Highly Unsaturated Fatty Acid Synthesis in Atlantic Salmon: Characterization of ELOVL5- and ELOVL2-like Elongases

Abstract

Running title: Characterisation of salmon ELOVL5-and ELOVL2 elongases Abbreviations: aa, amino acid; ARA, arachidonic acid (20:4n-6); DHA, docosahexaenoic acid (22:6n-3); EPA, eicosapentaenoic acid (20:5n-3); ER, endoplasmic reticulum; FA, fatty acid; FO, fish oil; HUFA, highly unsaturated fatty acids (carbon chain length ≥ C 20 with ≥ 3 double bonds); LO, linseed oil; ORF, open reading frame; PUFA, polyunsaturated fatty acids; Q-PCR, quantitative (real-time) polymerase chain reaction; RACE, rapid amplification of cDNA ends; RO, rapeseed oil; SO, soybean oil; UTR, untranslated region; VO, vegetable oil. 2Abstract Fish species vary in their capacity to biosynthesize the n-3 long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of LC-PUFA involves enzyme-mediated fatty acyl desaturation and elongation. Previously, a cDNA for an elongase, now termed elovl5a, had been cloned from Atlantic salmon. Here we report on the cloning of two new elongase cDNAs: a second elovl5b elongase, corresponding to a 294 aa protein, and an elovl2-like elongase, coding for a 287 aa protein, characterized for the first time in a nonmammalian vertebrate. Heterologous expression in yeast showed that the salmon Elovl5b elongated C18 and C20 PUFA, with low activity towards C22, while Elovl2 elongated C20 and C22 PUFA with lower activity towards C18 PUFA. All three transcripts showed predominant expression in the intestine and liver, followed by the brain. Elongase expression showed differential nutritional regulation. Levels of elovl5b and particularly of elovl2, but not of elovl5a, transcripts were significantly increased in liver of salmon fed vegetable oils (VO) compared to fish fed fish oil (FO). Intestinal expression showed a similar pattern.Phylogenetic comparisons indicate that, in contrast to salmon and zebrafish, Acanthopterygian fish species lack elovl2 which is consistent with their neglible ability to biosynthesise LC-PUFA and to adapt to VO dietary inclusion, compared to predominantly freshwater salmonids. Thus the presence of elovl2 in salmon explains the ability of this species to biosynthesise LC-HUFA and may provide a biotechnological tool to produce enhanced levels of LC-PUFA, particularly DHA, in transgenic organisms.3

Biosynthesis of very long-chain fatty acids (C>24) in Atlantic salmon: Cloning, functional characterisation, and tissue distribution of an Elovl4 elongase

“…Elovl4 proteins are highly conserved through evolution (Leonard et al, 2004). Consistent with that, the phylogenetic analysis revealed that the salmon elovl4 cDNA encodes a protein more similar to other Elovl4 orthologues from mammals, birds and fish, than the other Elovl family proteins characterised previously in Atlantic salmon, the Elovl5_a and Elovl5_b, and Elovl2 Morais et al, 2009 nigroviridis and Atlantic salmon Elovl4. This clustering pattern is consistent with the differing functional activities between the two fish Elovl4 groups.…”

“…showing high elongation efficiency towards C18 and C20 PUFA, whereas Elovl2 produced C24 PUFA from C20 and C22 PUFA and, in all cases, the production of longer PUFA was negligible Morais et al, 2009). Interestingly, a recent study has shown that mice ELOVL2 participates in the biosynthesis of n-6 VLC-PUFA that are critical in male fertility, and that 24:5n-6 appears a preferred substrate for murine ELOVL2 (Zadravec et al, 2011).…”

“…The search for a more sustainable candidate to replace fish oil is currently a major concern in aquaculture due to the dramatic depletion of wild fish stocks, which paradoxically are the source of traditional dietary lipid used in aquaculture. Therefore, it is important to understand the molecular mechanisms underlying the biosynthesis of PUFA and VLC-FA in salmon, and significant advances have been made in recent years with the molecular cloning and functional characterisation of several genes encoding fatty acyl desaturases (Fad) Monroig et al, 2010b), and elongases including Elovl5 and Elovl2 Morais et al, 2009). …”

Linoleic and α-linolenic acid as precursor and inhibitor for the synthesis of long-chain polyunsaturated fatty acids in liver and brain of growing pigs

“…Unfortunately, we did not have the standards in order to establish whether this was paralleled by a positive effect on the content of c24:5 n-3 and c24:4 n-6 as well. Fatty acid-dependent changes in hepatic ELOVL2 expression have also been reported in rat and salmon (Igarashi et al, 2007a;Morais et al, 2009).…”

Insights into the phylogenetic and molecular evolutionary histories of Fad and Elovl gene families in Actiniaria

“…Research investigating the Fad and Elovl gene families that desaturate and elongate LC‐PUFAs has largely been restricted to bilaterian taxa (Carmona‐Antoñanzas et al., 2011; Castro et al., 2012; Fonseca‐Madrigal et al., 2014; Kabeya et al., 2017; Li et al., 2017; Li et al., 2010; Mohd‐Yusof et al., 2010; Monroig, Guinot, et al., 2012; Monroig, Navarro, et al., 2012; Monroig, Webb, et al., 2011; Monroig et al., 2010, 2016, 2017, 2013; Morais et al., 2009; Surm et al., 2015). We have investigated the phylogenetic and molecular evolutionary histories of the Fad and Elovl gene families in phylum Cnidaria, to provide insights into evolution and the distribution of these gene families in Metazoa outside of bilaterian taxa.…”