Abstract
Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.
Highlights
Biological sulfation is a widespread reversible covalent modification found throughout nature [1]
We propose that appropriately validated small-molecule ligands might become invaluable probes for rapid cellular inhibition of heparan sulfate (HS) 2-O-sulfotransferase (HS2ST), and that further iteration could lead to the discovery and synthesis of small molecules, including compound classes currently employed as kinase inhibitors, to probe cellular HS2ST function
We purified a recombinant HS2ST catalytic domain fused to an N-terminal maltose-binding protein (MBP) tag to near homogeneity (Figure 1B) and evaluated its thermal denaturation profile with the MBP tag still attached in the presence of adenosine 30-phosphate 50-phosphosulfate (PAPS), heparin or maltose (Figure 1C)
Summary
Biological sulfation is a widespread reversible covalent modification found throughout nature [1]. FGF–receptor interactions and intracellular signalling to the ERK pathway are blunted in the absence of appropriate 2-O sulfation driven by heparan sulfate (HS)-modifying enzymes [4,5,6,7,8,9], while sulfation of the tetrasaccharide Sialyl LewisX antigen on glycolipids controls leukocyte adhesion to the endothelium during inflammation [10,11]. Inappropriate glycan sulfation can underlie aspects of abnormal signalling, infection, inflammation and, increasingly, human neuropathies [12], suggesting that targeting of carbohydrate sulfation dynamics using small-molecule enzyme inhibitors may be of value in both basic and translational research [13]. The current limited chemical toolbox to rapidly modify and study glycan sulfation is based around small-molecule inhibitors of sulfatase-2 (Sulf-2), such as OKN-007 [14] or heparanase inhibitors and HS mimics, including roneparstat and PG545, which have been employed for basic and clinical investigation [15]
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