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.

N-聚糖显示在细胞表面蛋白上，可以与膜结合和分泌的聚糖结合蛋白 (GBP) 进行直接结合相互作用。聚糖介导的相互作用的生化鉴定和表征通常因低结合亲和力而变得困难。在这里，我们描述了将二氮丙啶光交联剂代谢引入细胞表面 N-连接糖蛋白的 N-乙酰葡糖胺 (GlcNAc) 残基上。我们描述了掺入二氮杂环丙烷修饰的 GlcNAc 的位点，以及对聚糖结构的适度扰动。我们表明，二氮丙啶修饰的 GlcNAc 可用于将两个细胞外 GBPs、半乳糖凝集素 1 和霍乱毒素亚基 B​​ 共价交联到细胞表面 N 连接糖蛋白。交联的程度与 GBP 的首选聚糖配体的显示相关。此外，可以分离共价交联的复合物，并通过蛋白质组学分析鉴定交联的 N-连接糖蛋白的蛋白质成分。该方法可用于发现和表征依赖于 N-聚糖的结合相互作用。