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Cell-type-specific labeling and profiling of glycans in living mice

Nature Chemical Biology volume 18, pages 625–633 (2022)Cite this article

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Abstract

Metabolic labeling of glycans with clickable unnatural sugars has enabled glycan analysis in multicellular systems. However, cell-type-specific labeling of glycans in vivo remains challenging. Here we develop genetically encoded metabolic glycan labeling (GeMGL), a cell-type-specific strategy based on a bump-and-hole pair of an unnatural sugar and its matching engineered enzyme. N-pentynylacetylglucosamine (GlcNAl) serves as a bumped analog of N-acetylglucosamine (GlcNAc) that is specifically incorporated into glycans of cells expressing a UDP-GlcNAc pyrophosphorylase mutant, AGX2F383G. GeMGL with the 1,3-di-O-propionylated GlcNAl (1,3-Pr2GlcNAl) and AGX2F383G pair was demonstrated in cell cocultures, and used for specific labeling of glycans in mouse xenograft tumors. By generating a transgenic mouse line with AGX2F383G expressed under a cardiomyocyte-specific promoter, we performed specific imaging of cardiomyocyte glycans in the heart and identified 582 cardiomyocyte O-GlcNAcylated proteins with no interference from other cardiac cell types. GeMGL will facilitate cell-type-specific glycan imaging and glycoproteomics in various tissues and disease models.

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Fig. 1: A bump–hole substrate–enzyme pair for cell-type-specific glycan labeling.
Fig. 2: Cell-type-specific MGL in cocultures.
Fig. 3: Cell-specific MGL in mouse xenograft models.
Fig. 4: Cell-type-specific MGL in transgenic mice.
Fig. 5: Cardiac O-GlcNAcylated proteins identified by GeMGL in living mice.

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Data availability

The data that support the findings of this study are available within the paper and Supplementary Information. The proteomic raw data are deposited on ProteomeXchange with the dataset identifier PXD030663. Source data are provided with this paper.

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Acknowledgements

This project is supported by the National Key Research and Development Program of China (grant no. 2018YFA0507600) and the National Natural Science Foundation of China (grant nos. 22037001, 9215330 and 91753206).

Author information

Author notes

  1. These authors contributed equally: Xinqi Fan, Qitao Song.

Authors and Affiliations

  1. College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Xinqi Fan, Qitao Song, De-en Sun, Yi Hao, Jingyang Wang, Chunting Wang & Xing Chen

  2. Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China

    Xinqi Fan, De-en Sun, Yi Hao, Jingyang Wang, Chunting Wang & Xing Chen

  3. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Qitao Song & Xing Chen

  4. Synthetic and Functional Biomolecules Center, Peking University, Beijing, China

    Xing Chen

  5. Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China

    Xing Chen

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Contributions

X.F., Q.S. and X.C. conceived the project. X.F. and Q.S. prepared all samples and performed biochemistry, cell biology and animal experiments. D.S., Q.S. and X.F. performed fluorescence imaging experiments. Y.H. and X.F. generated stable cell lines. X.F., Q.S. and J.W. performed chemoproteomics experiments. X.F. and C.W. synthesized organic compounds. X.F., Q.S. and X.C. analyzed the data and wrote the manuscript.

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Correspondence to Xing Chen.

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Nature Chemical Biology thanks Wesley Zandberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Fluorescence imaging of MCF7 cells metabolically labeled with GlcNAz and 1,3-Pr2GlcNAz.

a-i, MCF7-T2A-GFP cells (a and b), MCF7-AGX2-T2A-GFP cells (c and d), MCF7-AGX2F381G-T2A-GFP cells (f and g), MCF7–AGX2F383G-T2A-GFP cells (h and i), or MCF7 cells transiently expressing AGX1-GFP or AGX2-GFP (e) were incubated with vehicle, 1 mM GlcNAz, or 100 μM 1,3-Pr2GlcNAz for 48 h. For imaging of cell-surface glycans (a, c, f, and h), the cells were directly reacted with alkyne-TAMRA, followed by imaging by confocal fluorescence microscopy. For imaging of intracellular glycans (b, d, e, g and i), the cells were fixed, permeabilized, reacted with alkyne-TAMRA, and visualized using confocal fluorescence microscopy. The nuclei were stained with Hoechst 33342 (blue). In a, the fluorescence intensity of TAMRA in the boxed areas was adjusted to 3 times, so that the weak cell-surface labeling can be observed. Scale bars, 20 μm. Representative results are shown from three independent experiments.

