Evolution and Functional Characteristics of the Novel elovl8 That Play Pivotal Roles in Fatty Acid Biosynthesis

Abstract

Elongation of very long-chain fatty acid (Elovl) proteins are key enzymes that catalyze the rate-limiting step in the fatty acid elongation pathway. The most recently discovered member of the Elovl family, Elovl8, has been proposed to be a fish-specific elongase with two gene paralogs described in teleosts. However, the biological functions of Elovl8 are still to be elucidated. In this study, we showed that in contrast to previous findings, elovl8 is not unique to teleosts, but displays a rather unique and ample phylogenetic distribution. For functional determination, we generated elovl8a (elovl8a−/−) and elovl8b (elovl8b−/−) zebrafish using CRISPR/Cas9 technology. Fatty acid composition in vivo and zebrafish liver cell experiments suggest that the substrate preference of Elovl8 overlapped with other existing Elovl enzymes. Zebrafish Elovl8a could elongate the polyunsaturated fatty acids (PUFAs) C18:2n-6 and C18:3n-3 to C20:2n-6 and C20:3n-3, respectively. Along with PUFA, zebrafish Elovl8b also showed the capacity to elongate C18:0 and C20:1. Gene expression quantification suggests that Elovl8a and Elovl8b may play a potentially important role in fatty acid biosynthesis. Overall, our results provide novel insights into the function of Elovl8a and Elovl8b, representing additional fatty acid elongases not previously described in chordates.

Molecular Characterization, Tissue Distribution and Differential Nutritional Regulation of Three n-3 LC-PUFA Biosynthesis-Related Genes in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

“…For instance, Elovl8b could elongate C18 (18:2n-6, 18:3n-3 and 18:4n-3) and C20 (20:4n-6 and 20:5n-3) polyunsaturated fatty acids (PUFAs) to longer-chain polyunsaturated fatty acids (LC-PUFAs), whereas Elovl8a lacked this ability in rabbitfish. However, in the study of zebrafish, Elovl8a activity was specific to C18-C20 PUFAs, just as Elovl4, Elovl5 and Elovl8b activity was specific to C18:0 and C20:1 MUFAs similar to Elovl1, Elovl3, and Elovl7 [9,19]. Thus, further studies need to explore Elovl8 functions in LC-PUFA synthesis in this hybrid grouper.…”

“…Multiple sequences alignment showed that elovl8 shared a closer relationship with elovl4 [18]. Afterwards, Sun et al conducted a phylogenetic analysis to establish the orthology of the newly identified elovl8 gene and revealed that the similar orthologs from some fish species which were all annotated as elovl4 or elovl4-like could be misidentified due to the sequence similarity to elovl4 genes [9]. Thus, the previously similar elovl genes that have been annotated as "elovl4" (or "elovl4-like") in many fish species were considered to be elovl8 [9].…”

“…Afterwards, Sun et al conducted a phylogenetic analysis to establish the orthology of the newly identified elovl8 gene and revealed that the similar orthologs from some fish species which were all annotated as elovl4 or elovl4-like could be misidentified due to the sequence similarity to elovl4 genes [9]. Thus, the previously similar elovl genes that have been annotated as "elovl4" (or "elovl4-like") in many fish species were considered to be elovl8 [9]. Similarly, amino acid sequence alignment with elovl4, elovl5 and elovl8 from other species showed that the new isolated "elovl4" gene from hybrid grouper was highly similar to those of elovl8b, and exhibited high identity with other teleost elovl8b, particularly Perciformes, including Siganus canaliculatus Elovl8b (86%), Salmo salar Elovl8b (83%), Oncorhynchus mykiss Elovl8b (83%), Ictalurus punctatus Elovl8b (81%) and Danio rerio Elovl8b (80%).…”

“…Elovls are the initial and rate-limiting enzymes responsible for the elongation reaction required for the de novo biosynthesis of LC-PUFA. At present, the Elovls family was found to have Elovl1-8, in which Elovl2, Elovl4, Elovl5 and Elovl8 are involved in the elongation of LC-PUFA [8][9][10]. Elovl2 is preferentially responsible for the elongation step from C22 to C24 LC-PUFA.…”

“…Accordingly, it is expected that this enzyme could compensate for Elovl2 activity in some marine fish species, such as rabbitfish (Siganus canaliculatus) [18]. Recently, two Elovl8 isoforms, "Elovl8a" and "Elovl8b", have been found in zebrafish [9] and rabbitfish [19]. Further functional characterization by heterologous expression in yeast showed that Elovl8b could elongate C18 (18:2n-6, 18:3n-3 and 18:4n-3) and C20 (20:4n-6 and 20:5n-3) polyunsaturated fatty acids (PUFAs) to longer-chain polyunsaturated fatty acids (LC-PUFAs), whereas Elovl8a lacked this ability in rabbitfish.…”