Substrate Promiscuity of CHST1 Enables Efficient Synthesis of Galactose‐6‐O‐Sulfated Glycans for Siglec Recognition Profiling

ABSTRACT The galactose‐6‐O‐sulfation (Gal‐6‐su) is a prevalent glycan modification that influences Siglec recognition across diverse glycan structures. In this study, we achieved high‐level expression of an active truncated CHST1 (carbohydrate sulfotransferase 1, a Gal‐6‐O‐sulfotransferase) and conducted systematic substrate specificity studies. We identified remarkable substrate promiscuity toward β‐galactosides with activity significantly enhanced by α2,3‐sialylation. Most other modifications on Gal are tolerated by CHST1 except for those at its C4‐hydroxyl position, and the activity is strongly influenced by proximal residues and linkage type. We also observed an unexpected activity toward β‐ N ‐acetyl‐galactosamine (GalNAc) glycosides including chondroitin, resembling the activity of CHST3. Exploiting this substrate flexibility, chemoenzymatic modular strategies were developed to prepare novel sulfated gangliosides and O‐glycans for profiling Siglec‐glycan interactions. Glycan microarray analyses, using synthesized sulfated glycans alongside their non‐sulfated counterparts, demonstrate that Gal‐6‐su broadly enhances binding across Siglec‐1, ‐3, ‐5, ‐7, ‐8, ‐15, and murine Siglec‐E and ‐F, while inhibiting binding to Siglec‐9. A clear Siglec preference to α2,3‐sialylated and 6‐O‐sulfated β1,4‐galactosides was also observed. Together, these findings define key determinants of CHST1 specificity, expand the glycan synthesis toolbox, and provide new insight into how Gal‐6‐su modulates Siglec recognition, enabling future therapeutic and biochemical applications.