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Notum deacylates Wnt proteins to suppress signalling activity.

Kakugawa S, Langton PF, Zebisch M, Howell SA, Chang TH, Liu Y, Feizi T, Bineva G, O'Reilly N, Snijders AP, Jones EY, Vincent JP - Nature (2015)

Bottom Line: 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.They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate.

View Article: PubMed Central - PubMed

Affiliation: MRC's National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

ABSTRACT
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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Wnt-deacylation by Notuma, LC-MS analysis of mWnt3A protein treated with hNotumcore or a mock solution. By comparison to mock treatment (light label), addition of hNotum (heavy label) caused a significant increase in the signal intensity of unlipidated CHGLSGSCEVK b, LC-MS peak areas from panel a.; shown as mean +/− s.e.m. (n=3) c, d, Quantification from MALDI analysis of synthetic lipid-bearing peptides treated with hNotumcore or its S232A variant; shown as mean +/− s.e.m. (n=3). Palmitoleoylated hWnt3A peptide, but not palmitoylated hSonic Hedgehog peptide, was specifically deacylated by the wild type enzyme. e, Close-up view on the seryl-palmitoleate active site complex of hNotum. The experimental omit electron density is contoured at 2σ. f, Feedback control by Notum. Notum deacylates Wnt in a Glypican-assisted fashion.
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Figure 5: Wnt-deacylation by Notuma, LC-MS analysis of mWnt3A protein treated with hNotumcore or a mock solution. By comparison to mock treatment (light label), addition of hNotum (heavy label) caused a significant increase in the signal intensity of unlipidated CHGLSGSCEVK b, LC-MS peak areas from panel a.; shown as mean +/− s.e.m. (n=3) c, d, Quantification from MALDI analysis of synthetic lipid-bearing peptides treated with hNotumcore or its S232A variant; shown as mean +/− s.e.m. (n=3). Palmitoleoylated hWnt3A peptide, but not palmitoylated hSonic Hedgehog peptide, was specifically deacylated by the wild type enzyme. e, Close-up view on the seryl-palmitoleate active site complex of hNotum. The experimental omit electron density is contoured at 2σ. f, Feedback control by Notum. Notum deacylates Wnt in a Glypican-assisted fashion.

Mentions: To test directly Notum-mediated Wnt deacylation we turned to LC-MS analysis. mWnt3A was purified from conditioned medium (CM), treated with recombinant hNotumcore or a mock solution, differentially isotope labelled, and trypsinised. No significant identification could be obtained for the predicted palmitoleoylated tryptic peptide, indicating incompatibility with the LC-MS conditions. Importantly however, following treatment with hNotum, this peptide could be identified and quantified in non-acylated form (Fig. 5a, b and Extended data Fig. 9a, b). Replicate LC-MS measurements and label reversal consistently showed an increase in signal intensity for the hNotum-treated de-acylated peptide whereas control peptides showed no significant change (Extended Data Fig. 9c, d). This suggests that treatment of mWnt3A with hNotum removes the palmitoleic acid moiety thus rendering the relevant peptide more hydrophilic and detectable by LC-MS. Encouraged by these results, we proceeded to assess the activity of hNotum on synthetic peptides. The predicted tryptic peptide from hWnt3A was synthesised in a disulphide bonded form with a palmitoleate group on the relevant serine (Methods). These peptides were treated with recombinant hNotumcore, or with hNotumcoreS232A, which is predicted to be enzymatically inactive, and the reaction products were analysed by MALDI-TOF. No significant deacylation was detected in hNotumcoreS232A-treated samples while hNotum-treated peptides were found to be extensively deacylated (Fig. 5c, Extended Data Fig. 9e). We conclude from these assays that Notum catalyses the removal of palmitoleic acid, which is normally O-linked to S209 of hWnt3A. We also assayed the effect of hNotum on a synthetic peptide from hSonic Hedgehog, which is N-palmitoylated at the amino terminus. No change in the level of acylation could be detected (Fig. 5d, Extended Data Fig. 9f), confirming that Notum’s activity on Wnt is specific, in agreement with our genetic evidence.


