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.
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Protein Data Bank
The crystal structures reported in this paper have been deposited in the Protein Data Bank (PDB) under accession numbers 4WBH, 4UYU, 4UYW, 4UZL, 4UYZ, 4UZ1, 4UZ5, 4UZ6, 4UZ7, 4UZ9, 4UZA, 4UZQ, 4UZJ and 4UZK.
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We thank K. Dingwell for supplying purified mWnt3A, H. Bellen for anti-Senseless, C. Alexandre for plasmids and advice, W. Chai for glycosaminoglycan probes, T. Holder for suggestions, T. Malinauskas and C. Lorenz for advice and technical support, T. Walter for technical support with crystallization, W. Lu and Y. Zhao for help with tissue culture, and the organisers of the EMBO Wnt meeting 2012 where our collaboration began. We thank staff at Diamond Light Source beamlines (i02, i03, i04, i04-1, i24) for assistance with data collection (proposal mx8423). This work was supported by the MRC (U117584268 to J.-P.V.; G0900084 to E.Y.J.), the UK Research Council Basic Technology Initiative (Glycoarrays Grant GRS/79268 and EPSRC Translational Grant EP/G037604/1), the Wellcome Trust (Biomedical Resource Grants WT093378MA and WT099197MA) to T.F., the European Union (ERC grant WNTEXPORT; 294523 to J.-P.V., a Marie Curie IEF grant to M.Z.), Cancer Research UK (C375/A10976 to E.Y.J.), and the Japan Society for the Promotion of Science (to S.K.). T.-H.C. was funded by a Nuffield Department of Medicine Prize Studentship in conjunction with Clarendon and Somerville College Scholarships. The Wellcome Trust Centre for Human Genetics is supported by Wellcome Trust Centre grant 090532/Z/09/Z.
The authors declare no competing financial interests.
Extended data figures and tables
a, b, Overexpression of dNotum-V5 with the apterous-Gal4 driver, which is expressed in the dorsal compartment, prevents expression of Senseless (Sens) (b, middle), a Wingless target gene, but has little effect on phospho-Mad (pMad) immunoreactivity (b), an indicator of Dpp signalling. c, Loss of notum activity, achieved by generating large patches of notumKO tissue (see Methods), marked by the loss of GFP, leads to broadening of Senseless expression but does not affect pMad immunoreactivity. d–g, Strong, but not complete, reduction of notum activity led to ectopic wing margin bristles (compare insets in d and e) but had no significant effect on wing area, which is sensitive to Dpp signalling (f) (P = 0.26, Student’s t-test), or on the distance between L3 and L4 veins, which is affected by changes in Hedgehog signalling61 (g) (P = 0.41, Student’s t-test). In total, 19 control (notum141/+) and 17 mutant (notum141/KO) wings were analysed. Error bars in f and g are s.d.
a, b, Ectopic expression of Senseless caused by NRT-wingless, as well as endogenous Senseless, is suppressed by co-expression of dNotum. NRT-wingless and notum are expressed in a vertical band under the control of dpp-Gal4. c, Western blot analysis of phase-separated extracts of S2 cells transfected with a plasmid expressing HA-tagged Dally. In control extracts, Dally is found largely in the detergent (D) phase. Coexpression of dNotum–V5 from a plasmid had no effect, while treatment with PIPLC shifted all detectable Dally to the aqueous (A) phase. d, dNotum-V5 expression as in c was sufficient to suppress Wingless-induced TOPFlash activity. Cells were transfected with a dual luciferase TOPFlash reporter60 along with a mock plasmid (−), tubulin::wingless (Wg), or tubulin::wingless + actin::notum-V5 (Wg + Notum). e–h, Extracellular Dlp in control (e, g), PIPLC-treated (f) or apterous-Gal4 UAS-notum-V5 (h) imaginal discs. i–l, Extracellular anti-GFP staining of imaginal discs from gene trap line expressing Dally–GFP fusion protein. Discs were treated with a mock solution (i) or PIPLC (j) (same discs as in e or f, respectively, but here showing Dally protein). In a separate experiment, dNotum was overexpressed with apterous-Gal4 in the Dally-GFP background (l). No change in the distribution of extracellular GFP could be seen compared to that in control discs (k, no apterous-Gal4).
a, Wingless and Senseless expression in a dally−/− wing imaginal disc expressing NRT-wingless and notum under the control of dpp-Gal4. Some senseless expression remains, indicating that, in the absence of Dally, dNotum is a poor inhibitor of NRT-Wingless-induced (as well as endogenous) signalling. b–d, Anterior margin of wings from control, spalt (sal)-Gal4 UAS-notum-V5, and sal-Gal4 UAS-notum-V5 dally−/− animals. Removal of dally rescues the loss of margin bristles caused by dNotum overexpression.
