Elongation of very Long-Chain (>C24) Fatty Acids in Clarias gariepinus: Cloning, Functional Characterization and Tissue Expression of elovl4 Elongases

Abstract

Elongation of very long-chain fatty acid 4 (Elovl4) proteins participate in the biosynthesis of very long-chain (>C₂₄) saturated and polyunsaturated fatty acids (FA). Previous studies have shown that fish possess two different forms of Elovl4, termed Elovl4a and Elovl4b. The present study aimed to characterize both molecularly and functionally two elovl4 cDNA from the African catfish Clarias gariepinus. The results confirmed that C. gariepinus possessed two elovl4-like elongases with high homology to two previously characterized Elovl4 from Danio rerio, and thus they were termed accordingly as Elovl4a and Elovl4b. The C. gariepinus Elovl4a and Elovl4b have open reading frames (ORF) of 945 and 915 base pairs, respectively, encoding putative proteins of 314 and 304 amino acids, respectively. Functional characterization in yeast showed both Elovl4 enzymes have activity towards all the PUFA substrates assayed (18:4n-3, 18:3n-6, 20:5n-3, 20:4n-6, 22:5n-3, 22:4n-6 and 22:6n-3), producing elongated products of up to C₃₆. Moreover, the C. gariepinus Elovl4a and Elovl4b were able to elongate very long-chain saturated FA (VLC-SFA) as denoted by increased levels of 28:0 and longer FA in yeast transformed with elovl4 ORF compared to control yeast. These results confirmed that C. gariepinus Elovl4 play important roles in the biosynthesis of very long-chain FA. Tissue distribution analysis of elovl4 mRNAs showed both genes were widely expressed in all tissues analyzed, with high expression of elovl4a in pituitary and brain, whereas female gonad and pituitary had the highest expression levels for elovl4b.

Keywords: Biosynthesis; Clarias gariepinus; Essential fatty acids; Very long-chain fatty acids; elovl4.

Fig. 1

Phylogenetic tree comparing the deduced amino acid sequences of Clarias gariepinus Elovl4a and Elovl4b (highlighted in bold) with Elovl4, Elovl2 and Elovl5 sequences from a range of vertebrates. The tree was constructed using the neighbor-joining method [27] with the MEGA 6.0 software. The numbers represent the frequencies (%) with which the tree topology presented was replicated after 1000 iterations. The Mortierella alpina PUFA elongase was included in the analysis as outgroup sequence to construct the rooted tree

Fig. 2

ClustalW amino acid alignment of the deduced Clarias gariepinus Elovl4 proteins with orthologues from Danio rerio (Elovl4a, gb|NP_957090.1|; Elovl4b, gb |NP_956266.1|), Nibea mitsukurii (gb|AJD80650.1|) and Clupea harengus (gb|XP_012692914.1|). Identical residues are shaded black and similar residues (based on the Blosum62 matrix, using ClustalW default parameters) are shaded grey. Indicated are four (i–iv) conserved motif of elongases: (i) KXXEXXDT, (ii) QXXFLHXXHH, (iii) NXXXHXXMYXYY and (iv) TXXQXXQ. The putative endoplasmic reticulum (ER) retrieval signal RXKXX at C-terminus is also indicated [25]

Fig. 3

Functional characterization of the newly cloned Clarias gariepinus Elovl4a (a, b) and Elovl4b (c, d) in yeast (Saccharomyces cerevisiae). The fatty acid profiles of yeast transformed with pYES2 containing the coding sequence of elovl4a and elovl4b were determined after the yeast were grown in the presence of one of the exogenously added substrates 22:5n-3 (a, c), and 22:4n-6 (b, d). The first peak (with asterisk) is derived from the exogenously added substrates. The elongation products are indicated accordingly in each panel

Fig. 4

Tissue distribution of Clarias gariepinus elovl4a and elovl4b transcripts. Expression levels quantified for each target gene were normalized with the expression of the reference gene 28 s rRNA. Data are reported as mean values with their standard errors (n = 4). Within each target gene, different letters indicate statistically significant differences in expression level among tissues (ANOVA and Tukey’s HSD post hoc tests). ABO accessory breathing organ, Adipose T. adipose tissue, F. Gonad female gonad, M. Gonad male gonad