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. 2020 Apr 17;15(4):1059-1066.
doi: 10.1021/acschembio.0c00074. Epub 2020 Mar 2.

Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells

Daniel H Ramirez  1 , Chanat Aonbangkhen  1 , Hung-Yi Wu  1 , Jeffrey A Naftaly  1 , Stephanie Tang  1 , Timothy R O'Meara  1 , Christina M Woo  1
Affiliations

Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells

Daniel H Ramirez et al. ACS Chem Biol. .

Abstract

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.

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Figures

Figure 1.
Figure 1.
Overview of proximity-directed OGT strategy. A. Schematic of dynamic O-GlcNAc modification of protein substrates. B. Linear representation of natural OGT isoforms ncOGT, mOGT, and sOGT. C. Strategy for selective induction of O-GlcNAc using a proximity-directed nanobody-OGT to transfer O-GlcNAc to the target protein. D. Linear representation of nanobody-OGT(13) and nanobody-OGT(4) fusion proteins.
Figure 2.
Figure 2.
Characterization of nanobody-OGT(13) as proximity-directing glycosyltransferase constructs. A. Linear representation of full-length OGT(13), RFP(13), and nGFP(13). B. Subcellular localization of OGT(13), RFP(13), and nGFP(13) constructs expressed in HEK293T cells by confocal fluorescent microscopy. Scale bars represent 10 μm. C. Western blot of O-GlcNAc levels on GFP-Flag-JunB-EPEA after immunoprecipitation with EPEA-beads from HEK293T cells. The expression of the various constructs was verified by Western blot analysis (10% input). D. Quantification of OGT expression. E. Quantification of O-GlcNAc levels on GFP-Flag-JunB-EPEA after normalization to OGT expression. F. Quantification of O-GlcNAc levels in whole cell lysate after normalization to OGT expression. Data are representative of three biological replicates per experiment. Error bars represent standard deviation, * represents a p-value <0.05 under a two-tailed t-test. Full blot and confocal images can be found in Figure S1–S2.
Figure 3.
Figure 3.
Characterization of nanobody-OGT(4) as proximity-directing glycosyltransferase constructs. A. Linear representation of TPR truncated OGT(4), RFP(4), nGFP(4), nEPEA(4), and catalytically inactive mutants. B. Subcellular localization of OGT(4), nGFP(4), and nEPEA(4) in HEK293T cells by confocal fluorescent microscopy. Scale bars represent 10 μm. C. Western blot and quantification of O-GlcNAc levels on GFP-Flag-JunB-EPEA after immunoprecipitation with EPEA-beads. The expression of the various constructs was verified by Western blot analysis (10% input). D. Western blot and quantification of O-GlcNAc levels on GFP-Flag-JunB-EPEA after immunoprecipitation with EPEA-beads. The expression of the various constructs was verified by Western blot analysis (10% input). E. Western blot and quantification of O-GlcNAc levels on JunB-Flag-EPEA after immunoprecipitation with EPEA-beads. The expression of the various constructs was verified by Western blot analysis (10% input). F. Western blot and quantification of O-GlcNAc levels on GFP-Flag-JunB-EPEA after immunoprecipitation with EPEA-beads. The expression of the various constructs was verified by Western blot analysis (10% input). G. Western blot and quantification of O-GlcNAc levels on JunB-Flag-EPEA, cJun-Flag-EPEA, and Nup62-Flag-EPEA after immunoprecipitation with EPEA-beads from α-syn knocked-out (KO) HEK293T cells co-transfected with the indicated nanobody-OGT fusion protein and target protein. The expression of the various constructs was verified by Western blot analysis (10% input). At least three biological replicates were performed per experiment. Error bars represent standard deviation, * represents p ≤ 0.05, ** represents p ≤ 0.01, *** represents p ≤ 0.001, **** represents p ≤ 0.0001 under a two-tailed t-test or one-way ANOVA. Full blot and confocal images can be found in Figures S4–S9.
Figure 4.
Figure 4.
Quantitative proteomics and glycoproteomics of the global O-GlcNAc proteome from α-syn KO HEK293T cells. A. Quantitative proteomics of enriched O-GlcNAcylated proteins from α-syn KO HEK293T cells after co-expression of nEPEA(4) (highlighted in blue) and JunB-Flag-EPEA (highlighted in red) compared to expression of JunB-Flag-EPEA alone (control). B. Venn diagram and list of unambiguous glycosite assignments for JunB-Flag-EPEA. C. Venn diagram and list of unambiguous glycosite assignments for Nup62-Flag-EPEA. The target protein was co-expressed with the indicated OGT fusion protein in α-syn KO HEK293T cells, immunoprecipitated, and analyzed by MS. Only mono-glycosylated peptides with unambiguous assignments and a PSM count > 2 across biological replicates are shown. Three biological replicates were performed per experiment.
Figure 5.
Figure 5.
Western blot and quantification of O-GlcNAc induced to α-synuclein by a mass shift assay. The indicated nanobody-OGT construct was expressed in HEK293T cells, the cells were lysed, chemoenzymatically labeled, and analyzed by mass shift assay. Global O-GlcNAc levels and the expression of the nanobody-OGT constructs was verified by Western blot analysis (10% input). Six biological replicates were performed per experiment. Error bars represent standard deviation, ns represents p ≥ 0.05, * represents p ≤ 0.05, ** represents p ≤ 0.01, **** represents p ≤ 0.0001 under a two-tailed t-test or one-way ANOVA. Full blots can be found in Figure S10.
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