Abstract
Elongation of very long-chain fatty acid 4 (Elovl4) proteins participate in the biosynthesis of very long-chain (>C24) saturated and polyunsaturated fatty acids (FA). Previous studies have shown that fish possess two different forms of Elovl4, termed Elovl4a and Elovl4b. The present study aimed to characterize both molecularly and functionally two elovl4 cDNA from the African catfish Clarias gariepinus. The results confirmed that C. gariepinus possessed two elovl4-like elongases with high homology to two previously characterized Elovl4 from Danio rerio, and thus they were termed accordingly as Elovl4a and Elovl4b. The C. gariepinus Elovl4a and Elovl4b have open reading frames (ORF) of 945 and 915 base pairs, respectively, encoding putative proteins of 314 and 304 amino acids, respectively. Functional characterization in yeast showed both Elovl4 enzymes have activity towards all the PUFA substrates assayed (18:4n-3, 18:3n-6, 20:5n-3, 20:4n-6, 22:5n-3, 22:4n-6 and 22:6n-3), producing elongated products of up to C36. Moreover, the C. gariepinus Elovl4a and Elovl4b were able to elongate very long-chain saturated FA (VLC-SFA) as denoted by increased levels of 28:0 and longer FA in yeast transformed with elovl4 ORF compared to control yeast. These results confirmed that C. gariepinus Elovl4 play important roles in the biosynthesis of very long-chain FA. Tissue distribution analysis of elovl4 mRNAs showed both genes were widely expressed in all tissues analyzed, with high expression of elovl4a in pituitary and brain, whereas female gonad and pituitary had the highest expression levels for elovl4b.
Highlights
Elongation of very long-chain fatty acid (Elovl) proteins catalyze the condensation reaction, regarded as the first and rate-limiting step of four sequential reactions required for the elongation of fatty acids (FA) [1, 2]
It was shown that two Elongation of very long-chain fatty acid 4 (Elovl4) genes, termed Elovl4a and Elovl4b, were present, in contrast to mammals in which only a single Elovl4 had been reported [7]. Both D. rerio Elovl4 showed an ability to elongate saturated fatty acids, but only Elovl4b appeared to have a role in the biosynthesis of very long-chain (>C24) polyunsaturated fatty acids (VLC-PUFA) [6]
polymerase chain reaction (PCR) fragments corresponding to the open reading frame (ORF) of C. gariepinus newly cloned elovl4 complementary DNA (cDNA) were amplified from a mixture of cDNA synthesized from eye and brain RNA, using the high fidelity Pfu DNA polymerase (Promega, USA) with primers containing BamHI and XhoI restriction sites (Table 1)
Summary
Elongation of very long-chain fatty acid (Elovl) proteins catalyze the condensation reaction, regarded as the first and rate-limiting step of four sequential reactions required for the elongation of fatty acids (FA) [1, 2]. It was shown that two Elovl genes, termed Elovl4a and Elovl4b, were present, in contrast to mammals in which only a single Elovl had been reported [7] Both D. rerio Elovl showed an ability to elongate saturated fatty acids, but only Elovl4b appeared to have a role in the biosynthesis of very long-chain (>C24) polyunsaturated fatty acids (VLC-PUFA) [6]. Since this pioneer study in fish, further elovl cDNA sequences have been studied in a variety of species including Atlantic salmon, Nibe croaker, orange-spotted grouper and rabbitfish [8,9,10,11]. We characterized, both molecularly and functionally, two elovl cDNA from C. gariepinus and investigated their tissue expression patterns
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