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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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Enzymatic activity of hNotuma, Activity of hNotumcore and its S232A variant on p-nitrophenyl (pNP) acetate (pNP2) and activity of hNotumcore on other chromogenic substrates. pNPS = pNP-sulfate (sulfatase substrate); pNPP=pNP-phosphate (phosphatase substrate); pNPPC=pNP-phosphorylcholine (phospholipase C substrate); pNAA=p-Nitroacetanilide (amidase/protease substrate). b, mWnt3A inactivation by hNotum. After the indicated time, hNotumcore or its S232A variant was removed with cobalt affinity beads and residual Wnt3A activity measured with TOPFlash. PC = no hNotum removal. Results are normalised to those from identically treated mock samples. c, Activity of hNotum and hAPT1 on chromogenic p-nitrophenyl ester substrates of different lengths. d, Inhibition of hNotum by various carboxylic acids. pNP8 was used as substrate at a concentration of 1 mM as were the carboxylic acids. c, t: cis or trans C9-C10 double bond. All graphs show the mean +/− s.d. (n=4).
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Figure 4: Enzymatic activity of hNotuma, Activity of hNotumcore and its S232A variant on p-nitrophenyl (pNP) acetate (pNP2) and activity of hNotumcore on other chromogenic substrates. pNPS = pNP-sulfate (sulfatase substrate); pNPP=pNP-phosphate (phosphatase substrate); pNPPC=pNP-phosphorylcholine (phospholipase C substrate); pNAA=p-Nitroacetanilide (amidase/protease substrate). b, mWnt3A inactivation by hNotum. After the indicated time, hNotumcore or its S232A variant was removed with cobalt affinity beads and residual Wnt3A activity measured with TOPFlash. PC = no hNotum removal. Results are normalised to those from identically treated mock samples. c, Activity of hNotum and hAPT1 on chromogenic p-nitrophenyl ester substrates of different lengths. d, Inhibition of hNotum by various carboxylic acids. pNP8 was used as substrate at a concentration of 1 mM as were the carboxylic acids. c, t: cis or trans C9-C10 double bond. All graphs show the mean +/− s.d. (n=4).

Mentions: The α/β hydrolase superfamily includes proteases, lipases, esterases, dehalogenases, peroxidases and epoxide hydrolases34. To identify which of these activities relate most closely to Notum’s, we compared the structure of hNotum to those of all known α/β hydrolases (PDBe Fold server35). The search returned many weak homologs, including human esterase D36 and acyl-protein thioesterase 1 (APT1)37 (Extended Data Fig. 7a). A structure-based search for function using the ProFunc Server38 also suggested that Notum is a carboxylesterase. Furthermore, the closest non-animal homologs of Notum, the pectin acetyl esterases (PAEs) of angiosperms (sequence identity to hNotum 22%, Extended Data Fig. 7b) are carboxylesterases. We assessed the functional significance of these observations by measuring the activity of hNotumcore on p-nitrophenyl (pNP) acetate (pNP2), a chromogenic carboxylesterase substrate39. Significant activity could be detected (Fig. 4a). This activity was strongly inhibited by Triton X-100, and by phenylmethanesulfonyl fluoride (PMSF), a compound known to covalently modify the catalytic serine of serine esterases and proteases (Extended Data Fig. 8a, b). In contrast, there was no measurable hNotum activity on representative sulfatase, phosphatase, phospholipase C or amidase/protease substrates (Fig. 4a). Addition of SOS or heparin resulted in a modest increase in Notum carboxylesterase activity (Extended Data Fig 8a). The possibility that GAGs also contribute to Notum function by allosteric activation requires further investigation.


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)

Enzymatic activity of hNotuma, Activity of hNotumcore and its S232A variant on p-nitrophenyl (pNP) acetate (pNP2) and activity of hNotumcore on other chromogenic substrates. pNPS = pNP-sulfate (sulfatase substrate); pNPP=pNP-phosphate (phosphatase substrate); pNPPC=pNP-phosphorylcholine (phospholipase C substrate); pNAA=p-Nitroacetanilide (amidase/protease substrate). b, mWnt3A inactivation by hNotum. After the indicated time, hNotumcore or its S232A variant was removed with cobalt affinity beads and residual Wnt3A activity measured with TOPFlash. PC = no hNotum removal. Results are normalised to those from identically treated mock samples. c, Activity of hNotum and hAPT1 on chromogenic p-nitrophenyl ester substrates of different lengths. d, Inhibition of hNotum by various carboxylic acids. pNP8 was used as substrate at a concentration of 1 mM as were the carboxylic acids. c, t: cis or trans C9-C10 double bond. All graphs show the mean +/− s.d. (n=4).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4376489&req=5

Figure 4: Enzymatic activity of hNotuma, Activity of hNotumcore and its S232A variant on p-nitrophenyl (pNP) acetate (pNP2) and activity of hNotumcore on other chromogenic substrates. pNPS = pNP-sulfate (sulfatase substrate); pNPP=pNP-phosphate (phosphatase substrate); pNPPC=pNP-phosphorylcholine (phospholipase C substrate); pNAA=p-Nitroacetanilide (amidase/protease substrate). b, mWnt3A inactivation by hNotum. After the indicated time, hNotumcore or its S232A variant was removed with cobalt affinity beads and residual Wnt3A activity measured with TOPFlash. PC = no hNotum removal. Results are normalised to those from identically treated mock samples. c, Activity of hNotum and hAPT1 on chromogenic p-nitrophenyl ester substrates of different lengths. d, Inhibition of hNotum by various carboxylic acids. pNP8 was used as substrate at a concentration of 1 mM as were the carboxylic acids. c, t: cis or trans C9-C10 double bond. All graphs show the mean +/− s.d. (n=4).
Mentions: The α/β hydrolase superfamily includes proteases, lipases, esterases, dehalogenases, peroxidases and epoxide hydrolases34. To identify which of these activities relate most closely to Notum’s, we compared the structure of hNotum to those of all known α/β hydrolases (PDBe Fold server35). The search returned many weak homologs, including human esterase D36 and acyl-protein thioesterase 1 (APT1)37 (Extended Data Fig. 7a). A structure-based search for function using the ProFunc Server38 also suggested that Notum is a carboxylesterase. Furthermore, the closest non-animal homologs of Notum, the pectin acetyl esterases (PAEs) of angiosperms (sequence identity to hNotum 22%, Extended Data Fig. 7b) are carboxylesterases. We assessed the functional significance of these observations by measuring the activity of hNotumcore on p-nitrophenyl (pNP) acetate (pNP2), a chromogenic carboxylesterase substrate39. Significant activity could be detected (Fig. 4a). This activity was strongly inhibited by Triton X-100, and by phenylmethanesulfonyl fluoride (PMSF), a compound known to covalently modify the catalytic serine of serine esterases and proteases (Extended Data Fig. 8a, b). In contrast, there was no measurable hNotum activity on representative sulfatase, phosphatase, phospholipase C or amidase/protease substrates (Fig. 4a). Addition of SOS or heparin resulted in a modest increase in Notum carboxylesterase activity (Extended Data Fig 8a). The possibility that GAGs also contribute to Notum function by allosteric activation requires further investigation.

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