Nature 519, 187–192 (2015)

Dev. Cell 32, 719–730 (2015)

Credit: NATURE

Wnts are secreted signaling molecules that are covalently linked to the monounsaturated fatty acid palmitoleic acid, which directly inserts into the hydrophobic cleft of the Frizzled receptor. Wnts also interact with particular heparin sulfate proteoglycans called glypicans to mediate extracellular distribution and signaling efficacy of the Wnt ligands. There are a number of secreted inhibitors, such as Notum, that modulate Wnt signaling. Notum was initially thought to act as a phospholipase that hydrolyzes the GPI anchor of glypicans to restrict the extracellular distribution of Wnts through glypican-mediated sequestration. Kakugawa et al. and Zhang et al. tested this putative mechanism and found that Notum does not cleave the GPI anchor of glypicans. Instead, as shown by Kakugawa et al., Notum interacts with the sulfated glycosaminoglycan chains. Loss of glypican activity reduces Notum-mediated inhibition and cell-surface localization. To identify the target of Notum's activity, Kakugawa et al. determined the crystal structure of human Notum, revealing a canonical α/β hydrolase fold and two distinct ligand-binding pockets. One pocket contained a heparin saccharide–binding site, which was confirmed through SPR assays to mediate the direct glypican-Notum interactions, whereas the other was a large hydrophobic pocket that could potentially accommodate unsaturated fatty acids such as palmitoleic acid. Another crystal structure from Kakugawa et al., showing Notum bound to a palmitoleoylated Wnt7a peptide, confirmed that the fatty acid occupied the hydrophobic pocket, with the ester bond located close to the catalytic center. Both groups of authors suspected that Notum is a carboxylesterase that cleaves the ester bond connecting palmitoleate to Wnt. Kakugawa et al. used mass spectrometry analysis whereas Zhang et al. used palmitic analogs to detect Notum-mediated cleavage of Wnt's palmitoleic acid linkage. Finally, Zhang et al. found that Wnt3a lacking palmitoleic acid formed soluble oligomers linked by intermolecular disulfide bonds that were unable to interact with the Frizzled receptor. Overall, these findings identify Notum as a novel extracellular deacylase.