Table 1.
Primers designed for cloning and expression analysis of goat.
Fig 1.
Nucleotide and deduced amino acid sequences of goldfish GOAT.
The full-length sequence corresponds to GOAT-V1, and the same sequence without the shaded nucleotides and amino acids represents GOAT-V2. Both sequences are accessible through GenBank (accession numbers KX953158 and KX953159). Encoding region of sequences extends from the first methionine residue to the stop codon (asterisk, *). The red star indicates the union point between the first exon (exon 1’) and exon 2. The sequence between these two exons comprises two introns (1’ and 1) separated by exon 1. The green star indicates the intron between the exons 2 and 3. The 5’ and 3’ extremities of these intronic sequences are indicated in lowercase letters, and their size is indicated between brackets. The proposed catalytic residues of GOAT (asparagine 273 and histidine 304) are boxed.
Fig 2.
Exon:intron structure and proposed transcription pattern of the goat gene in goldfish.
(A) Schematic illustration representing the proposed model for the exon-intron structure of the gene encoding GOAT in goldfish and its pattern of transcription into mRNA. Exons are indicated by boxes and introns by lines. The length (pb) of exons and introns is indicated inside the boxes (exons) or above lines (introns). (B—C) Alignment of the nucleotide sequences of the intron 1 (between exons 1 and 2, B) and intron 2 (between exons 2 and 3, C) of GOAT-V1 and GOAT-V2 DNAs with the corresponding intron sequences inferred from the common carp genome. Multiple sequence alignments were conducted using Clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and edited using the BioEdit Sequence Alignment Editor. Identical amino acids between the two sequences are colored. The common name of the species used for the alignment is given on the right side, and the species name and GenBank accession number of the common carp is as follows: Cyprinus carpio, LHQP01003245.1(80257–80106….78407–78390).
Fig 3.
Alignment of the deduced amino acid sequences from goldfish GOAT-V1 and GOAT-V2 with GOAT from common carp, zebrafish, mouse and human.
Multiple sequence alignment was conducted using Clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and edited using the BioEdit Sequence Alignment Editor. Dashed lines represent voids introduced to optimize the alignment. Identical amino acids among sequences are colored, and the proposed catalytic residues of GOAT (asparagine and histidine) are marked by asterisks. The common name of the species used for the alignment is given on the right side, and the species names and GenBank accession numbers are as follows: common carp, Cyprinus carpio, LHQP01003245.1(80257–80106….78407–78390); human, Homo sapiens, ACB05873.2; mouse, Mus musculus, ACB05874.1; zebrafish, Danio rerio, CABZ01049031.1(6992–7150…18211–18228).
Fig 4.
Phylogenetic analysis of GOAT sequences.
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 [36]. 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, Alligator sinensis, XP_006035341.1; Asian arowana, Scleropages formosus, JARO02002481.1(36315–36436….36746–36978….37427–38368); Atlantic herring, Clupea harengus, JZKK01021833.1(58124–58006….52933–52709….50211–49272); Atlantic salmon, Salmo salar, XP_014016526.1; channel catfish, Ictalurus punctatus, XP_017306886.1; chimpanzee, Pan troglodytes, ENSPTRT00000037288; cock, Gallus gallus, NP_001186218.1; coelacanth, Latimeria chalumnae, BK009986; common carp, Cyprinus carpio, (1) LHQP01003245.1(78599–78478…78401–78176…78096–77163) and (2) LHQP01015814.1(64651–64772…64875–65084…65162–65417…67215–67900); damselfish, Stegastes partitus, XP_008292386.1; elephant shark, Callorhinchus milii, BK009985; frog, Xenopus tropicalis, XP_002936505.2; goldfish, Carassius auratus, (V1) APD26025 and (V2) APD26026; horned