Inhibition of ghrelin O-acyltransferase (GOAT) by octanoylated pentapeptides

Abstract

The discovery of ghrelin O-acyltransferase (GOAT) opens the way to the design of drugs that block the attachment of an octanoyl group to the appetite-stimulating peptide hormone ghrelin, potentially preventing obesity. Here, we develop a biochemical assay that uses membranes from insect cells infected with baculovirus encoding mouse GOAT. The GOAT-containing membranes transferred the [(3)H]octanoyl group from [(3)H]octanoyl CoA to recombinant proghrelin in vitro. Transfer depended on the serine at residue 3 of proghrelin, which is the known site of acylation. GOAT also transferred [(3)H]octanoyl to a pentapeptide containing only the N-terminal five amino acids of proghrelin. GOAT activity could be inhibited by an octanoylated ghrelin pentapeptide, and its potency was enhanced 45-fold when the octanoylated serine-3 was replaced by octanoylated diaminopropionic acid. The data suggest that GOAT is subjected to end-product inhibition and this inhibition is better achieved with substrates having the octanoyl group attached through an amide linkage rather than the corresponding ester. These insights may facilitate the future design of useful inhibitors of GOAT.

Fig. 1.

Establishment of in vitro octanoylation assay. (A) GOAT activity in membranes from Sf9 insect cells. On day 0, Sf9 cells were set up for experiments and infected on day 1 with baculovirus encoding wild-type GOAT or the H338A-mutant GOAT, both containing a N-terminal His₁₀ tag. After infection for 48 h, the 100,000 × g membrane fraction from the uninfected cells (control) and the infected cells were prepared as described in Experimental Procedures. Each 50-μl reaction mixture contained 50 μg of membrane protein from the indicated cells, 5 μg of proghrelin-His₈, 50 μg of myristyl ether CoA, and 1 μM [³H]octanoyl CoA (11 dpm/fmol). After incubation for 5 min at 37°C, the amount of [³H]octanoyl transferred to proghrelin-His₈ was quantified by nickel chromatography and scintillation counting as described in Experimental Procedures. Each value represents the average of triplicate assays. (B) Immunoblot analysis of GOAT expressed in Sf9 cells. Aliquots of the membranes used in A (75 μg of protein) were subjected to immunoblot analysis with 1 μg/ml monoclonal anti-His antibody. The asterisk denotes an irrelevant cross-reacting band present in the membranes of Sf9 cells. Film was exposed for 10 s. (C) Autoradiography of [³H]octanoyl proghrelin-His₈ formed in the in vitro assay. The reaction products from replicate assays in A were precipitated with 80% acetone, loaded onto 16% Tricine SDS/PAGE, transferred to a PVDF membrane, and subjected to autoradiography for 5 days as described in SI Experimental Procedures.

Fig. 2.

Action of long-chain acyl-CoAs in stimulating GOAT activity (A and B) by inhibiting deacylation of [³H]octanoyl CoA (C and D). Membranes from Sf9 cells infected with baculovirus encoding His₁₀-GOAT were prepared as in Fig. 1A. Each 50-μl reaction mixture contained 50 μg of membrane protein, 5 μg of proghrelin-His₈, 1 μM [³H]octanoyl CoA (11 dpm/fmol), and the indicated compound. (A and B) After incubation for 5 min at 37°C, the amount of [³H]octanoyl transferred to proghrelin-His₈ was quantified by nickel chromatography and scintillation counting. Each value represents the average of duplicate assays. (C and D) After incubation at 37°C for the indicated time (C) or 5 min (D), the amount of [³H]octanoate formed in the reaction was quantified by TLC analysis as described in SI Experimental Procedures.

Fig. 3.

Characterization of GOAT activity in vitro. Membranes from Sf9 cells infected with baculovirus encoding His₁₀-GOAT were prepared as in Fig. 1A. Unless otherwise indicated, each 50-μl reaction mixture contained 50 μg of membrane protein, 5 μg (424,000 fmol) of proghrelin-His₈, 50 μM palmitoyl CoA, and 1 μM [³H]octanoyl CoA (50,000 fmol, 11 dpm/fmol). After incubation for 5 min at 37°C, the amount of [³H]octanoyl attached to proghrelin-His₈ was quantified by nickel chromatography. (A) Time course. (B) Concentration curve of membrane protein. (C) Concentration curve of [³H]octanoyl CoA. (D) Concentration curve of proghrelin-His₈. Each value represents the average of duplicate assays.

Fig. 4.

