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Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.

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Fig. 1: Design and development of a nanobody-directed split OGA for O-GlcNAc removal in a protein-selective manner.
Fig. 2: Design and optimization of nanobody-fused split OGA for protein-selective deglycosylation on GFP–Nup62.
Fig. 3: The nanobody-fused split OGA is generalizable for protein-selective deglycosylation on various O-GlcNAcylated proteins.
Fig. 4: Multiple nanobody–tag pairs are applicable for protein-selective deglycosylation.
Fig. 5: Nanobody-fused split OGA facilitates the functional attribution of O-GlcNAc on c-Jun to protein stability and on c-Fos to AP-1 transcriptional activity.

Data availability

The main data generated or analyzed during this study are included in this published article and its Supplementary Information. The MS proteomics data were searched against the UniProt/SwissProt human (Homo sapiens) protein database (19 August 2016; http://www.uniprot.org/proteomes/UP000005640) and have been deposited to the ProteomeXchange Consortium via the PRIDE51 partner repository with the dataset identifiers PXD022347 (Fig. 3d,e) and PXD018914 (Fig. 3f,g). Source data are provided with this paper.

Code availability

Code used to analyze the MS proteomics data has been deposited in GitHub and is available for download at https://github.com/harvardinformatics/quantproteomics/tree/master/PEA.

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Acknowledgements

We thank P. Schwein and Z. Lin for helpful discussions and B. Budnik (principal scientist of the Harvard University Proteomics Facility). Support from the National Institutes of Health (U01CA242098-01, C.M.W.), the Burroughs Wellcome Fund, a Career Award at the Scientific Interface (C.M.W.), the Sloan Foundation (C.M.W.), the Merck Fellowship Fund and Harvard University is gratefully acknowledged.

Author information

Affiliations

Authors

Contributions

Y.G. and C.M.W. conceived the project. Y.G., D.H.R. and C.M.W. designed the experiments. Y.G. and S.W. performed the experiments. B.Y. and A.K.D. helped with quantitative MS analyses. C.A. contributed reagents and technical advice on confocal imaging analysis. Y.G. and C.M.W. analyzed the data and wrote the paper with input from all authors.

Corresponding author

Correspondence to Christina M. Woo.

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Competing interests

Harvard University has filed a patent application (US Provisional Application No. 63/087,773, filed 5 October 2020) including work described herein. C.M.W., Y.G. and D.H.R. are inventors of this patent. All other authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks Jing Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data

Extended Data Fig. 1 Schematic representation of OGA and target protein constructs used in this study.

a, Schematic of the structures of human OGA and other truncations. Catalytic domain, stalk domain, HAT domain, and intrinsic disordered regions are shown in pink, cyan, orange and white, respectively. GS linker represents a 15-residue glycine and serine linker. b, Depiction of the strategy to fuse the nanobody on OGAs to achieve protein specificity. nGFP, nanobody against GFP. c, Design of GFP-fused, Ubc tag-fused and BC2 tag-fused proteins of interest used in this study. For GFP-fused proteins, GFP and a Flag tag are placed on the N-terminus and the EPEA tag is in the C-terminus unless otherwise noted. For Ubc tag-fused proteins, the 14-residue peptide tag, Flag tag and EPEA tag are sequentially placed in the C-terminus unless otherwise noted. For BC2 tag-fused proteins, the 12-residue peptide tag is placed on the N-terminus and Flag, EPEA tag are in the C-terminus. Peptide sequences of Ubc and BC2 were shown. d, Symbols used in this manuscript to represent the indicated split OGA constructs. Source data

Extended Data Fig. 2 Identification of the minimal OGA for nanobody-directed deglycosylation on the target protein.

a, Enzymatic activities of OGA and its truncations are evaluated on GFP-Nup62. GFP-Nup62 was coexpressed with indicated constructs, enriched by anti-EPEA beads, and analyzed by immunoblotting to visualize the protein level and O-GlcNAc modification level, respectively. b, Evaluation of enzymatic activities of nGFP-OGA fusion proteins on GFP-Nup62. Expression levels of the indicated proteins and degree of O-GlcNAc modification were quantified by immunoblotting. The ratio equals to the intensity of anti-O-GlcNAc immunoblot normalized by the intensity of anti-Flag immunoblot. WCL, whole-cell lysate. The data are representative of two biological replicates. Source data

