Abstract
Metabolic incorporation of chemically tagged monosaccharides is a facile means of tagging cellular glycoproteins and glycolipids. However, since the monosaccharide precursors are often shared by several pathways, selectivity has been difficult to attain. For example, N-linked glycosylation is a chemically complex and ubiquitous posttranslational modification, with three distinct classes of GlcNAc-containing N-glycan structures: oligomannose, hybrid and complex. Here we describe the synthesis of 1,3-Pr2-6-OTs GlcNAlk (MM-JH-1) as a next-generation metabolic chemical reporter for the selective labeling of hybrid N-glycan structures. We first developed a general strategy for defining the selectivity of labeling with chemically tagged monosaccharides. We then applied this approach to establish that MM-JH-1 is selectively incorporated into hybrid N-glycans. Using this metabolic chemical reporter as a detection tool, we performed imaging and fractionation to define features of the intracellular localization and trafficking of target proteins bearing hybrid N-glycan structures.
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Data availability
All raw data, including western blots and statistical source data files, are available within the paper and Supplementary Information files. For glycomics analysis, Byonic software (Protein Metrics; v.4.1) was used to search the acquired MS/MS data. The search was performed against the reviewed UniProt Human protein database (20,433 entries, http://http://www.uniprot.org) supplemented with a decoy database. Software used for molecular modeling are described in the Methods section within the paper. Source data are provided with this paper.
References
Zhang, J. & Wang, Y. Emerging roles of O-GlcNAcylation in protein trafficking and secretion. J. Biol. Chem. 300, 105677 (2024).
Kiessling, L. L. & Splain, R. A. Chemical approaches to glycobiology. Annu. Rev. Biochem. 79, 619–653 (2010).
Wulff-Fuentes, E. et al. The human O-GlcNAcome database and meta-analysis. Sci. Data 8, 25 (2021).
Bond, M. R. & Hanover, J. A. A little sugar goes a long way: the cell biology of O-GlcNAc. J. Cell Biol. 208, 869–880 (2015).
Weerapana, E. & Imperiali, B. Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology 16, 91R–101R (2006).
Walsh, C. T., Garneau-Tsodikova, S. & Gatto, G. J. Jr Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Ed. Engl. 44, 7342–7372 (2005).
Esmail, S. & Manolson, M. F. Advances in understanding N-glycosylation structure, function, and regulation in health and disease. Eur. J. Cell Biol. 100, 151186 (2021).
Stanley, P., Moremen, K. W., Lewis, N. E., Taniguchi, N. & Aebi, M. in Essentials of Glycobiology 4th edn, Vol. 9 (eds Varki, A. et al.) Ch. 9 (Cold Spring Harbor Laboratory Press, 2022).
Feng, T. et al. Glycosylation of viral proteins: implication in virus-host interaction and virulence. Virulence 13, 670–683 (2022).
Flynn, R. A. et al. Small RNAs are modified with N-glycans and displayed on the surface of living cells. Cell 184, 3109–3124 e3122 (2021).
Kanda, Y. et al. Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types. Glycobiology 17, 104–118 (2007).
Hall, M. K. et al. Predominant expression of hybrid N-glycans has distinct cellular roles relative to complex and oligomannose N-glycans. Int. J. Mol. Sci. 17, 925 (2016).
Hanzawa, K., Suzuki, N. & Natsuka, S. Structures and developmental alterations of N-glycans of zebrafish embryos. Glycobiology 27, 228–245 (2017).
Vocadlo, D. J., Hang, H. C., Kim, E.-J., Hanover, J. A. & Bertozzi, C. R. A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc. Natl Acad. Sci. USA 100, 9116 (2003).
Mukherjee, M. M. et al. Tools and tactics to define specificity of metabolic chemical reporters. Front Mol. Biosci. 10, 1286690 (2023).
McKay, C. S. & Finn, M. G. Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 21, 1075–1101 (2014).
Sletten, E. M. & Bertozzi, C. R. From mechanism to mouse: a tale of two bioorthogonal reactions. Acc. Chem. Res. 44, 666–676 (2011).
