Recent Advances in Protein‐Specific Glycan Imaging and Editing

Zheng, Xiaocui; He, Boqiang; Meng, Liying; Xie, Ran

doi:10.1002/cbic.202200778

Cited by 5 publications

(4 citation statements)

References 114 publications

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“…The use of metabolic chemical reporters has become a widely accessible and easy-to-use approach to study glycoconjugates in a cell or organism. This method allows researchers to utilize probes for imaging, enrichment, profiling, and targeting of glycans (Kiessling and Splain, 2010;Woo et al, 2015;Palaniappan and Bertozzi, 2016;Zheng et al, 2023). Typically, synthetic sugars containing a chemical reporter are given to cells or organisms and are incorporated into glycans by the endogenous biosynthetic machinery.…”

Section: Discussionmentioning

confidence: 99%

Tools and tactics to define specificity of metabolic chemical reporters

Mukherjee,

Bond,

Abramowitz

et al. 2023

Front. Mol. Biosci.

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Metabolic chemical reporters (MCRs) provide easily accessible means to study glycans in their native environments. However, because monosaccharide precursors are shared by many glycosylation pathways, selective incorporation has been difficult to attain. Here, a strategy for defining the selectivity and enzymatic incorporation of an MCR is presented. Performing β-elimination to interrogate O-linked sugars and using commercially available glycosidases and glycosyltransferase inhibitors, we probed the specificity of widely used azide (Ac4GalNAz) and alkyne (Ac4GalNAlk and Ac4GlcNAlk) sugar derivatives. Following the outlined strategy, we provide a semiquantitative assessment of the specific and non-specific incorporation of this bioorthogonal sugar (Ac4GalNAz) into numerous N- and O-linked glycosylation pathways. This approach should be generally applicable to other MCRs to define the extent of incorporation into the various glycan species.

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Section: Discussionmentioning

confidence: 99%

Tools and tactics to define specificity of metabolic chemical reporters

Mukherjee,

Bond,

Abramowitz

et al. 2023

Front. Mol. Biosci.

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“…FRET has already been used to evidence ligand–receptor binding but common strategies do not rely on the movement of ligands at the interface. They usually involve the simultaneous labeling of the ligand and receptor with a FRET pair and binding of the two promotes the energy transfer. − This approach requires the labeling of complex membrane receptors, which is not easily achieved, especially in complex biological samples such as primary cells. Numerous instances of FRET in particles have also been reported: inside of nanoparticules to demonstrate drug delivery or the formation of nanoparticles, , inside liposomes to study the organization of the membrane, or inside nanoemulsions or microemulsions. − Particles have also been reported as FRET donors to study protein-specific glycan binding, but this strategy again requires a labeled receptor and does not inform on the mobility and possible cooperation of ligand during receptor binding. , Herein, we propose a strategy to monitor the binding of particles to unlabeled multivalent receptors by monitoring the energy transfer induced by the clustering of fluorescent ligands upon protein binding at the interface of the particles (Figure ).…”

Section: Introductionmentioning

confidence: 99%

FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids

Michelis,

Pompili,

Niedergang

et al. 2024

ACS Appl. Mater. Interfaces

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Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand−receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface. Herein, we report on simple cell or bacterial mimics, based on liquid microparticles made of lipiodol functionalized with custom-designed fluorescent lipids. These lipids are targeted toward lectins or biotin membrane receptors, and the resulting particles can be specifically identified and internalized by cells, as demonstrated by their phagocytosis in primary murine bone marrow-derived macrophages. We also evidence the possibility to sense the binding of a multivalent lectin, concanavalin A, in solution by monitoring the energy transfer between two matching fluorescent lipids on the surface of the particles. We anticipate that these liquid particle-based sensors, which are able to report via Forster resonance energy transfer (FRET) on the movement of ligands on their interface upon protein binding, will provide a useful tool to study receptor binding and cooperation during adhesion processes such as phagocytosis.

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“…In recent years, there has been an expansion of chemical tools developed to study sialylation or fucosylation of cellular glycoconjugates through metabolic or exo-enzymatic glycan labeling. − Despite the biological importance of LacNAc structures, there have been limited reports of the development of analogous tools to study this specific glyco-motif in cells. Since the LacNAc disaccharide contains both GlcNAc and Gal residues, it is challenging to control the selective formation of LacNAc units on cells using commonly employed bioorthogonal labeling strategies like metabolic oligosaccharide engineering (MOE).…”

Section: Introductionmentioning

confidence: 99%

“…An alternative strategy to MOE that facilitates the specific display of glyco-motifs on cells is exogenous (exo)-enzymatic glyco-engineering. In this approach, modified nucleotide (NT)-sugars and glycosyltransferase enzymes are used to selectively edit cell-surface glycans with probes in a motif-specific manner for visualization, characterization, and interrogation of GBP recognition. , Selectivity arises by exploiting the specificity of the glycosyltransferase employed, enabling control over the glycosidic linkage, and often the glycan subclass, that is displaying the probe-modified monosaccharide. Prominent examples have used sialyl- and fucosyltransferases to install azide, alkyne, diazirine, biotin, and other modified NT-sugars onto cellular glycans. ,, …”

Section: Introductionmentioning

confidence: 99%

Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs

De León González,

Boddington,

Kofsky

et al. 2024

ACS Chem. Biol.

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Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups, which can be used to study glycans and their interactions with glycan-binding proteins. In recent years, strategies to edit cellular glycans by installing monosaccharides and their derivatives using glycosyltransferase enzymes have rapidly expanded. However, analogous methods to introduce chemical reporter-functionalized type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars bearing azide, alkyne, and diazirine functionalities on the C2-acetamido group using the mutant uridylyltransferase AGX1 F383A . The unnatural UDP-GlcNAc derivatives were examined as substrates for the human GlcNAc-transferase B3GNT2, where it was found that modified donors were tolerated for transfer, albeit to a lesser extent than the natural UDP-GlcNAc substrate. When the GlcNAc derivatives were examined as acceptor substrates for the human Gal-transferase B4GalT1, all derivatives were well tolerated and the enzyme could successfully form derivatized LacNAcs. B3GNT2 was also used to exo-enzymatically install GlcNAc and unnatural GlcNAc derivatives on cell-surface glycans. GlcNAc-or GlcNAz-engineered cells were further extended by B4GalT1 and UDP-Gal, producing LacNAc-or LacNAz-engineered cells. Our proof-of-concept glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural type 2 LacNAc motifs.

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Recent Advances in Protein‐Specific Glycan Imaging and Editing

Abstract: This article is part of the Special Collection ChemBioTalents2022. Please see our homepage for more articles in the collection.

Cited by 5 publications

References 114 publications

Tools and tactics to define specificity of metabolic chemical reporters

Tools and tactics to define specificity of metabolic chemical reporters

FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids

Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs

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