The Siglec family represents one of the major classes of human glycan-binding proteins (GBPs) that regulates a broad range of biological processes through engagement with their glycan ligands. Human Siglecs have attracted a great deal of attention in recent years, as they are regarded as new immune checkpoints. The disruption of Siglec-sialoglycan interactions represents a promising next-generation strategy for cancer immunotherapy. In addition to the sialic acid moiety, it has been well established that glycan sulfation plays an essential role in the ligand binding of human Siglecs. Therefore, there is an urgent need to thoroughly understand the subtle differences in the glycan scaffolds and sulfation patterns that govern Siglec binding. N-Glycans are the most abundant class of glycans occurring in living cells, while sulfation is the most widespread postglycosylation modification of N-glycans, indicating that sulfated N-glycans serve as key ligands for human Siglecs. However, the synthetic generation of large libraries of complex sulfated N-glycans is impractical as sulfation introduces additional challenges to the already demanding process of N-glycan synthesis. Herein, we describe a de novo chemoenzymatic approach for the efficient preparation of complex sulfated N-glycans. Using this approach, a comprehensive 72-member library of sulfated N-glycans encompassing the most typical complex structures was successfully prepared. With the well-defined library (comprising 72 sulfated N-glycans and 26 nonsulfated controls), we systematically investigated the binding activities of all human Siglecs. Beyond the synthetic breakthroughs, this work provides the most comprehensive profile to date for understanding how sulfation patterns and N-glycan scaffolds affect Siglec ligand binding, which will have an immediate impact on the development of Siglec-targeted immunotherapy strategies.
Ma et al. (Mon,) studied this question.