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...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Cell chemical biology 2021-07, Vol.29 (1), p.84-97.e8
Hauptverfasser: Wu, Han, Shajahan, Asif, Yang, Jeong-Yeh, Capota, Emanuela, Wands, Amberlyn M., Arthur, Connie M., Stowell, Sean R., Moremen, Kelley W., Azadi, Parastoo, Kohler, Jennifer J.
Format: Artikel
Sprache:eng
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
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.
ISSN:2451-9456
2451-9456
DOI:10.1016/j.chembiol.2021.07.007