golden-line barbel, Sinocyclocheilus rhinocerous, (1) XP_016428796.1 and (2) XP_016383356.1; human, Homo sapiens, ACB05873.2; Japanese eel, Anguilla japonica, AVPY01018663.1(5671–5450…5379–5155…4515–3518); lizard, Anolis carolinensis, XP_003224702.1; Mexican cavefish, Astyanax mexicanus, XP_007253942.1; mouse, Mus musculus, ACB05874.1; rabbit, Oryctolagus cuniculus, ENSOCUT00000014851; rainbow trout, Oncorhynchus mykiss, CDQ71181.1; rat, Rattus norvegicus, ACB05875.1; red pirahna, Pygocentrus nattereri, MAUM01004312.1(861–985…1063–1287…2692–3625); spotted gar, Lepisosteus oculatus, BK009987; stickleback, Gasterosteus aculeatus, AANH01001771.1(95917–95796….95445–95215….95100–94149); striped bass, Morone saxatilis, JTCL01001059.1(43688–43809….44568–44792….45768–46719); tilapia, Oreochromis niloticus, AERX01036891.2(7728–7607….6577–6353….4935–3987); wild boar, Sus scrofa, ADI55170.1; yellow croacker, Larimichthys crocea, XP_010729215.1; yellowbelly rockcod, Notothenia coriiceps, AZAD01045919.1(5788–5667….5115–4891….4240–3295); zebra mbuna, Maylandia zebra, XP_014262684.1; zebrafish, Danio rerio, ACB05876.1. Nucleotide sequences were translated to amino acids using Wise2 (http://www.ebi.ac.uk/Tools/psa/genewise/).
Fig 5.
Predicted topological organization of goldfish GOAT-V1 (A) and GOAT-V2 (B) in the membrane of the endoplasmic reticulum.
Topology was predicted using MemBrain prediction server [42,43]. 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.
Fig 6.
Tissue distribution of goat-V1 (A) and goat-V2 (B) mRNAs in goldfish.
Quantitative analysis of mRNA expression was performed by RT-qPCR considering elongation factor-1α (ef-1α) as reference gene. Data are expressed as mean + SEM (n = 4), relative to the tissue with the lowest mRNA expression. Bars not showing any coincident letter indicate that their difference in mean is statistically significant, as assessed by ANOVA and post-hoc SNK test (p<0.05).
Fig 7.
GOAT-like and ghrelin-like immunoreactivity in goldfish intestine detected by immunohistochemistry.
Figure shows transversal representative sections of intestine showing GOAT-like (A1 and A2, red) and ghrelin-like (B, green) immunoreactivity, and merged images of GOAT and ghrelin (C, yellow). Slides labeled only with secondary antibodies were used as negative controls (D). Arrowheads indicate cells stained with either GOAT or ghrelin, and solid arrows show cells that colocalize both GOAT and ghrelin. All images are merged with DAPI showing nuclei in blue. M, mucosa; SM, submucosa. Scale bars are indicated in each image.
Fig 8.
Effects of exposure of intestinal fragments to acylated ghrelin on GOAT mRNA and protein levels.
(A) Concentration-response curves for goat mRNA expression in goldfish cultured intestine treated with goldfish acylated ghrelin (0.1, 1 and 10 nM) during 30, 60 and 120 min. Data are shown as mean + SEM (n = 6). Asterisks denote statistical differences between control and treated groups assessed by ANOVA and post-hoc SNK test (** p<0.01, *** p<0.001). (B) Concentration-response curves for GOAT protein levels in goldfish cultured intestine treated with goldfish acylated ghrelin (0.1, 1 and 10 nM) during 30 min. Bands density was quantified from three blots and normalized to the density of vinculin protein. Resulting data is plotted as mean + SEM in the upper part of the figure. Asterisks denote statistical differences between control and treated groups assessed by ANOVA and post-hoc SNK test (* p<0.05, ** p<0.01). The lower part show representative blots from one goldfish.
Fig 9.
Relative expression of goat during a 24-h light/dark cycle in goldfish fed at midday (right panel) or at midnight (left panel).
(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).
Table 2.
Parameters defining the expression rhythms of goat in goldfish central and peripheral tissues.