Abstract
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 photo-cross-linker onto N-acetylglucosamine (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 cross-link two extracellular GBPs, galectin-1 and cholera toxin subunit B, to cell-surface N-linked glycoproteins. The extent of cross-linking correlates with display of the preferred glycan ligands for the GBPs. In addition, covalently cross-linked complexes could be isolated, and protein components of cross-linked 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.
Original language | English (US) |
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Pages (from-to) | 84-97.e8 |
Journal | Cell Chemical Biology |
Volume | 29 |
Issue number | 1 |
DOIs | |
State | Published - Jan 20 2022 |
Keywords
- N-glycan
- cholera
- cross-linking
- diazirine
- galectin
- glycosylation
- glycosyltransferase
ASJC Scopus subject areas
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Pharmacology
- Drug Discovery
- Clinical Biochemistry