Functional desaturase Fads1 (Δ5) and Fads2 (Δ6) orthologues evolved before the origin of jawed vertebrates

Abstract

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales.

Figure 1. Biosynthetic pathway of very long chain-PUFA (VLC-PUFA).

Figure 2. Maximum likelihood tree for the Fads gene family based on protein sequences. aLRT values are shown on each node.

Accession numbers of the sequences are given in table S1.

Figure 3. Synteny map of the Fads gene cluster in vertebrates.

Mammals (Hs-Homo sapiens, Mm-Mus musculus, Md-Monodelphis domestica), birds (Gg-Gallus gallus), reptiles (Ac-Anolis carolinensis), amphibians (Xt-Xenopus tropicalis) and teleosts (Ga-Gasterosteus aculeatus, Ol-Oryzias latipes, Dr-Danio rerio, Tr-Takifugu rubripes, Tn-Tetraodon nigroviridis). Double dash denotes gap.

Figure 4. Functional characterization of the newly cloned Scyliorhinus canicula fatty acyl desaturases FADS1.

(Panels A, C and E) and FADS2 (panels B, D and F) in transgenic yeast (Saccharomyces cerevisiae) grown in the presence of Δ6 substrates 18:3n-3 (A and B), Δ5 substrates 20:4n-3 (C and D) and Δ4 substrates (E and F). Fatty acids were extracted from yeast transformed with pYES2 vector containing the ORF of the putative fatty acyl desaturase cDNA as an insert. The first four peaks in all panels are the main endogenous fatty acids of S. cerevisiae, namely 16:0 (1), 16:1 isomers (2), 18:0 (3), and 18:1n-9 (4). Substrates (“*”) and their corresponding desaturated products are indicated accordingly in panels A–F. Vertical axis, FID response; horizontal axis, retention time.

Figure 5. Evolutionary model of Fads gene diversification along the vertebrate lineage.

Dotted line indicates physical linkage unknown. Gg-Gallus gallus, Ac-Anolis carolinensis, Xt-Xenopus tropicalis, Ga-Gasterosteus aculeatus, Ol-Oryzias latipes, Dr-Danio rerio, Tr-Takifugu rubripes, Tn-Tetraodon nigroviridis, Ss- Salmo salar, Sc- Siganus canaliculatus, and Sca- Scyliorhinus canicula.