Chemoenzymatic Synthesis of Sulfated N-Glycans Recognized by Siglecs and Other Glycan-Binding Proteins

Sulfated N-glycans are present in many glycoproteins, which are implicated in playing important roles in biological recognition processes. Here, we report the systematic chemoenzymatic synthesis of a library of sulfated and sialylated biantennary N-glycans and assess their binding to Siglecs and glycan-specific antibodies that recognize them as glycan ligands. The combined use of three human sulfotransferases, GlcNAc-6-O-sulfotransferase (CHST2), Gal-3-O-sulfotransferase (Gal3ST1), and keratan sulfate Gal-6-O-sulfotransferase (CHST1), resulted in asymmetric and symmetric branch-selective sulfation of the GlcNAc and/or Gal moieties of N-glycans. The extension of the sugar chain using alpha-2,3- and alpha-2,6-sialyltransferases afforded the sulfated and sialylated N-glycans. These synthetic glycans with different patterns of sulfation and sialylation were evaluated for binding to selected Siglecs and sulfoglycan-specific antibodies using glycan microarrays. The results confirm previously documented glycan-recognizing properties and further reveal novel specificities for these glycan-binding proteins, demonstrating the utility of the library for assessing the specificity of glycan-binding proteins recognizing sulfated and sialylated glycans.