A remarkably specific ligand reveals ghrelin O-acyltransferase interacts with extracellular peptides and exhibits unexpected cellular localization for a secretory pathway enzyme

Ghrelin O- acyltransferase (GOAT) plays a central role in the maturation and activation of the peptide hormone ghrelin, which performs a wide range of endocrinological signaling roles. Using a tight-binding fluorescent ghrelin-derived peptide designed for high selectivity for GOAT over the ghrelin receptor GHS-R1a, we demonstrate that GOAT interacts with extracellular ghrelin and facilitates ligand cell internalization in both transfected cells and prostate cancer cells endogenously expressing GOAT. Coupled with enzyme mutagenesis, ligand uptake studies provide the first direct evidence supporting interaction of the putative histidine general base within GOAT with the ghrelin peptide acylation site. Our work provides a new understanding of GOAT’s catalytic mechanism, establishes a key step required for autocrine/paracrine ghrelin signaling involving local reacylation by GOAT, and raises the possibility that other peptide hormones may exhibit similar complexity in their intercellular and organismal-level signaling pathways. ### Competing Interest Statement JLH has patent interests in ligands reported in this work. * GHS-R1a : growth hormone secretagogue receptor type 1a GOAT : ghrelin O -acyltransferase hGOAT, human ghrelin O -acyltransferase C8 : eight-carbon fatty acid octanoate ER : endoplasmic reticulum Dap : diaminopropionic acid PFPN : pentafluorophenyl naphthoate Aib : aminoisobutyric acid Inp : isonipecotic acid Nal-1 : 1-naphylalanine Nal-2 : 2-naphylalanine AcDan : acrylodan MAFP : methyl arachidonyl fluorophosphonate DMSO : dimethyl sulfoxide HEPES : 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Tris : 2-Amino-2-hydroxymethyl-propane-1,3-diol HPLC : high performance liquid chromatography MALDI : matrix-assisted laser desorption/ionization IC50 : half maximal inhibitory concentration DMF : N , N -dimethylformamide HCTU : O -(6-chlorobenzotriazol-1-yl)- N , N , N ′, N ′-tetramethyluronium hexafluorophosphate DIPEA : N , N -diisopropylethylamine TFA : trifluoroacetic acid TIS : triisopropylsilane TBME : tert -butyl methyl ether DMEM : Dulbecco’s modified eagle’s medium FBS : fetal bovine serum WT : wildtype EV : empty vector PBS : phosphate-buffered saline PBSAT : PBS + 1% bovine serum albumin-0.1% triton SDS : sodium dodecyl sulfate PVDF : polyvinylidene difluoride TBST : tris 10 buffered saline, DAPI, 4,6-diamidino-2-phenylindole HEK : human embryonic kidney fibroblasts.