Directed evolution of LaccID for cell surface proximity labeling and electron microscopy

Abstract

Enzymes that oxidize aromatic substrates have been harnessed for cell-based technologies including proximity labeling and electron microscopy; however, they are associated with drawbacks such as the need for toxic H₂O₂. Here, we explore multicopper oxidases (laccases) as a new enzyme class for proximity labeling and electron microscopy in mammalian cells. LaccID was generated through 11 rounds of directed evolution from an ancestral fungal laccase and catalyzes one-electron oxidation of diverse aromatic substrates using O₂ instead of toxic H₂O₂. Surprisingly, we found that LaccID is selectively active at the surface plasma membrane of both living and fixed cells. We use LaccID proximity labeling and mass spectrometry to map the changing surface proteome of T cells that engage with tumor cells through antigen-specific T cell receptors. In addition, we use LaccID as a genetically encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

Fig. 1. Directed evolution of LaccID.

a, LaccID is versatile, like APEX2, because it can be used for both proximity labeling and EM. LaccID is also minimally toxic, like TurboID, because it uses O₂ instead of toxic H₂O₂. B, biotin. b, AlphaFold2-predicted structure of LaccID. Four copper atoms (blue) are distributed in two copper centers (one Cu atom in type I and three Cu atoms in type II and III centers). Cu-coordinating side chains (H64, H109, H111, H396, H399, H401 and H457) are shown in yellow; residues involved in electron transfer (H451, C452 and H453) are shown in black; seven mutations introduced by directed evolution are shown in red. c, Initial screening of four laccase templates: LacAnc100 (ref. ), ChU-B, SCHEMA and KyLO. Enzymes were fused to the transmembrane (TM) domain of human CD4 for expression on the surface of HEK293T cells. Labeling was performed for 1 h with 500 μM biotin-phenol (BP), then cell lysates were analyzed by streptavidin blotting. For comparison, HRP labeling was performed for 1 min with 500 μM BP and 1 mM H₂O₂. *Self-labeling of LacAnc100. Arrows point to endogenous biotinylated proteins. Untrans., untransfected. This experiment was performed three times with similar results. d, Yeast surface display selection scheme. PE, phycoerythrin. e, Selection conditions used over three generations of directed evolution. The winning clone from each generation was named G1, G2 and G3, respectively. G3 is LaccID. f, FACS density plots summarizing the progress of directed evolution. Yeast cells displaying LacAnc100 or G1–G3 (LaccID) were compared by labeling with 50 μM BP for 30 min and then staining with streptavidin–PE and anti-MYC antibody. This experiment was performed three times with similar results. Percentages were calculated as a fraction of MYC⁺ cells with streptavidin–PE staining above background. g, Comparison of LacAnc100 and evolved clones on the surface of HEK293T cells. Expression of cell surface-targeted constructs was induced with doxycycline overnight and then labeling was performed for 1 h with 500 μM BP in EBSS. *Self-labeling of LaccID. Arrows point to endogenous biotinylated proteins. Uninduc., uninduced. Right, quantification of streptavidin blot data. Data are presented as the mean values ± s.d. of three biological replicates. Source data

Fig. 2. Characterization of LaccID.

a, LaccID labeling in different cell culture media. HEK293T cells expressing surface LaccID were labeled for 2 h with 500 μM BP. The molecular mass of LaccID is 53 kDa without glycosylation. This experiment was performed twice with similar results. b, LaccID labeling with BP versus BMP. HEK293T cells expressing surface LaccID were labeled for 1 h with 500 μM probe in EBSS. This experiment was performed three times with similar results. c, LaccID comparison to HRP and APEX2 on the surface of HEK293T cells. For LaccID, labeling was performed with 500 μM BP in EBSS or 500 μM BMP in RPMI. For HRP and APEX2, labeling was performed with 500 μM probe and 1 mM H₂O₂ in DMEM. This experiment was performed twice with similar results. d, Confocal imaging of cells labeled as in c. N-cadherin is a plasma membrane marker. Neutravidin detects biotinylated proteins. Anti-V5 antibody detects enzyme expression. This experiment was performed twice with similar results. e, Mutations impacting the HCH motif (H450A-C451A-H452A) abolish LaccID activity. HEK293T cells expressing LaccID or mutant LaccID were labeled for 1 h with 500 μM BP in EBSS. This experiment was performed three times with similar results. f, LaccID requires O₂. HEK293T cells expressing surface LaccID were labeled with 500 μM BP in PBS for 1 h. Glucose oxidase (GOase) and glucose were used to deplete oxygen in the culture medium. This experiment was performed twice with similar results. g, Thermal stability of LacAnc100 (template) and LaccID. Purified enzymes were incubated in citrate–phosphate pH 6.0 buffer at temperatures ranging from 30 to 70 °C and then assayed for activity at pH 4.0 with ABTS substrate. The dotted line shows T₅₀. Data are presented as the mean ± s.d. of three technical replicates. h, pH activity profile for LacAnc100 and LaccID. Purified enzymes were assayed with the substrate guaiacol at the indicated pH values. Conversion to product measured by absorbance at 470 nm. Additional data with other substrates (ABTS, DMP, p-coumaric acid, and sinapic acid) are presented in Supplementary Fig. 5c,d. Data are presented as the mean ± s.d. of three biological replicates. i, Kinetic constants for LacAnc100 and LaccID at pH 6.0 with guaiacol. Additional data with other substrates are presented in Supplementary Fig. 5e. Source data

