Ghrelin octanoylation mediated by an orphan lipid transferase

Abstract

The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.

Fig. 1.

Generation of octanoylated ghrelin peptides by octanoic acid treatment in TT cells. (A) Ghrelin immunoprecipitation MALDI-TOF MS (IPMS) analyses of TT cell culture media under control conditions. Des-acyl ghrelin 1–28 (m/z 3,244) and 1–27 (m/z 3,088) were observed within 6 days. (B) Exposure of TT cells to octanoic acid (125 μg/ml) induced production of octanoylated ghrelin 1–28 (m/z 3,370) and 1–27 (m/z 3,214) peptides by the cells. Treatment-dependent generation of octanoylated ghrelin peptides is denoted by downward arrows. Ghrelin peptide standards were added at the start of the culture period (m/z 3,187, 3,314, and 3,393 for rat des-acyl ghrelin, rat octanoylated ghrelin, and human SIL octanoylated ghrelin peptides).

Fig. 2.

GOAT is essential for ghrelin octanoylation in TT cells. (A) TT cells were exposed to targeting siRNAs (2 μg) specific for candidate 7 (Cand-7), MBOAT-1, MBOAT-2, MBOAT-3, MBOAT-5, human BB1, SOAT-1, or nontargeting control siRNAs and assayed for ghrelin octanoylation by using the ghrelin IPMS assay. Ghrelin octanoylation levels were normalized to cells treated with nontargeting siRNA control. (B) Dose-dependent decrease in octanoylated ghrelin levels in TT cells treated with candidate 7 gene siRNA 7-3. (C) Dose-dependent effects of candidate 7 gene siRNA 7-3 on normalized GOAT transcripts (GOAT/18s rRNA). (D) Exposure of TT cells to targeting siRNAs (2 μg) to five distinct regions of the candidate 7 transcript decrease the levels of octanoylated ghrelin (1–28) relative to siRNA control treatment.

Fig. 3.

GOAT octanoylates ghrelin peptide in HEK-293 cells. (A) HEK-293 cells transiently transfected with human preproghrelin cDNA secreted des-acyl ghrelin peptides 1–28 (m/z 3,244) and 1–27 (m/z 3,088). (B) Transient cotransfection of HEK-293 cells with human preproghrelin and GOAT cDNAs produced principally octanoylated ghrelin peptides 1–28 (m/z 3,370) and 1–27 (m/z 3,214). Ghrelin peptides standards were rat des-acyl ghrelin (m/z 3,188) and dog octanoyl des Q ghrelin (m/z 3,228). (C) MS fragmentation analyses showing the GOAT-mediated octanoyl modification of serine-3 in ghrelin. Immunoprecipitated ghrelin peptides from cotransfected cells were subjected to MS/MS analyses. (Upper) Fragmentation pattern for +5 ions (m/z 649.56) for des-acyl ghrelin denoting the presence of daughter y″ ions up to the unmodified serine-3 residue. (Lower) Fragmentation pattern for +5 ions (m/z 674.78) for octanoylated ghrelin. Daughter y″ ion fragmentation pattern for octanoylated ghrelin shows a shift for the modified serine-3 residue corresponding to the covalent octanoylation of this residue. Arrows denote mass shift at serine-3 for des-acyl and octanoylated ghrelin. The amino acid sequence for human octanoylated ghrelin is shown within the figure.

Fig. 4.

GOAT gene-null mice lack octanoylated ghrelin in circulation. Blood profiles for acylated and des-acyl ghrelin in either wild-type (Upper) or GOAT gene-disrupted (Lower) mice were determined by using the ghrelin IPMS assay. Arrow denotes location of octanoylated ghrelin. Ghrelin peptide standards were mouse SIL acyl (m/z 3,338) and des-acyl ghrelin (m/z 3,211) peptides, respectively.

Fig. 5.

GOAT and ghrelin transcript profiles in human tissues. Origene's TissueScan Real-Time 48 human tissue panel was used for these studies. Ghrelin and GOAT relative transcript levels were normalized to β-actin transcript amounts and then calibrated to stomach expression in each profile, which was given an arbitrary value of 1. Relative transcript levels for 22 major tissues are shown. (A) GOAT is expressed mainly in stomach and pancreas human tissues. (B) Ghrelin transcripts were abundantly detected in stomach and modestly in pancreas human tissues.