Binding of dNotum-V5 to a GAG oligosaccharide array, detected by immunofluorescence. CSA/B/C, chondroitin sulfate A/B/C; HA, hyaluronic acid, hep, heparin; HS, heparan sulfate. Details on the array are provided in the Methods.
a, Topology plot of hNOTUM. β-strands are shown as numbered triangles and α-helices as circles labelled in alphabetical order from the N to C terminus (NT to CT). Structural elements conserved among most α/β-hydrolases are outlined in grey. b, Comparison of the two most conformationally distinct hNOTUM structures (from crystal forms III and V). Crystal form III is the most structurally different. All other structures superimpose with root mean squared deviation (r.m.s.d.) of <0.7 Å. Circles highlight the most flexible regions. c, Comparison between the structures of hNOTUM (form V) and dNotum (form I). The circle highlights the lack of a cysteine bridge in dNotum.
a, Heparin affinity chromatography of wild-type hNOTUM and selected surface variants. b–e, Close-up views of additional sulfate binding sites on hNOTUM, crystal form III. Each view is accompanied with SPR heparin affinity data corresponding to each hNOTUM variant.
a, Comparison between hNOTUM and human esterase D (hESTD), showing structural relatedness. hNOTUM is also related to hAPT1, a cytosolic esterase used in this study as a positive control for fatty acid esterase activity. In the views shown here, the hNOTUM structure has been rotated by 90° around the x axis relative to the structure shown in Fig. 3b. b, Rootless phylogenetic tree of animal Notum proteins (red) and plant pectin acetylesterases (PAE, green). Extent of sequence identity to hNOTUM is shown next to species name. Percentages between branches indicate sequence identity between neighbours.
a, Inhibition of hNOTUM activity on pNP-butyrate (pNP4) by PMSF (30 min pre-incubation with 2 mM PMSF) as well as by Triton X-100 and CHAPS (0.5%). Presence of 20 mM SOS and 50 mg l−1 heparin results in a minor increase of esterase activity. The height of each bar represents activity relative to the mean of four control samples lacking the additives. b, Saturable inhibition of hNOTUM by Triton X-100. Triton X-100 inhibits many esterases owing to binding to the acyl binding pocket through its hydrophobic group. c, Lack of inhibition of Norrin-mediated β-catenin stabilization by Notum. Recombinant Norrin was pretreated with hNOTUMcore at a concentration sufficient to suppress Wnt3A-mediated signalling. d, e, Saturation kinetics of the action of hNOTUM on pNP-octanoate (pNP8, d) and pNP-butyrate (pNP4, e). The activity was normalized to the Amax calculated for hNOTUMcore. The activity values for the larger, full length protein were adjusted to compensate for the increased mass. Apparent Km values in d were corrected for the inhibition caused by Triton X-100. f, Saturation inhibition kinetics with myristoleic and palmitoleic acid. pNP8 was used at a concentration of 1 mM and 250 μM, respectively.
a, Mass spectra of CHGLSGSCEVK from trypsinized Wnt3A protein mock-treated or treated with hNOTUMcore. Left-hand graph is the same as that shown in Fig. 5a, while the right-hand side shows the results of a separate experiment performed with the labels reversed. b, Duplicate LC–MS peak areas with label reversal. Irrespective of the nature of the label (grey indicates light label, black indicates heavy label), hNOTUMcore triggered an increase in peak area of the delipidated Wnt3A tryptic peptide. c, d, Two control Wnt3A cysteine-containing peptides from the same data set were not affected by hNOTUMcore. e, Activity of hNOTUMcore and its Ser232Ala variant on a synthetic disulphide-bonded Wnt3A peptide (CHGLSGSCEVK) palmitoleoylated on the first serine. Both lipidated and unlipidated peptide could be detected by MALDI-TOF. Incubation with hNOTUMcore, but not its Ser232Ala variant, caused significant delipidation (peak corresponding to delipidated peptide is marked by asterisk). Quantification of triplicate experiments is shown in Fig. 5c. f, MALDI-TOF analysis shows that neither hNOTUMcore nor its Ser232Ala variant delipidated a synthetic SHH peptide (CGPGRGFGKRR) palmitoylated on its N-terminal cysteine. Quantification of triplicate experiments is shown in Fig. 5d (peak corresponding to lipidated peptide is marked by black triangle). g, Two-dimensional active site schematic relating to Fig. 5e. Additional hydrogen bonds and electron pair movements thought to occur during hydrolysis by the wild type protein are shown in grey. h, Close-up view on the myristoleate active site complex of hNOTUMcore (crystal form I). The experimental omit electron density is contoured at 2σ.
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Kakugawa, S., Langton, P., Zebisch, M. et al. Notum deacylates Wnt proteins to suppress signalling activity. Nature 519, 187–192 (2015) doi:10.1038/nature14259
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