Extended Data Fig. 3 nGFP-OGA(GS-∆HAT) has limited target protein selectivity and can alter subcellular localization of the target protein.

a, nGFP-OGA(GS-∆HAT) removes O-GlcNAc from Nup62 without GFP tag similar to the full length OGA (fl-OGA). HEK 293T whole cell lysates (WCL) and immunoprecipitation samples were analyzed by immunoblotting assays using the indicated antibodies. Results are representative of two biological replicates. b, nGFP colocalizes with nuclear transcription factor Sp1 with GFP and does not change the subcellular localization of GFP-Sp1 by immunofluorescence imaging. c, nGFP-OGA(GS-∆HAT) alters the subcellular localization of GFP-Sp1, but coexpression with fl-OGA does not change nuclear localization of GFP-Sp1 by immunofluorescence imaging. Channels are annotated on the top. Scale bar: 10 µm. Right: merged channel. Proteins coexpressed in each sample were labeled on the left side. Images are representative of at least three randomly selected frames. Source data

Extended Data Fig. 4 Optimization of nGFP-fused split OGA constructs in living cells.

a, Coexpressing N- and C-fragments of OGA reconstitutes deglycosidase activity in HEK 293T cells. b, Split OGA fragments, N2 and C3, instead of C4, associate with each other when coexpressed in HEK 293T cells. The asterisk indicated IgG heavy chain from anti-c-Myc magnetic beads. c, Comparison of nGFP-fused N- and C-terminal OGA fragments on GFP-Nup62 in HEK 293T cells. The pair of N2 and nGFP-fused C3 (N2 + nGFP-C3) shows the best deglycosylation performance. In a and c, activities of fragments alone or pairs with/without nGFP were evaluated on GFP-Nup62, which was enriched by beads against EPEA tag and blotted with RL2 antibody to reveal O-GlcNAc modification level. D174N, a catalytically impaired mutation on OGA. Anti-myc and anti-HA blots detect expression of full-length (fl-OGA) or N-terminal fragment, and C-terminal fragment, respectively. WCL, whole-cell lysates. The data in a-c are representative of at least two biological replicates. Source data

Extended Data Fig. 5 nGFP-splitOGA selectively deglycosylates the target protein without affecting the global O-GlcNAc proteome.

a, nGFP-splitOGA has little effect on endogenous glycoprotein CREB. HEK 293T cells coexpressing OGA constructs with GFP-Nup62 were subjected to mass shift assay. The intensities of O-GlcNAcylated and unmodified CREB were quantified. The ratios are shown below the anti-CREB blot. WCL, whole-cell lysates. b, Overexpression of selected split OGA constructs with target protein has little effect on global O-GlcNAcylation level. For OGT inhibition, cells were treated with 25 µM OSMI-4b for 30 h. Global O-GlcNAcylation level was evaluated by anti-O-GlcNAc (RL2) antibody. c, nGFP-splitOGA has minimal effect on protein levels of endogenous OGT, OGA and glycoprotein CREB. Anti-myc and anti-HA blots detect expression of N-terminal fragment, and C-terminal fragment, respectively. d, Comparison of overexpressed proteins with the corresponding endogenous proteins. The antibody against OGA recognizes both endogenous OGA and the overexpressed N-terminal fragment of split OGA. Endogenous Nup62(*) and OGA (**) are indicated. The data in a-d are representative of at least two biological replicates. Source data

Extended Data Fig. 6 Confocal imaging of intracellular distributions of GFP-Sp1 and the split OGAs in HEK 293T cells.

a, GFP-Sp1 localized in nucleus. b, Intracellular distributions of N2 and nGFP-C3 fragments when coexpressed in HEK 293T cells. Two fragments of nGFP-splitOGA were distributed on both cytoplasm and nucleus. c, Subcellular localizations of GFP-Sp1, N fragment and C fragment when expressed simultaneously in HEK 293T cells. Two fragments of split OGA without nGFP (c, upper row) were distributed on both cytoplasm and nucleus. C-terminal fragment of nGFP-splitOGA (c, bottom row) reveals better colocalization with nuclear protein GFP-Sp1, showing the binding between nGFP and GFP. Split OGAs do not change the subcellular localization of GFP-Sp1. Channels are annotated on the top. Scale bar: 10 µm. Right: merged channel. Proteins coexpressed in each sample were labeled on the left side. Images are representative of at least three randomly selected frames. Source data

