Enzyme-Sialylation-Controlled\nChemical Sulfation of\nGlycan Epitopes for Decoding the Binding of Siglec Ligands

Widely\ndistributed in nature, sulfated glycan epitopes\nplay important\nroles in diverse pathophysiological processes. However, due to their\nstructural complexity, the preparation of glycan epitopes with structurally\ndefined sulfation patterns is challenging, which significantly hampers\nthe detailed elucidation of their biological functions at the molecular\nlevel. Here, we introduce a strategy for site-specific chemical sulfation\nof glycan epitopes, leveraging enzymatic sialylation and desialylation\nprocesses to precisely control the regio-specificity of sulfation\nof disaccharide or trisaccharide glycan backbones. Using this method,\na sulfated glycan library covering the most common sialylated glycan\nepitopes was prepared in high yield and efficiency. By screening a\nmicroarray prepared with this glycan library, we systematically probed\ntheir binding specificity with human Siglecs (sialic acid-binding\nimmunoglobulin-type lectins), many of which function as glyco-immune\ncheckpoints to suppress immune system activation. Our investigation\nrevealed that sulfation and sialylation patterns serve as important\ndeterminants of Siglec binding affinity and specificity. Thus, these\nfindings offer new insights for the development of research tools\nand potential therapeutic agents targeting glyco-immune checkpoints\nby modulating the Siglec signaling pathway.