Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

Structural basis for sulfation-dependent self-glycan recognition by the human immune-inhibitory receptor Siglec-8

Siglec-8 is a human immune-inhibitory receptor that, when engaged by specific self-glycans, triggers eosinophil apoptosis and inhibits mast cell degranulation, providing an endogenous mechanism to down-regulate immune responses of these central inflammatory effector cells. Here we used solution NMR spectroscopy to dissect the fine specificity of Siglec-8 toward different sialylated and sulfated carbohydrate ligands and determined the structure of the Siglec-8 lectin domain in complex with its prime glycan target 6′-sulfo sialyl Lewisx. A canonical motif for sialic acid recognition, extended by a secondary motif formed by unique loop regions, recognizing 6-O–sulfated galactose dictates tight specificity distinct from other Siglec family members and any other endogenous glycan recognition receptors. Structure-guided mutagenesis revealed key contacts of both interfaces to be equally essential for binding. Our work provides critical structural and mechanistic insights into how Siglec-8 selectively recognizes its glycan target, rationalizes the functional impact of site-specific glycan sulfation in modulating this lectin–glycan interaction, and will enable the rational design of Siglec-8–targeted agonists to treat eosinophil- and mast cell-related allergic and inflammatory diseases, such as asthma.

Design and synthesis of active heparan sulfate-based probes

Synthesis of sulfated carbohydrate molecular probes and the utilities as specific inhibitors for carbohydrate sulfotransferases.

Easy protocol for making a good E-sinapinic acid matrix for neutral and sulfated carbohydrate MALDI-MS analysis

A Z- + E- mixture of cinnamics was prepared in a solution of commercial E-acid (i.e., E-SA) by photoisomerization. This “photo-made at home” MALDI matrix provides direct, rapid and sensitive detection of neutral and sulfated carbohydrates.