Abstract
Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin’s physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish.
Highlights
Ghrelin O-acyltransferase (GOAT) is a member of the membrane-bound O-acyltransferase (MBOAT) family [1], previously known as MBOAT4 [2,3,4,5]
The present study identifies the full-length sequence of goldfish ghrelin O-acyltransferase, revealing that this teleost has at least two highly homologous cDNAs, named goldfish goat-V1 and goat-V2, encoding this enzyme
These two forms of goat would be generated by the transcription of three exons, the first of which differs from the exon 1 that is commonly transcribed in the rest of vertebrates and which we are referred to as exon 1’
Summary
Ghrelin O-acyltransferase (GOAT) is a member of the membrane-bound O-acyltransferase (MBOAT) family [1], previously known as MBOAT4 [2,3,4,5] It is responsible for the attachment of a medium-chain fatty acid, typically octanoate, to the third aminoacid (a serine residue) of the N-terminal region of ghrelin, a peptide hormone mainly synthesized by the gut [6]. This posttranslational acyl modification of ghrelin is essential for the peptide to bind to its PLOS ONE | DOI:10.1371/journal.pone.0171874. Saskatchewan Health Research Foundation (SHRF) and John R. Evans Leaders Fund from the Canada Foundation for Innovation to SU. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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