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.