Notum deacylates Wnt proteins to suppress signalling activity.

Kakugawa S, Langton PF, Zebisch M, Howell SA, Chang TH, Liu Y, Feizi T, Bineva G, O'Reilly N, Snijders AP, Jones EY, Vincent JP - Nature (2015)

Wnt-deacylation by Notuma, LC-MS analysis of mWnt3A protein treated with hNotumcore or a mock solution. By comparison to mock treatment (light label), addition of hNotum (heavy label) caused a significant increase in the signal intensity of unlipidated CHGLSGSCEVK b, LC-MS peak areas from panel a.; shown as mean +/− s.e.m. (n=3) c, d, Quantification from MALDI analysis of synthetic lipid-bearing peptides treated with hNotumcore or its S232A variant; shown as mean +/− s.e.m. (n=3). Palmitoleoylated hWnt3A peptide, but not palmitoylated hSonic Hedgehog peptide, was specifically deacylated by the wild type enzyme. e, Close-up view on the seryl-palmitoleate active site complex of hNotum. The experimental omit electron density is contoured at 2σ. f, Feedback control by Notum. Notum deacylates Wnt in a Glypican-assisted fashion.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4376489&req=5

Figure 5: Wnt-deacylation by Notuma, LC-MS analysis of mWnt3A protein treated with hNotumcore or a mock solution. By comparison to mock treatment (light label), addition of hNotum (heavy label) caused a significant increase in the signal intensity of unlipidated CHGLSGSCEVK b, LC-MS peak areas from panel a.; shown as mean +/− s.e.m. (n=3) c, d, Quantification from MALDI analysis of synthetic lipid-bearing peptides treated with hNotumcore or its S232A variant; shown as mean +/− s.e.m. (n=3). Palmitoleoylated hWnt3A peptide, but not palmitoylated hSonic Hedgehog peptide, was specifically deacylated by the wild type enzyme. e, Close-up view on the seryl-palmitoleate active site complex of hNotum. The experimental omit electron density is contoured at 2σ. f, Feedback control by Notum. Notum deacylates Wnt in a Glypican-assisted fashion.
Mentions: To test directly Notum-mediated Wnt deacylation we turned to LC-MS analysis. mWnt3A was purified from conditioned medium (CM), treated with recombinant hNotumcore or a mock solution, differentially isotope labelled, and trypsinised. No significant identification could be obtained for the predicted palmitoleoylated tryptic peptide, indicating incompatibility with the LC-MS conditions. Importantly however, following treatment with hNotum, this peptide could be identified and quantified in non-acylated form (Fig. 5a, b and Extended data Fig. 9a, b). Replicate LC-MS measurements and label reversal consistently showed an increase in signal intensity for the hNotum-treated de-acylated peptide whereas control peptides showed no significant change (Extended Data Fig. 9c, d). This suggests that treatment of mWnt3A with hNotum removes the palmitoleic acid moiety thus rendering the relevant peptide more hydrophilic and detectable by LC-MS. Encouraged by these results, we proceeded to assess the activity of hNotum on synthetic peptides. The predicted tryptic peptide from hWnt3A was synthesised in a disulphide bonded form with a palmitoleate group on the relevant serine (Methods). These peptides were treated with recombinant hNotumcore, or with hNotumcoreS232A, which is predicted to be enzymatically inactive, and the reaction products were analysed by MALDI-TOF. No significant deacylation was detected in hNotumcoreS232A-treated samples while hNotum-treated peptides were found to be extensively deacylated (Fig. 5c, Extended Data Fig. 9e). We conclude from these assays that Notum catalyses the removal of palmitoleic acid, which is normally O-linked to S209 of hWnt3A. We also assayed the effect of hNotum on a synthetic peptide from hSonic Hedgehog, which is N-palmitoylated at the amino terminus. No change in the level of acylation could be detected (Fig. 5d, Extended Data Fig. 9f), confirming that Notum’s activity on Wnt is specific, in agreement with our genetic evidence.

Bottom Line: 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.They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate.

View Article: PubMed Central - PubMed

Affiliation: MRC's National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus