A photocrosslinking GlcNAc analog enables covalent capture of N-linked glycoprotein binding partners on cell surface
N-glycans are displayed on cell surface proteins, and can engage in direct binding interactions with membrane-bound and secreted glycan-binding proteins (GBPs). Biochemical identification and characterization of glycan-mediated interactions is often made difficult by low binding affinities. Here we...
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Veröffentlicht in: | Cell chemical biology 2021-07, Vol.29 (1), p.84-97.e8 |
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Sprache: | eng |
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Zusammenfassung: | N-glycans are displayed on cell surface proteins, and can engage in direct binding interactions with membrane-bound and secreted glycan-binding proteins (GBPs). Biochemical identification and characterization of glycan-mediated interactions is often made difficult by low binding affinities. Here we describe the metabolic introduction of a diazirine photocrosslinker onto GlcNAc residues of N-linked glycoproteins on cell surfaces. We characterize sites at which diazirine-modified GlcNAc is incorporated, as well as modest perturbations to glycan structure. We show that diazirine-modified GlcNAc can be used to covalently crosslink two extracellular GBPs, galectin-1 and cholera toxin subunit B (CTB), to cell surface N-linked glycoproteins. The extent of crosslinking correlates with display of the preferred glycan ligands for the GBPs. Additionally, covalently crosslinked complexes could be isolated, and protein components of crosslinked N-linked glycoproteins were identified by proteomics analysis. This method may be useful in the discovery and characterization of binding interactions that depend on N-glycans.
Wu et al. report that a photocrosslinking analog of GlcNAc can be incorporated into cellular N-glycans and used to covalently crosslink glycan-protein interactions in their native cellular context. This method can be used to identify novel glycan-protein interactions, and to evaluate the extent of glycan-protein binding occurring under diverse conditions. |
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ISSN: | 2451-9456 2451-9456 |
DOI: | 10.1016/j.chembiol.2021.07.007 |