Molecular Cloning, Functional Characterization and Nutritional Regulation of the Putative Elongase Elovl5 in the Orange-Spotted Grouper (Epinephelus coioides).

Songlin Li,Yuhui Yuan,Kangsen Mai,Tianjiao Wang,Qinghui Ai,Mingzhu Li,Wei Xu,Alexander Chong Shu-Chien

doi:10.1371/journal.pone.0150544

Abstract

The enzymes involved in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) are widely studied in fish species, as fish are the main source of n-3 LC-PUFAs for human beings. In the present study, a putative gene for elovl5, which encodes a key enzyme involved in LC-PUFA synthesis, was cloned and functionally characterized, and its transcription in response to dietary n-3 LC-PUFA exposure was investigated. Moreover, cell transfection and luciferase assays were used to explore the mechanism underlying the regulation of elovl5. The full-length cDNA of elovl5 was 1242 bp (excluding the polyA tail), including an 885 bp coding region encoding a 295 amino acid protein that possesses all of the characteristic features of elovl proteins. Functional characterization of heterologously expressed grouper Elovl5 indicated that it effectively elongates both C18 (18:2n-6, 18:3n-3, 18:3n-6 and 18:4n-3) and C20 (20:4n-6 and C20:5n-3) PUFAs, but not the C22 substrates. The expression of elovl5 was significantly affected by dietary n-3 LC-PUFA exposure: a high n-3 LC-PUFA level repressed the expression of elovl5 by slightly down-regulating the expression of sterol regulatory element-binding protein (SREBP)-1 and liver X receptor (LXR) α, which are major regulators of hepatic lipid metabolism. Promoter studies showed that grouper elovl5 reporter activity was induced by over-expression of LXRα but not SREBP-1. This finding suggests that elovl5 is a direct target of LXRα, which is involved in the biosynthesis of PUFAs via transcriptional regulation of elovl5. These findings may contribute to a further understanding of the mechanism underlying the regulation of LC-PUFA biosynthesis in marine fish species.

Highlights

N-3 long-chain polyunsaturated fatty acids (LC-PUFAs, C20 and double bonds3), docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), are essential nutrients for humans that play a variety of important roles in promoting cardiovascular health and immune function [1, 2] and in preventing metabolic disease [3]
Δ6 desaturase (Fads2), which catalyzes the first desaturation step in LC-PUFA synthesis, has been widely studied as the rate-limiting enzyme in the LC-PUFA biosynthetic pathway [9, 10], an important role for Elovl5 has been demonstrated in turbot, Scophthalmus maximus [11], and cod, Gadus morhua [12]
BLAST analysis of the deduced amino acid (AA) sequence of grouper Elovl5 indicated that Elovl5 in orange-spotted grouper shares sequence homology with Elovl5 of other teleosts, such as the large yellow croaker (Larimichthys crocea, 96%), the cobia (R. canadum, 95%), the Atlantic salmon (S. salar, 84%), and the zebrafish (D. rerio, 77%), as well as greater than 70% identity with Elovl5 of humans (H. sapiens, 70%), mice (M. musculus, 71%) and cattle (Bos taurus, 71%)

Summary

Introduction

N-3 long-chain polyunsaturated fatty acids (LC-PUFAs, C20 and double bonds3), docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), are essential nutrients for humans that play a variety of important roles in promoting cardiovascular health and immune function [1, 2] and in preventing metabolic disease [3]. The accepted LC-PUFA biosynthetic pathway in vertebrates involves consecutive desaturation and elongation reactions that convert linolenic acid (18:3n-3) and linoleic acid (18:2n-6) to LC-PUFAs; these reactions are catalyzed by the enzymes fatty acyl desaturase (Fad) and elongation of very long-chain fatty acids (Elovl) [7, 8]. Δ6 desaturase (Fads2), which catalyzes the first desaturation step in LC-PUFA synthesis, has been widely studied as the rate-limiting enzyme in the LC-PUFA biosynthetic pathway [9, 10], an important role for Elovl has been demonstrated in turbot, Scophthalmus maximus [11], and cod, Gadus morhua [12]. Given that limited elongation of C18 to C20 PUFAs, rather than limited Δ5 desaturation, accounts for the limited rate of conversion of 18:3n–3 to EPA in a turbot cell line, relatively low Elovl activity may be implicated in the cell’s poor ability to synthesize n-3LC-PUFA. The discovery of Δ8 desaturation may indicate the existence of a possible alternative pathway, “Elovl5—Δ8 desaturation—Δ5 desaturation” (the Δ8 desaturation pathway) [14, 15]

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