was normalized using BCA assay (Thermo Fisher). Afterwards, 20 µg protein from lysate was analyzed on 15% SDS-PAGE gels, fixed in buffer containing 30% ethanol and 10% acetic acid, and subsequently imaged using ChemiDoc TM MP Imaging System (BIO-RAD) with cy5 laser and filter setup.