Selective bioorthogonal probe for n-glycan hybrid structures

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. Access through your

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AZIDE AND ALKYNE TAGS ON BIOORTHOGONAL REPORTERS IN METABOLIC LABELING OF SIALYLATED GLYCOCONJUGATES: A COMPARATIVE STUDY Article Open access 22 December 2022 MUTANT GLYCOSIDASES FOR

LABELING SIALOGLYCANS WITH HIGH SPECIFICITY AND AFFINITY Article Open access 07 February 2025 SELENIUM-BASED METABOLIC OLIGOSACCHARIDE ENGINEERING STRATEGY FOR QUANTITATIVE GLYCAN DETECTION

Article Open access 13 December 2023 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.

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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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD, USA Mana Mohan Mukherjee, 

Devin Biesbrock, Lara K. Abramowitz & John A. Hanover * Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD, USA Matteo Pavan & Kenneth A.

Jacobson * Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, USA Bhoj Kumar & Parastoo Azadi * Clinical Mass Spectrometry Core, NIDDK, National Institutes of

Health, Bethesda, MD, USA Peter J. Walter Authors * Mana Mohan Mukherjee View author publications You can also search for this author inPubMed Google Scholar * Devin Biesbrock View author

publications You can also search for this author inPubMed Google Scholar * Lara K. Abramowitz View author publications You can also search for this author inPubMed Google Scholar * Matteo

Pavan View author publications You can also search for this author inPubMed Google Scholar * Bhoj Kumar View author publications You can also search for this author inPubMed Google Scholar *

Peter J. Walter View author publications You can also search for this author inPubMed Google Scholar * Parastoo Azadi View author publications You can also search for this author inPubMed 

Google Scholar * Kenneth A. Jacobson View author publications You can also search for this author inPubMed Google Scholar * John A. Hanover View author publications You can also search for

this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHOR Correspondence to John A. Hanover. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER

REVIEW PEER REVIEW INFORMATION _Nature Chemical Biology_ thanks Charlie Fehl, Matthew Pratt and Pamela Stanley for their contribution to the peer review of this work. ADDITIONAL INFORMATION

PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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. Source data 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. Source data 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. Source data 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. Source data 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). Source data 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. REPORTING SUMMARY 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 Unprocessed western blots. SOURCE DATA FIG. 2 Unprocessed

western blots. SOURCE DATA FIG. 3 Unprocessed western blots. SOURCE DATA FIG. 5 Unprocessed western blots. SOURCE DATA FIG. 6 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 1

Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 2 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 4 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 7

Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 8 Unprocessed western blots. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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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