Extended Data Fig. 2 In-gel fluorescence scanning analysis of lysates from cells incubated with GlcNAz or 1,3-Pr2GlcNAz.

a-d, MCF7-T2A-GFP (a), AGX2-T2A-GFP (b), AGX2F381G-T2A-GFP (c), and AGX2F383G-T2A-GFP cells (d) were treated with vehicle, GlcNAz or 1,3-Pr2GlcNAz at varied concentrations for 48 h, followed by reacted with alkyne-Cy5 and analysis by in-gel fluorescence scanning. Coomassie Brilliant Blue (CBB)-stained gels demonstrate equal loading. Western blots demonstrate comparable expression of AGX2F383G by using an antibody against the N-terminal Flag tag. Representative results are shown from three independent experiments.

Source data

Extended Data Fig. 3 Fluorescence imaging of MCF7 cells metabolically labeled with GlcNAl and 1,3-Pr2GlcNAl.

a-f, MCF7-T2A-GFP (a), MCF7-AGX2-T2A-GFP (b and c), MCF7-AGX2F383G-T2A-GFP (d), or MCF7-AGX2F381G-T2A-GFP cells (e and f) were incubated with vehicle, 1 mM GlcNAl, or 100 μM 1,3-Pr2GlcNAl for 48 h. For imaging of cell-surface glycans (a, b, d, and e), the cells were directly reacted with azide-TAMRA, followed by imaging by confocal fluorescence microscopy. For imaging of intracellular glycans (c and f), the cells were fixed, permeabilized, reacted with azide-TAMRA, and visualized by confocal fluorescence microscopy. The nuclei were stained with Hoechst 33342 (blue). Scale bars, 20 μm. Representative results are shown from three independent experiments.

Extended Data Fig. 4 In-gel fluorescence scanning analysis of lysates from cells incubated with GlcNAl or 1,3-Pr2GlcNAl.

MCF7-AGX2-T2A-GFP (a) and MCF7-AGX2F381G-T2A-GFP (b) cells were treated with vehicle, GlcNAl or 1,3-Pr2GlcNAl at varied concentrations for 48 h, followed by reacted with azide-Cy5 and analysis by in-gel fluorescence scanning. Coomassie Brilliant Blue (CBB)-stained gels demonstrate equal loading. Western blots demonstrate comparable expression of AGX2 or AGX2F381G by using an antibody against the N-terminal Flag tag. Representative results are shown from three independent experiments.

Source data

Extended Data Fig. 5 Enzymatic conversion of GlcNAl-1-P to UDP-GlcNAl in vitro.

a, TLC analysis of conversion from GlcNAl-1-P to UDP-GlcNAl. GlcNAl-1-P at 10 mM was incubated with 10 μg/mL AGX2 (2), AGX2F381G (3), or AGX2F383G (4) at 37 °C for 30 min, followed by heating at 90 °C for 2 min to quench the reaction. The TLC plates were developed using the solvent (n-butanol:acetic acid:H2O = 2:1:1), and stained with p-anisaldehyde. The standard nucleotide sugars GlcNAl-1-P and UDP-GlcNAl were shown in line 1 and 5, respectively. b, Mass spectra showing MW of the enzymatic production matching UDP-GlcNAl. c, Kinetic analysis of different enzymes in catalyzing formation of UDP-GlcNAl. AGX2, AGX2F381G, or AGX2F383G was incubated with GlcNAl-1-P at varied concentrations for 30 min at 37 °C, quenched, and analyzed by HPLC. Error bars represent mean ± s.d.. Results are from three independent experiments.

Extended Data Fig. 6 Metabolism of 1,3-Pr2GlcNAl in cells.

a, HPAEC traces of extracts from MCF7-AGX2F383G-T2A-GFP cells fed with vehicle or 1,3-Pr2GlcNAl at varied concentrations and from MCF7-T2A-GFP cells treated with vehicle for 36 h. The nucleotides sugars were extracted by solid-phase extraction chromatography. ADP-glucose (ADP-Glc) was used as a spike-in control to normalize the UV absorbance between different runs. The inserted traces show the retention time of nucleotide sugar standards in separate runs. Representative results are shown from three independent experiments. b, Quantification of the cellular level of nucleotides. A.U., arbitrary units. Error bars represent mean ± s.d. from three independent experiments. The P values listed were calculated using the two-tailed Student's t-test. **P<0.01 (one-way ANOVA).

