Notum deacylates octanoylated ghrelin

Abstract

Objectives: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined.

Methods: We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme-substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation.

Results: Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet.

Conclusions: Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.

Keywords: Crystal structure; Deacylation; Ghrelin; Metabolism; Notum.

Figure 1

Mass spectra of AG treated with Notum. (A) The MALDI-TOF mass spectra of AG-biotin, with signal intensity (on the y axis) and m/z on the x axis. (B) Active Notum-treated AG-biotin. (C) Inactive Notum-treated AG-biotin.

Figure 2

Structure of the Notum-ghrelin complex. (A) Cartoon presentation of the Notum-ghrelin complex structure. Notum is shown in cartoon (enzyme core in dark grey and lid domain in light grey); ghrelin serine-3 is shown as brown sticks with the octanoyl lipid coloured green. (B) |Fo–Fc| annealing omit electron density map contoured at 3 σ for the octanoyl lipid and ghrelin serine-3 (OCT/S3). (C) The Notum enzyme pocket is shown (green mesh) with surrounding residues denoted as brown sticks and balls. Octanoyl-linked ghrelin serine-3 is shown as grey sticks. (D) Comparison of the Notum enzyme pocket occupied with octanoyl (OCT, green), myristoleate (MYZ, light blue), and palmitoleic acid (PAM, cyan). The pocket is shown as a grey surface with 50% transparency. The cavity detection radius cut-off was set at 4 solvents. (E) Notum-ghrelin lipid interaction details. The Notum structure is shown as the Cα trace, the interaction residues are denoted as grey sticks (Ser 232 to Ala mutation in cyan), and the hydrogen bonds are represented by dashed yellow lines. (F) Comparison of the Notum residue Y129 side chain conformation in the Apo (cyan) and complex (grey) structures; hydrophobic interactions are shown as dashed lines.

Figure 3

Immunofluorescence, Western blotting, and immunoprecipitation detection of Notum expression in the HA knock-in mice. (A) Liver section from a WT or (B) a homozygous Notum-HA mouse stained with anti-HA, anti-GS (terminal hepatic vein marker), and anti-PECAM-1 (endothelial cell marker). The scale bars represent 100 μm. (C) A low-magnification micrograph showing the central vein (CV) and portal vein (PV). The colour scheme is the same. The scale bar represents 500 μm. (D) Western blotting of mouse liver extracts stained with anti-HA. (E) Immunoprecipitation with HA antibody for mice plasma. WT, wild-type mice; N^HA/+ and N^HA/N^HA indicate heterozygous and homozygous Notum-HA knock-in mice, respectively. Molecular weight markers are shown in kDa. The bottom D and F panels show loading controls of β-actin and transferrin, respectively.

Figure 4

Comparison of mouse serum levels of ghrelin. (A) The ratio of AG over total ghrelin and (B) ratio of DAG over the total in the WT and liver-specific Notum knock-out mice (−/−) fed a SD or HFD. ∗∗∗ indicates statistical significance (P < 0.001) and ns indicates no statistically significant difference (P > 0.05).