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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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Structural and biophysical analysis of heparin bindinga, 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.
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Figure 11: Structural and biophysical analysis of heparin bindinga, 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.

Mentions: Seven sulfate binding sites were identified in hNotumcore crystal form III (Fig. 3c; Extended Data Fig. 6). Among them, one (Sulfate 1) was found by SPR to contribute substantially to Heparin-Notum interactions (Fig. 3d). In addition, co-crystals with short heparin oligomers or sucrose-octasulfate (SOS), a heparin mimic, were generated and analysed. These structural studies and additional biophysical analyses (described in Supplementary information and illustrated in Extended Data Fig. 6) delineated an extensive GAG-binding patch centred on a basic groove between the top of the β-sheet and helix αK (Fig. 3c). Importantly, the GAG-binding surface on Notum is distant from the catalytic triad, consistent with our earlier evidence that Notum binds to glypicans, but does not act on them enzymatically.


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)

Structural and biophysical analysis of heparin bindinga, 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.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 11: Structural and biophysical analysis of heparin bindinga, 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.
Mentions: Seven sulfate binding sites were identified in hNotumcore crystal form III (Fig. 3c; Extended Data Fig. 6). Among them, one (Sulfate 1) was found by SPR to contribute substantially to Heparin-Notum interactions (Fig. 3d). In addition, co-crystals with short heparin oligomers or sucrose-octasulfate (SOS), a heparin mimic, were generated and analysed. These structural studies and additional biophysical analyses (described in Supplementary information and illustrated in Extended Data Fig. 6) delineated an extensive GAG-binding patch centred on a basic groove between the top of the β-sheet and helix αK (Fig. 3c). Importantly, the GAG-binding surface on Notum is distant from the catalytic triad, consistent with our earlier evidence that Notum binds to glypicans, but does not act on them enzymatically.

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