Source data

Extended Data Fig. 7 Fluorescence imaging of the cocultured cells metabolically labeled with GlcNAl, 1,3-Pr2GlcNAl, GlcNAz, or 1,3-Pr2GlcNAz.

a-e, MCF7 cells stably expressing AGX2-T2A-GFP (a and c), AGX2F381G-T2A-GFP (b and d), and AGX2F383G-T2A-GFP (e) were cocultured with HeLa cells, respectively. The cocultures were incubated with vehicle, 1 mM GlcNAl, or 100 μM 1,3-Pr2GlcNAl (a and b); the cocultures were incubated with vehicle, 1 mM GlcNAz, or 100 μM 1,3-Pr2GlcNAz (c, d, and e). After incubation for 48 h, the cells were fixed, permeabilized, and reacted with azide-TAMRA or alkyne-TAMRA. The nuclei were stained with Hoechst 33342 (blue). Scale bars, 20 μm. Representative results are shown from three independent experiments.

Extended Data Fig. 8 Confocal fluorescence images of the cardiac tissue slices from the α-MHC-AGX2F383G mice administered with 1,3-Pr2GlcNAl, related to Fig. 4c.

The slices were click-labeled with azide-TAMRA, immunostained with an anti-cTnT antibody, and stained with Hoechst 33343. Magnified views of three boxed regions are shown in the three rows below, respectively. Scale bars, 20 μm. Representative results are shown from three independent experiments.

Extended Data Fig. 9 Evaluation of the effect of AGX2F383G expression on protein O-GlcNAcylation.

a,b, Immunoblots showing O-GlcNAcylated proteins of MCF7-T2A-GFP and MCF7-AGX2F383G-T2A-GFP cells (a), or from the heart tissue of WT and α-MHC-AGX2F383G mice (b). CTD110.6 is an O-GlcNAc-specific antibody. Immunoblots using an anti-Flag antibody show the expression of AGX2F383G. CBB-stained gels are shown as the loading control. Representative results are shown from three independent experiments.

Source data

Extended Data Fig. 10 Cardiac glycoproteins identified from α-MHC-AGX2F383G mice injected with 1,3-Pr2GlcNAl.

a,b,c Volcano plots displaying the average log2 enrichment ratios for proteins quantified in at least two out of three biological replicates (x axis) and their adjusted P values (y axis; empirical Bayes moderated t test, adjusted by the Benjamini-Hochberg method). Proteins with adjusted P value < 0.01 and a fold change of two or greater were considered as identified glycoproteins. 967 and 949 proteins were identified over the vehicle (heavy/light, H/L; a) and non-transgenic mice controls (heavy/medium, H/M; b), respectively. No protein was identified in non-transgenic mice (medium/light, M/L; c).

Supplementary information

Supplementary Information

Supplementary Figs. 1–10, Note 1 and Protocols.

Reporting Summary

Supplementary Table 1

Cardiac glycoproteins identified from α-MHC-AGX2F383G mice.

Supplementary Table 2

Identified O-GlcNAc sites in MCF7-AGX2F383G-T2A-GFP cells with 1,3-Pr2GlcNAl.

Supplementary Table 3

Identified S-glyco-modification sites in MCF7-AGX2F383G-T2A-GFP cells with 1,3-Pr2GlcNAl.

Supplementary Data 1

Statistical source data for Supplementary Fig. 5.

Source data

Source Data Fig. 1

Unprocessed western blots and gels of Fig. 1.

Source Data Fig. 3

Unprocessed western blots and gels of Fig. 3.

Source Data Fig. 4

Unprocessed western blots and gels of Fig. 4.

Source Data Extended Data Fig. 2

Unprocessed western blots and gels of Extended Data Fig. 2.

Source Data Extended Data Fig. 4

Unprocessed western blots and gels of Extended Data Fig. 4.

Source Data Extended Data Fig. 6

Statistical source data for Extended Data Fig. 6.

Source Data Extended Data Fig. 9

Unprocessed western blots and gels of Extended Data Fig. 9.

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Fan, X., Song, Q., Sun, De. et al. Cell-type-specific labeling and profiling of glycans in living mice. Nat Chem Biol 18, 625–633 (2022). https://doi.org/10.1038/s41589-022-01016-4

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