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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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dNotum requires Dally to inhibit Wingless signalinga, 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 NRTWingless-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.
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Figure 8: dNotum requires Dally to inhibit Wingless signalinga, 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 NRTWingless-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.

Mentions: Even though dNotum does not seem to modulate Wingless signalling by cleaving the GPI anchor of glypicans, genetic interactions between notum and dlp suggest a functional relationship31,32. We therefore investigated the role of Dlp or Dally in dNotum’s ability to suppress Wingless signalling. dNotum overexpression, along the anterior-posterior (A-P) boundary, led to complete and long range suppression of Senseless expression (Fig. 2a). In the absence of either Dally or Dlp, this activity was very much reduced, as indicated by the recovery of endogenous Senseless expression (Fig. 2b, c). Notably, Dally was also required for Notum to suppress signalling by membrane-tethered Wingless (Extended Data Fig. 3a). Since Dally is not essential for survival, this requirement could be confirmed in adult wings (Extended Data Fig. 3b-d). To address the relevance of glypican GPI anchorage, we created a transgene expressing Dlp-CD8 (34 C-terminal amino acids of Dlp replaced by the CD8 transmembrane domain) under control of the tubulin promoter. This transgene restored the ability of overexpressed dNotum to repress Wingless signalling in dlp mutant homozygotes (Fig. 2d; compare to Fig 2b), confirming the importance of glypicans but not their GPI anchor.


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)

dNotum requires Dally to inhibit Wingless signalinga, 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 NRTWingless-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.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 8: dNotum requires Dally to inhibit Wingless signalinga, 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 NRTWingless-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.
Mentions: Even though dNotum does not seem to modulate Wingless signalling by cleaving the GPI anchor of glypicans, genetic interactions between notum and dlp suggest a functional relationship31,32. We therefore investigated the role of Dlp or Dally in dNotum’s ability to suppress Wingless signalling. dNotum overexpression, along the anterior-posterior (A-P) boundary, led to complete and long range suppression of Senseless expression (Fig. 2a). In the absence of either Dally or Dlp, this activity was very much reduced, as indicated by the recovery of endogenous Senseless expression (Fig. 2b, c). Notably, Dally was also required for Notum to suppress signalling by membrane-tethered Wingless (Extended Data Fig. 3a). Since Dally is not essential for survival, this requirement could be confirmed in adult wings (Extended Data Fig. 3b-d). To address the relevance of glypican GPI anchorage, we created a transgene expressing Dlp-CD8 (34 C-terminal amino acids of Dlp replaced by the CD8 transmembrane domain) under control of the tubulin promoter. This transgene restored the ability of overexpressed dNotum to repress Wingless signalling in dlp mutant homozygotes (Fig. 2d; compare to Fig 2b), confirming the importance of glypicans but not their GPI anchor.

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