Tandem triplication of the fabp1 gene
Fatty acid binding proteins (Fabps) are important members of the intracellular lipid binding protein family (iLBP; Bass, 1988). Fabps can reversibly bind intracellular hydrophobic ligands (including the long-chain fatty acids, eicosanoids, bile salts and peroxisome proliferators) and participate in their transfer processes (Storch & Thumser, 2000). They are distributed in many tissues, such as intestine, liver, and heart muscle. From the gene family analysis, we observed a tandem triplication event of thefabp1 gene in the striped catfish genome. Three copies were located on the chromosome 10 (Fig. 4), and thereby named asfabp1-1 , -2 and-3 . The mRNA transcriptions of fabp1-1 and -3 were detectable at a high level by RNA-Seq (Table 3). The deduced protein sequences from fabp1-2 and -3 genes are much similar, but there are many mutation variances in the Fabp1-1 (Fig. 4).
In order to examine the phylogenetic relationships of these fabp1genes in various teleost species, we constructed a phylogenetic tree using PhyML (the left section in Fig. 5). A synteny region of 9 Siluriformes species for the fabp1 gene was identified, in which we observed the remarkable expansion with 3 copies of fabp1 in the stripped catfish (top in the right section of Fig. 5). Compared with other Siluriformes species, mutations were identified in the Fabp1 isotypes 2 and 3 of striped catfish, such as M117T, F124L, and R126T (Fig. 4).
Potential functional effects of these amino acid substitutions were evaluated by using PolyPhen-2 (Adzhubei et al., 2013) and PROVEAN (Choi & Chan, 2015; Table 4). Interestingly, the R126T in Fabp1-2 and -3 was predicted as “damaging” by both HumDiv and HumVar methods in PolyPhen-2 and “deleterious” by PROVEAN, suggesting that this mutation may affect both proteins’ functions. Based on homology, the Arg126 of Fabp1 in striped catfish corresponds to the Arg122 of human Fabp1, which has been proved to act as two fatty acid binding sites in relevant studies (Thompson et al., 1997). We therefore propose that the R126T mutation may potentially affect the fatty acid binding capacity of the Fabp1 isotypes 2 and 3.
Peroxisome proliferator activated receptors (PPARs), classified into three subtypes (α, β or δ, and γ), are nuclear receptors with functional regulation of reproduction, development, and metabolism (Braissant et al., 1996). They are proved to participate in the regulation process of several genes including fabp1 by heterodimerizing with the retinoid X receptor (RXR) and binding to a PPAR response element (PPRE) in the promoters (Hsu et al., 1998). The consensus sequence for the vertebrate PPREs is defined as 5′-CAAAACAGGTCANAGGTCA-3′. Based on various isotypes of PPAR, including PPARα and PPARγ, it seems that PPRE binds to different PPARs (Juge-Aubry et al., 1997). A PPRE with high sequence identity in the 5′ flanking region (5′FR; 5′-CAAAAC-3′) shows a higher binding activity with PPARα compared to PPARγ. On the contrary, a PPRE with low sequence identity in the 5′FR and high sequence identity in the direct repeat element (DR1; 5′-AGGTCANAGGTCA-3′) may exhibit PPARγ-selective (Juge-Aubry et al., 1997).
To find potential PPREs in the fabp1 promoter of striped catfish, we first extracted sequences of 3,000 bp from 5′ upstream of the transcription start sites (TSS) for the 3 isotypes. JASPAR 2020 (Fornes et al., 2020) was then applied for the prediction using two PPREs matrixes (PPARα-selective, Matrix ID: MA1148.1; PPARγ-selective, Matrix ID: MA0065.1), with the relative profile score threshold of 70%. After extraction of putative PPREs located in the sense strand, they were aligned to the consensus sequence of vertebrate PPRE using BLASTn (Altschul et al., 1990). Subsequently, PPREs with 7 or more consecutive nucleotide matches were selected, and then mapped to the promoter sequences (Fig. 6). For the 5 predicted PPREs, those regions highly homologous to the DR1 of the consensus PPRE were identified, but no region with high sequence identity to the 5′FR of the consensus PPRE was detected. It seems that all these putative PPREs within the fabp1 promoters are PPARγ-selective in striped catfish.