Fig 2: Endogenous hGOAT expression in prostate cancer cells supports GOAT ligand binding and uptake. (a) 22Rv1 cells were incubated with Cy5-ghrelin ligand 15 (purple) for 30 min and immunostained with anti-MBOAT4 (cyan) for GOAT expression detection. (b) LNCaP cells were incubated with Cy5-ghrelin ligand 15 (purple) for 30 min and immunostained with anti-MBOAT4 (cyan) for GOAT expression detection. (c) Quantification of cells showing uptake of ligand 15 (n = 50 cells, over n = 3 independent experiments) for HEK 293, HEK 293 transfected with hGOAT, 22Rv1, and LNCaP cells. (d) Dependence of ligand 15 uptake by 22Rv1 cells on ligand concentration in the cell media, as reflected by total cell fluorescence. One-way ANOVA comparison tests between all ligand concentrations indicated significance with adjusted P values of <0.0001 (****). (e) Temperature dependence of ligand 15 uptake in 22Rv1 cells. One-way ANOVA comparison tests between 37°, 21°, and 4° indicated significance at both lower temperatures with adjusted P values of <0.001 (***37 vs 21) and <0.0001 (****37 vs 4). (f) Incubation of 22Rv1 cells and ligand 15 in the presence of unlabeled competitor ligand 12 reduces ligand 15 uptake. (g) Quantification of ligand 15 uptake in 22Rv1 cells in the absence and presence of a competitor ligand, as indicated by total cell fluorescence. The reduction in the presence of the competitor ligand was determined to be significant with p < 0.05 (*) by an unpaired t test. Dotted lines denote the average values for each data group. Scale bars, 10 μm.

Fig 3: GOAT expression in prostate cancer cells detected by immunofluorescence. (a) GOAT imaged in LNCaP PCa cells by anti-MBOAT4 immunofluorescence. (b) GOAT imaged in 22Rv1 PCa cells by anti-MBOAT4 immunofluorescence. Cells were prepared for imaging as described in the Methods. Scale bars, 10 μm.

Fig 4: An expanded view of GOAT cellular localization and signaling roles. In ghrelin-producing cells, GOAT localized in the ER membrane catalyzes proghrelin octanoylation prior to final proteolytic processing and secretion. In cells presenting GOAT on their plasma membrane, GOAT could catalyze reacylation of desacyl ghrelin which can then activate GHSR signaling. GOAT could also facilitate cellular uptake of peptide ligands through either enzyme–ligand complex internalization or ligand transport through the GOAT internal channel. Steps in these pathways proposed but not proven are denoted by dashed arrows. Figure created using BioRender.

Fig 5: Interaction between the Dap amino group and H338 is essential for ligand uptake by GOAT-expressing cells. (a) HEK 293 cells were transfected with empty vector or FLAG-tagged hGOAT/mutant hGOAT as indicated. At 40 h post-transfection, cells were incubated with Cy5-ghrelin ligand 15 (purple) for 30 min and immunostained with anti-FLAG (cyan) for GOAT expression detection. Wild type, N307A, and R304A hGOAT expressing cells showed binding and internalization of ligand 15. Empty vector and H338A hGOAT variants do not support peptide uptake. Scale bar, 10 μm. (b) Proposed model for the interaction between the ghrelin serine 3 side chain hydroxyl group and the ligand 15 Dap side chain amine with H338 leading to general base catalysis and tighter ligand binding, respectively. (c) Quantification of percent hGOAT-positive cells showing the uptake of ligand 15 (n = 100 cells, over n = 3 experiments) for wild type, R304A, N307A, and H338A hGOAT variants. The locations of R304 (olive green), N307 (forest green), and H338 (orange) are shown within the hGOAT structure. Dotted lines denote the average percent of each group. One-way ANOVA comparison test between wild type and hGOAT variants indicated significance with adjusted P values of <0.01 (**N307A), <0.001 (***R304A), and <0.0001 (****H338A).