Switchable glycan probe enables pH-dependent activity modulation of UDP-glucose: glycoprotein glucosyltransferase through a responsive aglycone

Abstract

Fluorescently labeled glycans are widely used as chemical probes to study glycoprotein processing and quality control; however, they are generally regarded as passive reporters of enzymatic activity. In contrast, the development of glycan probe that can actively control enzymatic activity remains an important challenge in chemical biology. Herein, we report a switchable glycan probe that actively modulates UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) activity through a pH-responsive aglycone design. A Man₉GlcNAc₂-Asn conjugate bearing fluorescein was synthesized, in which the fluorescein aglycone undergoes reversible pH-dependent structural interconversion, altering its physicochemical character. In vitro UGGT1 assays revealed that the switchable glycan probe exhibits glucose transfer activity in the order of pH 6.0 > pH 8.5, while a unresponsive control probe showed no pH-dependent change in activity. Furthermore, UGGT1 activity could be dynamically switched during the reaction by changing the pH. These results demonstrate that aglycone structural populations can actively regulate UGGT1 activity, providing the first example of controlling UGGT1-mediated glucose transfer using a single, environmentally switchable glycan probe. This work establishes an innovative molecular design strategy, opening a conceptual framework to probe and manipulate ER glycoprotein quality control mechanism using chemically programmable glycan probes.