Fig. 3. LaccID is selectively active on the cell surface.

a, Confocal imaging of enzymes targeted to the cell surface or ER lumen of HEK293T cells. LaccID samples were labeled with BP for 1 h in EBSS. HRP and APEX samples were labeled for 1 min with BP and H₂O₂ in DMEM. The cells were then fixed and stained with anti-V5 antibody to detect expression, neutravidin–AF647 to detect biotinylated proteins and organelle markers as indicated. This experiment was performed twice with similar results. b, Promiscuous biotinylation activity of enzymes at the surface (S) or in the ER lumen of HEK293T cells. Cells were labeled with BP as in a and then blotted with streptavidin and anti-V5 antibody. The molecular mass of APEX2 is 30.9 kDa and the higher band may be a crosslinked dimer. This experiment was performed twice with similar results. c, Western blot detection of proteins enriched by LaccID, HRP or APEX, targeted to the cell surface or ER lumen. Lysates from HEK293T cells labeled as in a were enriched with streptavidin beads and eluates were blotted for a surface marker protein (N-cadherin) or ER lumen marker protein (calreticulin). This experiment was performed twice with similar results. Source data

Fig. 4. LaccID is a genetically encoded EM tag.

a, LaccID generates EM contrast by catalyzing the oxidative polymerization of diaminobenzidine (DAB), which recruits electron-dense osmium that appears dark on EM micrographs. b, LaccID-catalyzed DAB staining of fixed cells. HEK293T cells expressing surface LaccID, HRP or APEX2 were fixed using 2% (v/v) glutaraldehyde, stained with DAB for indicated times and imaged by brightfield microscopy. This experiment was performed twice with similar results. c, EM imaging of HEK293T cells expressing LaccID targeted to the cell surface through fusion to the TM domain of CD4. Cells were fixed using 2% glutaraldehyde, incubated with DAB for 30 min and then postfixed with 1% osmium tetroxide for 1 h. Images are representatives of >3 fields of view. PM, plasma membrane; Nuc, nucleus; Mito, mitochondria. Right, negative control with LaccID omitted. d, Generation of neuron-specific LaccID transgenic flies. e, Schematic of the larval Drosophila central nervous system and the innervation of body wall muscles by motor neuron axons. LaccID is expressed in neurons of the brain and VNC. Inset shows motor neuron axons leaving the VNC to innervate muscles at NMJs, which are visualized by confocal microscopy in f. f, Representative confocal images of anti-V5 immunostained LaccID (epitope tagged with V5) in the VNC (left) or NMJ (right). Neuronal nuclei and membranes were labeled with anti-Elav and anti-HRP, respectively. Boxed regions are magnified to show LaccID expression in neuronal cell bodies and neuropil in the VNC (left) and at a motor neuron terminal (right). This experiment was performed twice with similar results. g, Representative EM images of DAB-stained fly VNC. Fixed VNC tissues from fly larvae were stained with Ce-DAB2 for 75 min, followed by postfixation with 1% osmium tetroxide for 1 h. Red arrows indicate plasma membrane (PM) labeling in LaccID-expressing samples (Elav > LaccID). Additional fields of view, as well as cerium detection by EELS and EFTEM, are shown in Supplementary Fig. 8c,d. This experiment was performed once.

Fig. 5. LaccID mapping of the T cell surface proteome in the presence of tumor cells.

a, Cocultures were established between LaccID-expressing T cells and A375 melanoma tumor cells. The tumor cells expressed the NY-ESO-1 tumor antigen, while T cells expressed a CAR (orange) or TCR (blue) directed toward NY-ESO-1. In a control sample (green), T cells did not express CAR or TCR. This image was created with BioRender.com. b, Design of 18-plex TMT proteomic experiment. All samples were treated with BMP for 2 h before quenching, lysis and streptavidin enrichment of LaccID-labeled proteins. Controls omit TCR and CAR (T cell samples, green), omit LaccID, or are not cocultured (monoculture samples). c, LaccID enriches cell surface proteins in all three monoculture samples. Golgi-resident and ER-resident proteins (lacking cell surface annotation) are de-enriched. d, Volcano plot comparing LaccID-labeled TCR-T cell cocultures to monocultures. Proteins with previous literature connections to T cell activation are colored red, those with indirect connections are colored orange, and previously- annotated downregulated proteins are colored blue (Supplementary Table 3). P values were computed using moderated t-tests without multiple-testing correction (cutoff: P < 0.01). e, GO terms enriched in top 50 TCR-T cell coculture versus monoculture dataset. GO enrichment analysis was performed using g:Profiler, which applies a cumulative hypergeometric test with Benjamini–Hochberg FDR correction for multiple comparisons.