Fluorogenic Coupled Assays Reveal Catalytic Properties, Inhibition Constants and Cellular Location of Mucin-Active Carbohydrate Sulfatases.

Sulfated glycans play a central role in human health and influence cell signaling, cancer progression, pathogen invasion, and host-microbiome interactions. Metabolism of these glycans requires a specialized class of enzymes termed carbohydrate sulfatases. These enzymes are particularly important in the human gut where sulfated colonic mucin is produced and subsequently degraded by colonic bacteria. Despite the biological importance of carbohydrate sulfatases, there is currently a lack of chemical tools to study their activity, substrate selectivity, inhibition, and the discovery of novel enzymes. To address this, we have synthesized new chemical tools to rapidly and quantitatively determine the activity and selectivity of carbohydrate sulfatases in plate-based coupled assays. We have synthesized 3-O-sulfated fluorogenic glycosides using efficient synthetic routes and combined these fluorogenic substrates with a glycosidase that selectively cleaves unsulfated glycosides, allowing sensitive detection of sulfatase activity on both purified protein and cell lysate from the S1_20 subfamily sulfatases. Furthermore, we show that the assay enables differentiation and quantification of substrate specificity, identification of sulfatase inhibitors, and determination of sulfatase (sub-)cellular location for two S1_20 subfamily sulfatases. Collectively, we anticipate that these tools will further our understanding of the interplay between carbohydrate sulfatases, sulfated glycans, and human health.