Defining Substrate Specificities of N-Acetylglucosamine-6-O-Sulfotransferases for Enzymatic Modular Assembly of Sulfated O-Glycans

Abstract

Sulfated O-glycans are widely distributed and play key roles in a wide range of physiological and disease processes, yet their synthesis remains challenging due to difficulties in regioselective sulfation. Here, we systematically characterized two human GlcNAc-6-O-sulfotransferases, CHST2 and CHST6, revealing their strong preferences towards O-glycans (including O-GalNAc and O-mannosyl glycans) over N-glycans and poly-LacNAc chains. Both enzymes favored β1-6branched GlcNAc residues, with CHST6 showing higher activity and broader substrate tolerance than CHST2. Guided by these insights, we established a modular enzymatic assembly platform for efficient synthesis of 32 well-defined sulfated O-GalNAc, O-mannosyl glycans, and O-glycopeptides. This streamlined strategy enables versatile access to sulfated O-glycans and provides a general route for constructing other classes of sulfated glycan.