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

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.