Heparan Sulfate (HS) is a critical cell signaling molecule linked to pathological processes ranging from cancer to viral entry, yet fundamental aspects of its biosynthetic mechanism remain incompletely understood. Here, the binding preferences of the 2-O-sulfotransferase (2OST) towards well-defined variably-sulfated hexasaccharides are examined. Surprisingly, higher-sulfated oligosaccharides, associated with later-acting sulfotransferases, bind more tightly to 2OST than oligosaccharides that correspond to its natural substrate or product. Binding analyses performed with Biolayer Interferometry indicate affinity correlates simply with degree of oligosaccharide sulfation, consistent with non-specific binding. However, inhibition assays show that the higher-sulfated oligosaccharides also display proportionally-higher IC50 values relative to the natural product of 2OST, indicating their specific interaction with the active site. Molecular docking and dynamics simulations predict a mode of inhibition, in which a 6-sulfate in the higher-sulfated oligosaccharides occupies the canonical binding site of the cofactor 3'-phosphoadenosine-5'-phosphosulfate. The unexpected finding that oligosaccharides associated with later stages in HS biosynthesis inhibit 2OST indicates that the enzyme must be separated temporally and or spatially from downstream products during biosynthesis in vivo, and highlights a challenge for the enzymatic synthesis of lengthy HS chains in vitro.

Unexpected binding specificity of 2OST: Implications for heparan sulfate biosynthesis

255th National Meeting (2018)

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