Kim, E. J. Advances in strategies and tools available for interrogation of protein O-GlcNAcylation. Chem. Bio. Chem. 22, 3010–3026 (2021).
Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).
Kim, E. J. Chemical reporters and their bioorthogonal reactions for labeling protein O-GlcNAcylation. Molecules 23, 2411 (2018).
Pedowitz, N. J. & Pratt, M. R. Design and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteins. RSC Chem. Biol. 2, 306–321 (2021).
Palaniappan, K. K. & Bertozzi, C. R. Chemical glycoproteomics. Chem. Rev. 116, 14277–14306 (2016).
Konzman, D. et al. O-GlcNAc: regulator of signaling and epigenetics linked to X-linked intellectual disability. Front. Genet. 11, 605263 (2020).
Devaraj, N. K. The future of bioorthogonal chemistry. ACS Cent. Sci. 4, 952–959 (2018).
Breidenbach, M. A. et al. Targeted metabolic labeling of yeast N-glycans with unnatural sugars. Proc. Natl Acad. Sci. USA 107, 3988–3993 (2010).
Kim, E. J., Perreira, M., Thomas, C. J. & Hanover, J. A. An O-GlcNAcase-specific inhibitor and substrate engineered by the extension of the N-acetyl moiety. J. Am. Chem. Soc. 128, 4234–4235 (2006).
Kim, E. J. et al. Distinctive inhibition of O-GlcNAcase isoforms by an α-GlcNAc thiolsulfonate. J. Am. Chem. Soc. 129, 14854–14855 (2007).
Kim, E. J. et al. Optimizing the selectivity of DIFO-based reagents for intracellular bioorthogonal applications. Carbohydr. Res. 377, 18–27 (2013).
Kim, E. J. et al. Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors. Bioconjugate Chem. 25, 1025–1030 (2014).
Mukherjee, M. M. et al. Development of 9-Pr4ManNAlk phenanthrene derivative as fluorescent-raman bioorthogonal probe to specifically interrogate the biosynthesis of gangliosides. Glycobiology 33, 980–980 (2023).
Darabedian, N. et al. O-acetylated chemical reporters of glycosylation can display metabolism-dependent background labeling of proteins but are generally reliable tools for the identification of glycoproteins. Front. Chem. 8, 318 (2020).
Tan, H. Y. et al. Direct one-step fluorescent labeling of O-GlcNAc-modified proteins in live cells using metabolic intermediates. J. Am. Chem. Soc. 140, 15300–15308 (2018).
Chuh, K. N., Zaro, B. W., Piller, F., Piller, V. & Pratt, M. R. Changes in metabolic chemical reporter structure yield a selective probe of O-GlcNAc modification. J. Am. Chem. Soc. 136, 12283–12295 (2014).
Tsukiji, S. & Hamachi, I. Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applications. Curr. Opin. Chem. Biol. 21, 136–143 (2014).
Hays, J. B., Sussman, M. L. & Glass, T. W. Inhibition by 6-O-tosyl galactosides of beta-galactoside phosphorylation and transport by the lactose phosphotransferase system of Staphylococcus aureus. J. Biol. Chem. 250, 8834–8839 (1975).
Batt, A. R., Zaro, B. W., Navarro, M. X. & Pratt, M. R. Metabolic chemical reporters of glycans exhibit cell-type-selective metabolism and glycoprotein labeling. Chem. Bio. Chem. 18, 1177–1182 (2017).
Fan, X. et al. Cell-type-specific labeling and profiling of glycans in living mice. Nat. Chem. Biol. 18, 625–633 (2022).
Qin, W. et al. Artificial cysteine S-glycosylation induced by per-O-acetylated unnatural monosaccharides during metabolic glycan labeling. Angew. Chem. Int. Ed. Engl. 57, 1817–1820 (2018).
Elbein, A. D. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu. Rev. Biochem. 56, 497–534 (1987).
