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

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

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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 languageEnglish (US)
Pages (from-to)84-97.e8
JournalCell Chemical Biology
Volume29
Issue number1
DOIs
StatePublished - 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

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