Identification of amino acids in proghrelin required for octanoylation. (A) Amino acid sequence of the ghrelin portion of mouse proghrelin. The asterisk denotes the octanoylated serine-3 residue. (B) Octanoylation of mutant proghrelins in vitro. Membranes from Sf9 cells infected with baculovirus encoding mouse His₁₀-GOAT were prepared as described in Fig. 1A. Each 50-μl reaction mixture contained 50 μg of membrane protein, 50 μM palmitoyl CoA, 1 μM [³H]octanoyl CoA (11 dpm/fmol), and 5 μg of wild-type proghrelin-His₈ or the indicated mutant version. Each value represents the average of duplicate assays. (B Inset) SDS/PAGE of purified wild-type and single amino acid mutants of proghrelin-His₈. Each recombinant protein (5 μg) was subjected to 16% Tricine SDS/PAGE and visualized by Coomassie blue staining. (C) Octanoylation of mutant ghrelins in INS-1 cells. On day 0, 100-mm dishes of INS-1 cells were set up for experiments as described in Experimental Procedures. On day 2, the cells were cotransfected with 5 μg of pCMV-preproghrelin or its indicated mutant version and 0.1 μg of pCMV-GOAT. On day 4, each dish received a direct addition of 50 μM octanoate-albumin (final concentration). On day 5, three dishes of cells from each transfection were harvested and pooled, after which peptides were extracted from the cells, fractionated on reverse-phase chromatography, and subjected to immunoblot analysis with 1:1,000 dilution of rabbit anti-ghrelin antibody as described in Experimental Procedures. Film was exposed for 1 min.

Fig. 5.

Free N-terminal glycine residue of proghrelin is required for optimal GOAT activity. Membranes from Sf9 cells infected with baculovirus encoding His-10-GOAT were prepared as in Fig. 1A. Each 50-μl reaction mixture contained 50 μg of membrane protein, 5 μg of the indicated wild-type or mutant proghrelin-His₈, 50 μM palmitoyl CoA, and 1 μM [³H]octanoyl CoA (11 dpm/fmol). After incubation at 37°C for the indicated time (A) or for 5 min (B), the amount of [³H]octanoyl attached to proghrelin-His₈ was quantified by nickel chromatography. Each value represents the average of duplicate assays. Mutant residues in proghrelin are shaded. (A Inset) SDS/PAGE of purified recombinant wild-type or mutant versions of proghrelin-His-8. Each recombinant protein (5 μg) was subjected to 16% Tricine SDS/PAGE and visualized by Coomassie blue staining.

Fig. 6.

Ability of desacyl-ghrelin peptides to compete for octanoylation of proghrelin. Membranes from Sf9 cells infected with baculovirus encoding mouse His-10-GOAT were prepared as described in Fig. 1A. Each 50-μl reaction mixture contained 50 μg of membrane protein, 5 μg of proghrelin-His₈, 50 μM palmitoyl CoA, 1 μM [³H]octanoyl CoA (11 dpm/fmol), and the indicated concentration of peptide. Each value represents the average of duplicate assays. The “100% of control values,” which represent the amount of [³H]octanoyl proghrelin formed in the absence of peptide, were 278 (A), 247 (B), and 244 (C) fmol per tube. (A Inset) Autoradiography of [³H]octanoyl ghrelin (1–28) formed in the in vitro assay. The reaction contained 50 μg of membrane protein from either uninfected Sf9 cells (control) or cells infected with baculovirus encoding GOAT as indicated, 50 μM palmitoyl CoA, 1 μM [³H]octanoyl CoA (11 dpm/fmol), and 20 μM desacyl-ghrelin (1–28) as indicated. After incubation for 5 min at 37°C, the samples were processed for autoradiography as in Fig. 1C, except that the film was exposed for 7 days.

Fig. 7.

Inhibition of GOAT activity by octanoyl-ghrelin (1–5)-NH₂ peptide. Membranes from Sf9 cells infected with baculovirus encoding mouse His₁₀-GOAT were prepared as described in Fig. 1A. Each 50-μl reaction mixture contained 50 μg of membrane protein, 5 μg of proghrelin-His₈, 50 μM palmitoyl CoA, 1 μM [³H]octanoyl CoA (11 dpm/fmol), and the indicated concentration of peptide in a final concentration of 3% (vol/vol) DMSO. Each value represents the average of duplicate assays. The 100% of control values, which represent the amount of [³H]octanoyl proghrelin formed in the absence of peptide, were 205 fmol per tube (A) and 214 fmol per tube (B).

Fig. 8.

Inhibition of GOAT activity by [Dap³]octanoyl-ghrelin (1–28) (A) and [Dap³]octanoyl-ghrelin (1–5)-NH₂ (B). Assays were carried out as described in the legend to Fig. 7 except for the presence of different ghrelin peptides as indicated. [Dap³] denotes the substitution of a (S)-2,3-diaminopropionic acid residue for serine-3 in ghrelin (1–28) and ghrelin (1–5)-NH₂. The insets show an expanded scale for the concentration curve of [Dap³]octanoyl-ghrelin (1–28) (A) and [Dap³]octanoyl-ghrelin (1–5)-NH₂ (B).