Stanley P., Taniguchi N. & Aebi M. in Essentials of Glycobiology 3rd edn (eds Varki, A. & Esko, J. D.) 2015–2017 (Cold Spring Harbor Laboratory Press, 2017).
Moore, S. E. & Spiro, R. G. Demonstration that Golgi endo-alpha-d-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins. J. Biol. Chem. 265, 13104–13112 (1990).
Choi, H. Y. et al. N-glycan remodeling using mannosidase inhibitors to increase high-mannose glycans on acid alpha-glucosidase in transgenic rice cell cultures. Sci. Rep. 8, 16130 (2018).
Tarentino, A. L., Trimble, R. B. & Plummer, T. H. in Methods in Cell Biology Vol. 32. (ed. Tartakoff, A. M.) 111–139 (Academic, 1989).
Carpentier, M. et al. Nucleolin undergoes partial N- and O-glycosylations in the extranuclear cell compartment. Biochemistry 44, 5804–5815 (2005).
Zhang, L. et al. Boronic acid functionalized core-satellite composite nanoparticles for advanced enrichment of glycopeptides and glycoproteins. Chemistry 15, 10158–10166 (2009).
Sawicki, S. G. & Godman, G. C. On the differential cytotoxicity of actinomycin D. J. Cell Biol. 50, 746–761 (1971).
Snaar, S., Wiesmeijer, K., Jochemsen, A. G., Tanke, H. J. & Dirks, R. W. Mutational analysis of fibrillarin and its mobility in living human cells. J. Cell Biol. 151, 653–662 (2000).
Du, J. et al. Metabolic glycoengineering: sialic acid and beyond. Glycobiology 19, 1382–1401 (2009).
Hao, Y. et al. Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells. Nat. Commun. 10, 4065 (2019).
Choi, J. et al. Engineering orthogonal polypeptide GalNAc-transferase and UDP-sugar pairs. J. Am. Chem. Soc. 141, 13442–13453 (2019).
Schumann, B. et al. Bump-and-hole engineering identifies specific substrates of glycosyltransferases in living cells. Mol. Cell 78, 824–834 e815 (2020).
Kadirvelraj, R. et al. Human N-acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite. Proc. Natl Acad. Sci. USA 115, 4637–4642 (2018).
Wu, H. et al. A photo-cross-linking GlcNAc analog enables covalent capture of N-linked glycoprotein-binding partners on the cell surface. Cell. Chem. Biol. 29, 84–97 (2022).
Pederson, T. Protein mobility within the nucleus—what are the right moves? Cell 104, 635–638 (2001).
Taniguchi, N. & Kizuka, Y. Glycans and cancer: role of N-glycans in cancer biomarker, progression and metastasis, and therapeutics. Adv. Cancer Res. 126, 11–51 (2015).
Munkley, J. & Elliott, D. J. Hallmarks of glycosylation in cancer. Oncotarget 7, 35478–35489 (2016).
Shivatare, V. S. et al. Unprecedented role of hybrid N-glycans as ligands for HIV-1 broadly neutralizing antibodies. J. Am. Chem. Soc. 140, 5202–5210 (2018).
Zhou, J., Gao, H., Xie, W. & Li, Y. FcgammaR-binding affinity of monoclonal murine IgG1s carrying different N-linked Fc oligosaccharides. Biochem. Biophys. Res. Commun. 520, 8–13 (2019).
Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal. Bioanal. Chem. 390, 2089–2097 (2008).
Zaro, B. W., Yang, Y. Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc. Natl Acad. Sci. USA 108, 8146–8151 (2011).
Nakajima, K. et al. Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC. Glycobiology 20, 865–871 (2010).
Sun, X., Mahajan, D., Chen, B., Song, Z. & Lu, L. A quantitative study of the Golgi retention of glycosyltransferases. J. Cell Sci. 134, jcs258564 (2021).
Shajahan, A., Heiss, C., Ishihara, M. & Azadi, P. Glycomic and glycoproteomic analysis of glycoproteins–a tutorial. Anal. Bioanal. Chem. 409, 4483–4505 (2017).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
The UniProt Consortium UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res. 51, D523–D531 (2023).
Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).
Gordon, R. D. et al. X-ray crystal structures of rabbit N-acetylglucosaminyltransferase I (GnT I) in complex with donor substrate analogues. J. Mol. Biol. 360, 67–79 (2006).
Jacobson, M. P., Friesner, R. A., Xiang, Z. & Honig, B. On the role of the crystal environment in determining protein side-chain conformations. J. Mol. Biol. 320, 597–608 (2002).
Jacobson, M. P. et al. A hierarchical approach to all-atom protein loop prediction. Proteins Struct. Funct. Bioinf. 55, 351–367 (2004).
Lu, C. et al. OPLS4: improving force field accuracy on challenging regimes of chemical space. J. Chem. Theory Comput. 17, 4291–4300 (2021).
Grubmüller, H. & Groll, V. Solvate v.1.0.1 (Max Planck Institute, 2024).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).
Case, D. A. et al. AmberTools. J. Chem. Inf. Model. 63, 6183–6191 (2023).
Jakalian, A., Jack, D. B. & Bayly, C. I. Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. Parameterization and validation. J. Comput. Chem. 23, 1623–1641 (2002).
Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157–1174 (2004).
Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 (2015).
Harvey, M. J., Giupponi, G. & Fabritiis, G. D. ACEMD: accelerating biomolecular dynamics in the microsecond time scale. J. Chem. Theory Comput. 5, 1632–1639 (2009).
Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593 (1995).
Davidchack, R. L., Handel, R. & Tretyakov, M. V. Langevin thermostat for rigid body dynamics. J. Chem. Phys. 130, 234101 (2009).
Faller, R. & de Pablo, J. J. Constant pressure hybrid molecular dynamics–Monte Carlo simulations. J. Chem. Phys. 116, 55–59 (2002).
Friesner, R. A. et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 47, 1739–1749 (2004).
Friesner, R. A. et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein−ligand complexes. J. Med. Chem. 49, 6177–6196 (2006).
Genheden, S. & Ryde, U. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities. Expert Opin. Drug Discov. 10, 449–461 (2015).
Acknowledgements
We thank the members of the Hanover laboratory for discussion and advice, as well as former laboratory member I. Akan for general assistance and valuable suggestions. This work was conducted in the Laboratory of Cell and Molecular Biology section in NIDDK, National Institutes of Health and supported by the NIDDK, National Institutes of Health, intramural research program (grant no. DK060101-18). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Glycomics analysis was performed at the Complex Carbohydrate Research Center and was supported in part by the National Institutes of Health (NIH)-funded R24 (grant no. R24GM137782) awarded to P.A. Molecular modeling analysis was performed at the Laboratory of Cell Biology section in NIDDK, National Institutes of Health, and supported by the NIDDK, National Institutes of Health, intramural research program (grant no. ZIADK031116) awarded to K.A.J. The graphical abstract was created with BioRender.com. Grant nos. DK060101-18 to J.A.H. R24GM137782 to P.A. and ZIADK031116 to K.A.J.
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J.A.H. conceived the project, supervised the study, wrote and edited the manuscript and arranged necessary funding. M.M.M. conceptualized, synthesized and characterized the chemical compounds, performed most of the experiments, analyzed data, wrote the manuscript and coordinated with other coauthors. D.B. performed the immunoblotting, analyzed data and wrote the manuscript. L.K.A. performed the immunoblotting, provided feedback on the project and wrote and edited the manuscript. M.P. and K.A.J. conducted molecular modeling, bioinformatics analysis and wrote a section of the manuscript. B.K. and P.A. performed the glycomics analysis and wrote sections of the manuscript. P.J.W. conducted the UPLC–HRMS analysis of the nucleotide sugars.
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Extended data
Extended Data Fig. 1 Assessment of concentration dependent labeling of MM-JH-1 in HeLa cells.
