Characterization of Ghrelin O-Acyltransferase (GOAT) in goldfish (Carassius auratus)

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

Optimizing NECEEM-Based Aptamer Selection Using Emulsion PCR To Obtain Potential DNA Inhibitors For The Human Dealkylating Enzyme ABH2

The oldest chemotherapeutic drugs still in use today are the alkylating agents. In many cases, their effectiveness is hindered by the ability of cancerous cells to develop resistance using the cell’s innate DNA repair enzymes, such as hABH2. Targeted inhibition of such enzymes can potentially greatly improve chemotherapy. Aptamers are more efficient, versatile and theoretically easier to discover than conventional small molecule inhibitors. However, Aptamer selection is regularly unsuccessful and screening inhibitors is a lengthily process. Here we optimize the aptamer selection process using Emulsion PCR to improve the amplification efficiency post NECEEM separation and rapidly select high affinity aptamers to unmodified hABH2 in 4 rounds. We then show the efficiency and robustness of the recently introduced direct CE-based approach to rapidly measure the demethylation activity of hABH2 and hABH3 towards ssDNA 3-meC substrates and discuss its potential as method to identify aptamers able to selectively inhibit hABH2

Relative expression of <i>goat</i> during a 24-h light/dark cycle in goldfish fed at midday (right panel) or at midnight (left panel).

<p>(A and B) Telencephalon, (C and D) Hypothalamus, (E and F) Vagal lobe, (G and H) Pituitary, and (I and J) Intestinal bulb. Relative mRNA amounts were quantified by RT-qPCR. Data are expressed as mean ± SEM (n = 6/time point). The grey area indicates the dark phase of the daily photocycle, and the arrow indicates the scheduled feeding time (ZT-6 or ZT-18). Dashed lines represent the periodic sinusoidal functions determined by the cosinor analysis when a significant rhythm was detected. Different letters indicate significant differences by ANOVA and post-hoc SNK test (p<0.05).</p

Phylogenetic analysis of GOAT sequences.

<p>A phylogenetic tree showing the evolutionary relationships of the deduced amino acid sequences of goldfish GOAT with those of other species was inferred by the neighbor-joining method using MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171874#pone.0171874.ref036" target="_blank">36</a>]. The numbers at tree nodes refer to percentage of trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The scale bar indicates the average number of substitutions per position (a relative measure of evolutionary distance). The common name of the species used for the alignment is given on the right side of the tree, among which goldfish is shaded. Species names and GenBank and Ensembl accession numbers of the sequences used are as follows: alligator, <i>Alligator sinensis</i>, XP_006035341.1; Asian arowana, <i>Scleropages formosus</i>, JARO02002481.1(36315–36436….36746–36978….37427–38368); Atlantic herring, <i>Clupea harengus</i>, JZKK01021833.1(58124–58006….52933–52709….50211–49272); Atlantic salmon, <i>Salmo salar</i>, XP_014016526.1; channel catfish, <i>Ictalurus punctatus</i>, XP_017306886.1; chimpanzee, <i>Pan troglodytes</i>, ENSPTRT00000037288; cock, <i>Gallus gallus</i>, NP_001186218.1; coelacanth, <i>Latimeria chalumnae</i>, BK009986; common carp, <i>Cyprinus carpio</i>, (1) LHQP01003245.1(78599–78478…78401–78176…78096–77163) and (2) LHQP01015814.1(64651–64772…64875–65084…65162–65417…67215–67900); damselfish, <i>Stegastes partitus</i>, XP_008292386.1; elephant shark, <i>Callorhinchus milii</i>, BK009985; frog, <i>Xenopus tropicalis</i>, XP_002936505.2; goldfish, <i>Carassius auratus</i>, (V1) APD26025 and (V2) APD26026; horned golden-line barbel, <i>Sinocyclocheilus rhinocerous</i>, (1) XP_016428796.1 and (2) XP_016383356.1; human, <i>Homo sapiens</i>, ACB05873.2; Japanese eel, <i>Anguilla japonica</i>, AVPY01018663.1(5671–5450…5379–5155…4515–3518); lizard, <i>Anolis carolinensis</i>, XP_003224702.1; Mexican cavefish, <i>Astyanax mexicanus</i>, XP_007253942.1; mouse, <i>Mus musculus</i>, ACB05874.1; rabbit, <i>Oryctolagus cuniculus</i>, ENSOCUT00000014851; rainbow trout, <i>Oncorhynchus mykiss</i>, CDQ71181.1; rat, <i>Rattus norvegicus</i>, ACB05875.1; red pirahna, <i>Pygocentrus nattereri</i>, MAUM01004312.1(861–985…1063–1287…2692–3625); spotted gar, <i>Lepisosteus oculatus</i>, BK009987; stickleback, <i>Gasterosteus aculeatus</i>, AANH01001771.1(95917–95796….95445–95215….95100–94149); striped bass, <i>Morone saxatilis</i>, JTCL01001059.1(43688–43809….44568–44792….45768–46719); tilapia, <i>Oreochromis niloticus</i>, AERX01036891.2(7728–7607….6577–6353….4935–3987); wild boar, <i>Sus scrofa</i>, ADI55170.1; yellow croacker, <i>Larimichthys crocea</i>, XP_010729215.1; yellowbelly rockcod, <i>Notothenia coriiceps</i>, AZAD01045919.1(5788–5667….5115–4891….4240–3295); zebra mbuna, <i>Maylandia zebra</i>, XP_014262684.1; zebrafish, <i>Danio rerio</i>, ACB05876.1. Nucleotide sequences were translated to amino acids using Wise2 (<a href="http://www.ebi.ac.uk/Tools/psa/genewise/" target="_blank">http://www.ebi.ac.uk/Tools/psa/genewise/</a>).</p

Predicted topological organization of goldfish GOAT-V1 (A) and GOAT-V2 (B) in the membrane of the endoplasmic reticulum.

<p>Topology was predicted using MemBrain prediction server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171874#pone.0171874.ref042" target="_blank">42</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171874#pone.0171874.ref043" target="_blank">43</a>]. Transmembrane helices are represented by blue rectangles spanning the endoplasmic reticulum membrane (gray). Black lines represent loops, and their size indicates the relative loop length (although they are not precisely scaled). GOAT-V2 is predicted to contain a peptide signal, which is shaded in the N-terminus. Positions of the two conserved catalytic residues asparagine and histidine are shown with red and yellow stars, respectively.</p

Parameters defining the expression rhythms of <i>goat</i> in goldfish central and peripheral tissues.

<p>Parameters defining the expression rhythms of <i>goat</i> in goldfish central and peripheral tissues.</p

Primers designed for cloning and expression analysis of <i>goat</i>.

<p>Primers designed for cloning and expression analysis of <i>goat</i>.</p