Molecular and Functional Characterization of Elovl4 Genes in Sparus aurata and Solea senegalensis Pointing to a Critical Role in Very Long-Chain (>C24) Fatty Acid Synthesis during Early Neural Development of Fish

Abstract

Very long-chain fatty acids (VLC-FA) play critical roles in neural tissues during the early development of vertebrates. However, studies on VLC-FA in fish are scarce. The biosynthesis of VLC-FA is mediated by elongation of very long-chain fatty acid 4 (Elovl4) proteins and, consequently, the complement and activity of these enzymes determines the capacity that a given species has for satisfying its physiological demands, in particular for the correct development of neurophysiological functions. The present study aimed to characterize and localize the expression of elovl4 genes from Sparus aurata and Solea senegalensis, as well as to determine the function of their encoded proteins. The results confirmed that both fish possess two distinct elovl4 genes, named elovl4a and elovl4b. Functional assays demonstrated that both Elovl4 isoforms had the capability to elongate long-chain (C_20-24), both saturated (SFA) and polyunsaturated (PUFA), fatty acid precursors to VLC-FA. In spite of their overlapping activity, Elovl4a was more active in VLC-SFA elongation, while Elovl4b had a preponderant elongation activity towards n-3 PUFA substrates, particularly in S. aurata, being additionally the only isoform that is capable of elongating docosahexaenoic acid (DHA). A preferential expression of elovl4 genes was measured in neural tissues, being elovl4a and elovl4b mRNAs mostly found in brain and eyes, respectively.

Keywords: Elovl4; Gilthead seabream; Senegalese sole; functional characterization; neural tissue development; tissue expression; very long-chain polyunsaturated fatty acid.

Figure 1

Biosynthetic pathways of long-chain (LC-PUFA; C_20–24) and very long-chain polyunsaturated fatty acids (VLC-PUFA; >C₂₄) in fish. Desaturation reactions are mediated by fatty acyl desaturases (Fads), whereas elongation reactions are catalyzed by elongation of very long-chain fatty acid (Elovl) proteins. Microsomal β-oxidation reactions are denoted as “β-ox”. Two pathways for docosahexaenoic acid (DHA; 22:6n-3) biosynthesis from docosapentaenoic acid (DPA; 22:5n-3) are indicated, namely the Sprecher pathway (green background) and the Δ4 pathway (orange background). Elongation reactions leading to VLC-PUFA biosynthesis of up to C₃₆ are indicated with blue arrows. Note the fish species studied herein (Sparus aurata and Solea senegalensis) lack elovl2 in their genomes [2].

Figure 2

Phylogenetic tree comparing Sparus aurata and Solea senegalensis Elovl4a and Elovl4b proteins (highlighted in bold) with Elovl2, Elovl4 and Elovl5 proteins from other vertebrates. The tree was constructed while using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model. The numbers in branches represent the frequencies (%) of each node after 1000 iterations by bootstrapping. The Mortierella alpina PUFA elongase was included in the analysis as an outgroup, to construct the rooted tree.

Figure 3

Tissue distribution of elovl4a and elovl4b transcripts in Sparus aurata (A) and Solea senegalensis (B) determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (n = 1 fish). Expression of housekeeping gene 18s is also shown. Expression in selected tissues of Sparus aurata elovl4a (C) Solea senegalensis elovl4a (D), Sparus aurata elovl4b (E) and Solea senegalensis elovl4b (F) transcripts were also determined by qPCR. The results, shown as relative index, are β-actin normalized values (gene copy number/β-actin copy number). Bars represent means and standard deviations (n = 3 fish). Different letters denote significant differences (ANOVA and Tukey HSD test, p ≤ 0.05) among tissues.