Extended Data Fig. 7 Mass spectrometry analysis on the activity and selectivity of nGFP-splitOGA on GFP-Nup62.

a, Schematic representation of the workflow of O-GlcNAcylated protein enrichment and mass spectrometry-based identification. Proteins with O-GlcNAc modification were labeled with GalNAz by GalT(Y289L)-mediated chemoenzymatic labeling, followed by a click reaction with an alkyne-biotin probe. Biotin-labeled proteome was enriched by streptavidin beads and digested by trypsin. Released peptides were labeled by TMT reagents and compiled into a single pool. Proteins were identified and quantified by LC-MS. bd, Reproducibility of the TMT experiments of O-GlcNAcylated proteome shown in Fig. 3d,e. The signal abundances of the corresponding TMT channels for each protein were extracted and were log10 transformed for full-length OGA treatment (b, fl-OGA), nGFP-splitOGA treatment (c) and its inactive form [N2(D174N) + nGFP-C3] treatment (d) groups (n = 2 independent biological replicates). Source data

Extended Data Fig. 8 Peptide tag BC2 and its nanobody can be adapted to split OGA to achieve protein-selective deglycosylation.

a, Schematic of nanobodies against BC2 and EPEA tag adapted to split OGA. BC2 tag refers to a 12-residue peptide epitope, which is functional irrespective of its position on the target protein. b, nBC2-splitOGA is able to remove O-GlcNAc from Nup62 tagged with BC2 and EPEA in a similar manner to nEPEA-splitOGA. Symbols represent the corresponding OGA constructs as indicated in Extended Data Fig. 1. Anti-myc and anti-HA blots detect expression of full-length (fl-OGA) or N-terminal and C-terminal fragment, respectively. WCL, whole-cell lysate. The data are representative of two biological replicates. Source data

Extended Data Fig. 9 Modulation of O-GlcNAc modification level and validation of its functional contribution on the stability of GFP-c-Jun.

a, O-GlcNAc level on GFP-c-Jun and endogenous CREB were evaluated by the mass-shift assay. GFP-c-Jun was coexpressed with indicated OGA constructs. The intensities of O-GlcNAcylated and unmodified c-Jun and CREB were quantified. Quantification is shown as mean ± s.d. of n = 3 independent biological experiments. All ratios were normalized by the Blank samples. Unpaired two-tailed Student’s t tests were used for statistical analysis. n.s., not significant. b, Whole cell lysates (WCL) were analyzed by immunoblotting assays using the indicated antibodies. Anti-myc and anti-HA blots detect expression of full-length (fl-OGA) or N-terminal fragment, and C-terminal fragment, respectively. The data in a and b are representative of at least three biological replicates. c, The stability of GFP-c-Jun was enhanced by OGA inhibition (Thiamet-G treatment) and impeded by OGT inhibition (Ac45SGlcNAc treatment). HEK 293T cells expressing GFP-c-Jun pre-treated with DMSO or Ac45SGlcNAc or Thiamet-G were incubated with 50 μM CHX for up to 12 h, during which the protein level of GFP-c-Jun and global O-GlcNAcylation level were monitored. Results in c are representative of two biological replicates. Source data

Extended Data Fig. 10 Modulation of O-GlcNAc modification level with nUbc-splitOGA on c-Fos-Ubc in comparison to OGT inhibition.

Immunoblotting analysis of protein expression and O-GlcNAcylation status of c-Fos-Ubc and endogenous c-Jun under the indicated treatments corresponding to Fig. 5d,e. by either enrichment against EPEA-tag (a) or chemoenzymatic labeling followed with Biotin-IP (b). Endogenous c-Jun shows negligible changes on O-GlcNAcylation status with the coexpression of nUbc-splitOGA but shows reduced O-GlcNAc modification upon OGT inhibition with OSMI-4b. No detectable endogenous c-Fos was observed in HEK 293T cells. The data in a and b are representative of two biological replicates.

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Ge, Y., Ramirez, D.H., Yang, B. et al. Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase. Nat Chem Biol 17, 593–600 (2021). https://doi.org/10.1038/s41589-021-00757-y

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