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

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.