A photo-cross-linking GlcNAc analog enables covalent capture of N-linked glycoprotein-binding partners on the cell surface

Wu, Han-Chung; Shajahan, Asif; Yang, Jeong‐Yeh; Capota, Emanuela; Wands, Amberlyn M.; Arthur, Connie M.; Stowell, Sean R.; Moremen, Kelley W.; Azadi, Parastoo; Kohler, Jennifer J.

doi:10.1016/j.chembiol.2021.07.007

Cited by 25 publications

(31 citation statements)

References 75 publications

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“…Several groups have developed sugar monophosphate analogs with protecting groups on the sugar and phosphate moieties to promote cellular uptake for eukaryotic glycoconjugate labeling experiments. 283,284 These approaches have been inspired by the synthesis of nucleoside monophosphate prodrugs that contain biolabile protecting groups that "mask" the charge of attached phosphates to promote intracellular delivery of several clinically used drugs, particularly antivirals, which are deprotected and activated by cellular enzymes. 285 While acetyl protecting groups are known to be removed by cytosolic esterases upon cell entry in eukaryotes, there are few examples of the utility of this approach in bacteria.…”

Section: ■ Chemical and Biomolecular Reagents For Detection Of Gram-n...mentioning

confidence: 99%

“…For instance, it is not known if the natural precursors to most NDP-sugars, negatively charged monophosphorylated sugars, can bypass the cell membranes of different bacterial strains. Several groups have developed sugar monophosphate analogs with protecting groups on the sugar and phosphate moieties to promote cellular uptake for eukaryotic glycoconjugate labeling experiments. , These approaches have been inspired by the synthesis of nucleoside monophosphate prodrugs that contain biolabile protecting groups that “mask” the charge of attached phosphates to promote intracellular delivery of several clinically used drugs, particularly antivirals, which are deprotected and activated by cellular enzymes . While acetyl protecting groups are known to be removed by cytosolic esterases upon cell entry in eukaryotes, there are few examples of the utility of this approach in bacteria. , One possible solution is to promote cellular uptake by caging sugar phosphates with photolabile groups, which has been successfully employed in plant cells .…”

Section: Chemical and Biomolecular Reagents For Detection Of Gram-neg...mentioning

confidence: 99%

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Chemical Biology Tools for Modulating and Visualizing Gram-Negative Bacterial Surface Polysaccharides

Zheng

Epstein

et al. 2021

ACS Chem. Biol.

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Bacterial cells present a wide diversity of saccharides that decorate the cell surface and help mediate interactions with the environment. Many Gram-negative cells express O-antigens, which are long sugar polymers that makeup the distal portion of lipopolysaccharide (LPS) that constitutes the surface of the outer membrane. This review highlights chemical biology tools that have been developed in recent years to facilitate the modulation of O-antigen synthesis and composition, as well as related bacterial polysaccharide pathways, and the detection of unique glycan sequences. Advances in the biochemistry and structural biology of O-antigen biosynthetic machinery are also described, which provide guidance for the design of novel chemical and biomolecular probes. Many of the tools noted here have not yet been utilized in biological systems and offer researchers the opportunity to investigate the complex sugar architecture of Gram-negative cells.

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Section: ■ Chemical and Biomolecular Reagents For Detection Of Gram-n...mentioning

confidence: 99%

Section: Chemical and Biomolecular Reagents For Detection Of Gram-neg...mentioning

confidence: 99%

Chemical Biology Tools for Modulating and Visualizing Gram-Negative Bacterial Surface Polysaccharides

Zheng

Epstein

et al. 2021

ACS Chem. Biol.

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“…15,18,20 Diazirine-conjugated sialic acids have been incorporated into cellular glycoproteins and glycolipids through MOE using mannosamine or sialic acid derivatives as unnatural biosynthetic precursors. [15][16][17][18]20,21 While MOE is a powerful approach for the incorporation of photo-cross-linking and other chemical reporter probes, 12 it is limited by the tolerance of the appended probe by cellular metabolic machinery. The incorporation efficiency of probemodified monosaccharides is highly variable and many cell types, including primary cells, are unable to incorporate these probes, limiting the interactions that can be studied.…”

Section: ■ Introductionmentioning

confidence: 99%

Exo-Enzymatic Cell-Surface Glycan Labeling for Capturing Glycan–Protein Interactions through Photo-Cross-Linking

Babulic

Capicciotti

2022

Bioconjugate Chem.

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Tools to interrogate glycoconjugate–protein interactions in the context of living cells are highly attractive for the identification of critically important functional binding partners of glycan-binding proteins. These interactions are challenging to study due to the low affinity and rapid dissociation rates of glycan–protein binding events. The use of photo-cross-linkers to capture glycan–protein interaction complexes has shown great promise for identifying binding partners involved in these interactions. Current methodologies use metabolic oligosaccharide engineering (MOE) to incorporate photo-cross-linking sugars. However, these MOE strategies are not amenable to all cell types and can result in low incorporation and cell-surface display of the photo-cross-linking probe, limiting their utility for studying many types of interactions. We describe here an exo-enzymatic strategy for selectively introducing photo-cross-linking probes into cell-surface glycoconjugates using the recombinant human sialyltransferase ST6GAL1 and a diazirine-linked CMP-Neu5Ac derivative. Probe introduction is highly efficient, amenable to different cell types, and resulted in improved cross-linking when compared to MOE. This exo-enzymatic labeling approach can selectively introduce the photo-cross-linking sugar onto specific glycan epitopes and subclasses by harnessing the specificity of the sialyltransferase employed, underscoring its potential as a tool to interrogate and identify glycoconjugate ligands for diverse glycan-binding proteins.

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“…Notwithstanding these low affinities, glycan recognition events can be highly specific, often through multivalent presentation of glycans and/or their ligands on cell surfaces. , Typical biochemical methods for interaction discovery break apart cells, resulting in disruption of and scrambling of glycan–protein interactions. To solve this challenge, we and others have used metabolic glycan engineering approaches to introduce photoactivatable crosslinking groups (photocrosslinkers) onto cellular glycans. − These strategies involve preparing monosaccharide analogs functionalized with photocrosslinkers. Such photocrosslinking sugars can be metabolized by cells and incorporated into glycoconjugates in place of normal monosaccharides.…”

Section: Introductionmentioning

confidence: 99%

Exo-Enzymatic Addition of Diazirine-Modified Sialic Acid to Cell Surfaces Enables Photocrosslinking of Glycoproteins

et al. 2022

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Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ; however, use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here, we report an exoenzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves the chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAzylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

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A photo-cross-linking GlcNAc analog enables covalent capture of N-linked glycoprotein-binding partners on the cell surface

Cited by 25 publications

References 75 publications

Chemical Biology Tools for Modulating and Visualizing Gram-Negative Bacterial Surface Polysaccharides

Chemical Biology Tools for Modulating and Visualizing Gram-Negative Bacterial Surface Polysaccharides

Exo-Enzymatic Cell-Surface Glycan Labeling for Capturing Glycan–Protein Interactions through Photo-Cross-Linking

Exo-Enzymatic Addition of Diazirine-Modified Sialic Acid to Cell Surfaces Enables Photocrosslinking of Glycoproteins

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