Identification of Fatty Acid Desaturase 6 in Golden Pompano Trachinotus Ovatus (Linnaeus 1758) and Its Regulation by the PPARαb Transcription Factor

Abstract

Fatty acid desaturases are rate-limiting enzymes in long-chain polyunsaturated fatty acid biosynthesis. The transcription factor peroxisome proliferator-activated receptor alpha b (PPARαb) regulates lipid metabolism in mammals, however, the mechanism whereby PPARαb regulates fatty acid desaturases is largely unknown in fish. In this study, we report the full length cDNA sequence of Trachinotus ovatus fatty acid desaturase, which encodes a 380 amino acid polypeptide, possessing three characteristic histidine domains. Phylogenetic and gene exon/intron structure analyses showed typical phylogeny: the T. ovatus fatty acid desaturase contained a highly conserved exon/intron architecture. Moreover, functional characterization by heterologous expression in yeast indicated that T. ovatus desaturase was a fatty acid desaturase, with Δ4/Δ5/Δ8 Fad activity. Promoter activity assays indicated that ToFads6 desaturase transcription was positively regulated by PPARαb. Similarly, PPARαb RNA interference decreased ToPPARαb and ToFads6 expression at the mRNA and protein levels in a time-dependent manner. Mutation analyses showed that the M2 binding site of PPARαb was functionally important for protein binding, and transcriptional activity of the ToFads6 promoter was significantly decreased after targeted mutation of M2. Electrophoretic mobile shift assays confirmed that PPARαb interacted with the binding site of the ToFads6 promoter region, to regulate ToFads6 transcription. In summary, PPARαb played a vital role in ToFads6 regulation and may promote the biosynthesis of long-chain polyunsaturated fatty acids by regulating ToFads6 expression.

Keywords: PPARαb and Fads6; Trachinotus ovatus; fatty acid desaturases; heterologous expression; transcriptional activity.

Figure 1

Amino acid sequences of Trachinotus ovatus Fads6 and with other homologs. Sequences comparisons revealed three regions of conserved histidine (his) cluster motifs containing eight his residues sites: HXXXH, HXXXHH, and HXXHH. Eight conserved his residues sites which are putative di-iron ligands are marked in red. Membrane-FADS-like domains are underlined. Accession numbers of Fads6 sequences are listed in Table S3. Identical and similar residues are marked with ‘*’ and ‘:’, respectively.

Figure 2

Genome structure analysis of ToFads6 genes according to the phylogenetic relationship. Exon and intron lengths of each PPARα gene are displayed proportionally. Different color boxes and lines represent exons and introns, respectively. Identical color boxes represent homologous sequences.

Figure 3

Functional characterization of the putative ToFads6 in transgenic yeast. Fatty acid methyl esters (FAMEs) are extracted from yeast transformed with pYES2-Fads6, and grown in the presence of PUFA substrates C18:3n-3 (A), C18:2n-6 (C), C20:2n-6 (E), C20:3n-6 (G), C22:4n-6 (I), and C22:5n-3 (K). All left panels are yeast cells with empty vector. Based on retention times, additional peaks were identified as C18:3n-3 (B), C18:2n-6 (D), C20:3n-6 (F), C20:4n-6 (H), C22:5n-6 (J), and C22:6n-3 (L). Peaks 1–4 represent the main endogenous FAs in yeast, namely C16:0, C16:1 isomers, C18:0, and C18:1n-9, respectively. Moreover, peaks 5–14 represent exogenously supplemented FAs and the corresponding desaturation products, including C18:3n-3 (5), C18:2n-6 (6), C20:2n-6 (7), C20:3n-6 (8), C20:3n-6 (9), C20:4n-6 (10), C22:4n-6 (11), C22:5n-6 (12), C22:5n-3 (13), and C22:6n-3 (14), respectively.

Figure 4

Gene transcriptions of ToFads6 in various tissues. The tissues are small intestine (In), head-kidney (Ki), white muscle (Wm), stomach (St), female gonad (Fg), male gonad (Mg), brain (Br), liver (Li), gill (Gi), spleen (Sp), fin (Fi), blood (Bl), and snout (Sn). Different letters refer to significant differences.

Figure 5

Promoter activity analysis of ToFads6. pGL3-Basic-Fads6s were cotransfected with the transcription factor PPARαb into 293 T cells. All values are presented as the mean ± SD (n = 3). Asterisks indicate different values, with respect to controls (* p < 0.05 and ** p < 0.01). Bars on the same group with different letters are statistically significant from one another.

Figure 6

The nucleotide sequence and predicted binding sites for transcription factors in the core ToFads6 promoter region (A). Effects of transcription factor mutations on ToFads6-5 promoter activity (B). Binding sites are shown with boxes M1–M4. Mutations in promoter sequences are listed in Table 2. All values are presented as the mean ± SD (n = 3). Asterisks indicate values are different from controls (* p < 0.05 and ** p < 0.01). Bars on the same group with different letters are statistically significant from one another.

Figure 7

ToPPARαb upregulates ToFads6 expression. Western blot and real-time PCR analysis were used to detect ToPPARαb expression (A,C) and ToFads6 expression (B,D) after the transfection of either control RNA (control) or siRNA (RNAi), respectively. TOCF cells were stimulated with 0.1, 1, and 4 mM of PPARαb agonist (WY-14643) (E) and inhibition (GW6471) (F) for 24 h. The expression of ToPPARαb and ToFads6 was dramatically increased and decreased in a concentration-dependent manner, respectively. ToPPARαb expressions were described as Zhu et al. [27]. All values were expressed as the mean ± SD (n = 3). Bars on the same group with different letters are statistically significant from one another (p < 0.05).

Figure 8

Binding reactions of ToPPARαb and ToFads6 promoters. Biotin-labeled electrophoretic mobility shift assay (EMSA) probes were incubated with HEK293T lysates containing recombinant PPARαb protein. WT, wild-type probe; MT: mutated probe. 1, Fads6-WT; 2, Fads6-WT+Protein; 3, Fads6-WT+Flag; 4, Fads6-WT (cold 100×) + Protein; 5, Fads6-MT + protein.

Figure 9

The proposed synthesis pathway of PUFA in T. ovatus. Red arrows represent the pathway confirmed in T. ovatus [27], blue arrows indicate the pathway confirmed in other marine fish [8,10,23].