Notum deacylates Wnt proteins to suppress signalling activity

Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase. The biochemical activity of Notum as a carboxylesterase that removes an essential lipid moiety from Wnt proteins is uncovered; the interaction of Notum with glypicans is required to ensure localization at the cell surface, and Notum may provide a new target for therapeutic development in diseases with defective Wnt signalling. The secreted enzyme known as Notum is a feedback inhibitor of the Wnt signalling pathway, found in most metazoans including planarian worms and humans. It was thought to act as a phospholipase targeting heparan sulfate proteoglycans (glypicans), but how it achieved specificity for Wnt ligands was unclear. Jean-Paul Vincent and colleagues now report a novel biochemical activity for Notum as an extracellular carboxylesterase that removes an essential lipid moiety from Wnt proteins. Notum's interaction with glypicans is required to achieve its localization at the cell surface, rather than the enzyme–substrate relationship previously suspected. This activity of Notum may provide a new target for therapeutics in diseases with defective Wnt signalling.