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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.Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor.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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Related in: MedlinePlus

Additional mass spectrometric analysis of hNotum’s deacylase activitya, Mass spectra of CHGLSGSCEVK from trypsinised 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, Triplicate LC-MS peak areas with label reversal. Irrespective of the nature of the label (grey indicates light label and black, 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 dataset were not affected by hNotumcore. e, Activity of hNotumcore and its S232A 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 S232A variant, caused significant delipidation (peak corresponding to delipidated peptide is marked by asterisk). Quantification of duplicate such experiments is shown in Fig. 5c. f, MALDI-TOF analysis shows that neither hNotumcore nor its S232A variant delipidated a synthetic Sonic Hedgehog peptide (CGPGRGFGKRR) palmitoylated on its amino terminal Cysteine. Quantification of duplicate such experiments is shown on Fig 5d (peak corresponding to lipidated peptide is marked by black triangle). g, 2D active site schematic relating to Fig. 5e. Additional hydrogen bonds and electron pair movements thought to occur during hydrolysis by the wild type 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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Figure 14: Additional mass spectrometric analysis of hNotum’s deacylase activitya, Mass spectra of CHGLSGSCEVK from trypsinised 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, Triplicate LC-MS peak areas with label reversal. Irrespective of the nature of the label (grey indicates light label and black, 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 dataset were not affected by hNotumcore. e, Activity of hNotumcore and its S232A 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 S232A variant, caused significant delipidation (peak corresponding to delipidated peptide is marked by asterisk). Quantification of duplicate such experiments is shown in Fig. 5c. f, MALDI-TOF analysis shows that neither hNotumcore nor its S232A variant delipidated a synthetic Sonic Hedgehog peptide (CGPGRGFGKRR) palmitoylated on its amino terminal Cysteine. Quantification of duplicate such experiments is shown on Fig 5d (peak corresponding to lipidated peptide is marked by black triangle). g, 2D active site schematic relating to Fig. 5e. Additional hydrogen bonds and electron pair movements thought to occur during hydrolysis by the wild type 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.

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)

Additional mass spectrometric analysis of hNotum’s deacylase activitya, Mass spectra of CHGLSGSCEVK from trypsinised 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, Triplicate LC-MS peak areas with label reversal. Irrespective of the nature of the label (grey indicates light label and black, 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 dataset were not affected by hNotumcore. e, Activity of hNotumcore and its S232A 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 S232A variant, caused significant delipidation (peak corresponding to delipidated peptide is marked by asterisk). Quantification of duplicate such experiments is shown in Fig. 5c. f, MALDI-TOF analysis shows that neither hNotumcore nor its S232A variant delipidated a synthetic Sonic Hedgehog peptide (CGPGRGFGKRR) palmitoylated on its amino terminal Cysteine. Quantification of duplicate such experiments is shown on Fig 5d (peak corresponding to lipidated peptide is marked by black triangle). g, 2D active site schematic relating to Fig. 5e. Additional hydrogen bonds and electron pair movements thought to occur during hydrolysis by the wild type 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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Figure 14: Additional mass spectrometric analysis of hNotum’s deacylase activitya, Mass spectra of CHGLSGSCEVK from trypsinised 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, Triplicate LC-MS peak areas with label reversal. Irrespective of the nature of the label (grey indicates light label and black, 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 dataset were not affected by hNotumcore. e, Activity of hNotumcore and its S232A 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 S232A variant, caused significant delipidation (peak corresponding to delipidated peptide is marked by asterisk). Quantification of duplicate such experiments is shown in Fig. 5c. f, MALDI-TOF analysis shows that neither hNotumcore nor its S232A variant delipidated a synthetic Sonic Hedgehog peptide (CGPGRGFGKRR) palmitoylated on its amino terminal Cysteine. Quantification of duplicate such experiments is shown on Fig 5d (peak corresponding to lipidated peptide is marked by black triangle). g, 2D active site schematic relating to Fig. 5e. Additional hydrogen bonds and electron pair movements thought to occur during hydrolysis by the wild type 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.
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.Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor.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