HeLa Cells incubated with different concentrations (25 μM-500 μM) of MM-JH-1 were collected to check the extent of cellular uptake and the effect on O-GlcNAcylation. (a) HeLa cells collected by physical scraping. N = 4 independent biological replicates. (b) HeLa cells collected by trypsinization. N = 4 independent biological replicates. An ordinary one-way ANOVA test was performed, P values are shown, and all error bars represent standard deviation with mean as center. Quantifications are shown to the right of the images.
Extended Data Fig. 2 MM-JH-1 is enzymatically labeling HeLa cell lysates.
(a) Workflow to assess the enzymatic incorporation of MCRs in HeLa cells. HeLa cell lysates before and after heat deactivation were incubated with different MCRs for 2 hours at 37 oC, followed by a click reaction with TRITC-azide, after which the lysates were subjected to western blotting. (b) HeLa cell lysates before and after heat deactivation were incubated with DMSO, GlcNAlk, Ac4GlcNAlk, 1,3-Pr2 GlcNAlk, MM-JH-1, or 6-OTs GlcNAlk for 2 hours at 37 oC. N = 3 independent biological replicates.
Extended Data Fig. 3 UPLC-HRMS analysis of nucleotide sugar extract of DMSO and MM-JH-1 treated HeLa cells.
(a) UPLC-HRMS analysis of nucleotide sugar extract from DMSO treated HeLa cells. (b) UPLC-HRMS analysis of nucleotide sugar extracted from HeLa cells treated with 100 µM MM-JH-1 (showing 200–850 Da mass range). (c) UPLC-HRMS analysis of nucleotide sugar extracted from HeLa cells treated with 100 µM MM-JH-1 (showing 845–870 Da mass range).
Extended Data Fig. 4 MM-JH-1 is not labeling lipids, RNAs, or GPI-anchors in HeLa cells.
(a) Neither nonionic detergent Triton-X100, nor organic solvent acetone has any effect on the MM-JH-1 labeling. All quantification of images was done by normalizing mean fluorescent signal to DAPI (blue). N = 3 independent biological replicates; n = 25 individual cells chosen for quantification of the confocal images. An ordinary one-way ANOVA test was performed, P values are shown, and all error bars represent standard deviation with mean as center. Quantification is shown to the below right corner of the images. (b) RNAse treatment has no effect on MM-JH-1. All quantification of images was done by normalizing mean fluorescent signal to DAPI (blue). N = 3 independent biological replicates; n = 25 individual cells chosen for quantification of the confocal images. A two-sided unpaired t-test was performed, P values are shown, and error bars represent standard deviation with mean as center. Quantification is shown to the below of the images. (c) MM-JH-1 labeling in HeLa cells remained unchanged after phospholipase C (PLC) treatment. N = 4 independent biological replicates. A two-sided unpaired t-test was performed, P values are shown, and error bars represent standard deviation with mean as center. Quantification is shown to the below of the images.
Extended Data Fig. 5 Glucosidase inhibitor 1-deoxynojirimycin (DNJM) reduced MM-JH-1 labeling.
Increasing concentration of glucosidase inhibitor 1-deoxynojirimycin (DNJM) reduced MM-JH-1 labeling (green, detected with AF 488 staining on confocal images). Augmented signals of ConA (red, detected with ConA-Texas red staining on confocal images. All quantification of images was done by normalizing mean fluorescent signal to DAPI (blue). N = 3 independent biological replicates; n = 25 individual cells chosen for quantification of the confocal images. Scale bar = 50 mm and 10 mm for zoomed in images. An ordinary one-way ANOVA test was performed, P values are shown, and all error bars represent standard deviation with mean as center. Quantifications are shown to the right of the images.
Extended Data Fig. 6 MS/MS spectrum of Man6 N-glycans and MM-JH-1-derivatized hybrid N-Glycan structures.
(a) Full MS/MS spectrum of the Man6 N-glycan. The inset shows the structure of the Man6 N-glycan (mass 1538.754). Green circles represent mannose residues, blue squares represent N-acetylglucosamine residues. (b) Molecular structure of Man6 N-glycan. (c) MM-JH-1-derivatized hybrid N-Glycan.
Extended Data Fig. 7 MM-JH-1 incorporation in MGAT1 knockout HEK 293S GnTI- cells and MGAT1 mutated Lec1CHO cells.
Cell lysates collected from HEK 293S GnTI- or Lec1CHO cells incubated with different concentrations of MM-JH-1 were subjected to click reactions with TRITC-azide to check the extent of cellular uptake. No incorporation of MM-JH-1 was observed in HEK 293S GnTI⁻ cells collected by (a) physical scraping. N = 3 independent biological replicates. (b) trypsinization. N = 3 independent biological replicates. (c) MM-JH-1 incorporated into HEK 293 T cells in a concentration dependent manner, but labeling was not detected in HEK 293S GnTI- cells. N = 3 independent biological replicates. No incorporation of MM-JH-1 was observed in Lec1CHO cells collected by (d) physical scraping. N = 3 independent biological replicates. (e) trypsinization. N = 3 independent biological replicates. (f) MGAT1 cDNA rescue on Lec1CHO cells resulted in a return in signal for both MGAT1 (detected with anti-GFP antibody) and MM-JH-1 labeling (detected with anti TRITC antibody) on immunoblot. N = 4 independent biological replicates.
Extended Data Fig. 8 Cytoplasmic, nuclear and plasma membrane labeling by MM-JH-1 are N-linked.
(a–c) Extracts were fractionated, blotted, and subjected to PNGase F treatment. PNGase F removed TRITC signal from nuclear (a), cytoplasmic (b) and plasma membrane extracts (c) (N = 3 independent biological replicates). (d–f) Extracts were fractionated, blotted and subject to Endo H treatment. Endo H removed TRITC signal from nuclear (d), cytoplasmic (e) and plasma membrane extracts (f) (N = 3 independent biological replicates).
Extended Data Fig. 9 Change in colocalization between fibrillarin signal and MM-JH-1 with Actinomycin D treatment.
Actinomycin D treatment was used to disrupt the nucleolus. Colocalization of fibrillarin (red, detected with anti-nucleolar marker fibrillarin antibody) with MM-JH-1 (green, detected with AF 488 signal on confocal images) was examined. Colocalization was determined by Pearson’s R value as indicated to the right of the image. N = 3 independent biological replicates, n = 10 individual cells chosen for quantification of the confocal images. Error bars represent standard deviation with mean as center. Scale bar = 50 mm and 10 mm for zoomed in images.
Extended Data Fig. 10 Change in fibrillarin distribution with Brefeldin A (BFA) treatment in HeLa cells.
BFA treatment resulted in disrupted fibrillarin labeling with a detectable cytoplasmic accumulation of MM-JH-1 and fibrillarin. N = 3 independent biological replicates. Scale bar = 5 mm shown in images.
Supplementary information
Supplementary Information
Supplementary Figs. 1–15, Notes I and II, Tables 1–4 and source data for figures.
Supplementary Video 1
10 ns of molecular dynamics simulation on the MGAT1-UDP-MM-JH-1 putative complex.
Supplementary Video 2
10 ns of molecular dynamics simulation on the MGAT2-UDP-MM-JH-1 putative complex.
Supplementary Video 3
500 ns of molecular dynamics simulation on the MGAT2-UDP-MM-JH-1 putative complex.
Supplementary Data 1
Statistical source data for Supplementary figures.
Source data
Source Data Figs. 1–3, 5 and 6 and Extended Data Figs. 1, 4, 5 and 9
Numerical source data for Figs. 1–3, 5 and 6 and Extended Data Figs. 1, 4, 5 and 9.
Source Data Fig. 1
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Source Data Extended Data Fig. 1
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Source Data Extended Data Fig. 8
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Mukherjee, M.M., Biesbrock, D., Abramowitz, L.K. et al. Selective bioorthogonal probe for N-glycan hybrid structures. Nat Chem Biol 21, 681–692 (2025). https://doi.org/10.1038/s41589-024-01756-5
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DOI: https://doi.org/10.1038/s41589-024-01756-5
