Results Elongases Sequence analysis of Elovl2 and Elovl5. Amplification of the 840 bp Elovl2 ORF and subsequent alignment with the rat Elovl2 sequence in GenBank (NM_001109118) revealed the two sequences were identical. Amplification of the 900 bp Elovl5 ORF and subsequent alignment with the rat Elovl5 sequence in GenBank (NM_134382) revealed the two sequences had four nucleotide differences. The Elovl5 sequence has been deposited in GenBank as accession number HQ404314. The predicted Elovl2 and Elovl5 proteins of 279 and 299 amino acids, respectively, included all of the characteristic features of a microsomal fatty acyl elongase and had 56% identity. However, due to the nucleotide changes in the Elovl5 sequence, the predicted Elovl5 protein sequence included four residue differences from the rat Elovl5 sequence in GenBank (NP_599209). The Elovl2 and Elovl5 proteins are predicted to contain five and six transmembrane regions, respectively (SOSUI software, http://bp.nuap.nagoya-u. ac.jp/sosui/). Comparison of Elovl2 and Elovl5 substrate specificities. Recombinant S. cerevisiae cells expressing Elovl2 or Elovl5 were cultured in the presence of 100 mM of various C18- 22 PUFA to determine substrate specificities of the elongases. Elovl5 demonstrated elongase activity with C18 and C20, but not C22 PUFA (Fig. 1a). By contrast, Elovl2 demonstrated little activity with C18 PUFA, but was active with C20 and C22 PUFA substrates (Fig. 1b). The Elovl2 activity was 4.5- and 2.5-fold higher with the n-3 substrates EPA and DPA, respectively, compared with their n- 6 homologs, arachidonic acid and 22:4n-6. Competition between n-3 substrates for Elovl2 and Elovl5. Since both SDA and its downstream product, EPA, are substrates for Elovl5, there is the possibility of competition for enzyme activity. However, over the concentration range investigated there was no competitive inhibition of EPA elongation by SDA, or SDA elongation by EPA (Fig. 2). The elongation products of SDA and EPA were not detected in the empty vector dose response experiments (Fig. 2). Elovl2 also has Role of Elongase Reactions in LCPUFA Synthesis two n-3 substrates, EPA and DPA. However, attempts to examine possible competition between EPA and DPA were problematic due to Elovl2 performing sequential reactions with EPA such that DPA is both product and substrate. The sequential reactions with Elovl2 were examined further. Examination of product/substrate relationships for Elovl2 and Elovl5. The initial results indicate that EPA at 100 mM can be used efficiently as a substrate by both Elovl2 and Elovl5. For further comparisons, EPA dose response curves for the two enzymes were made. For Elovl5 there was a proportional increase in DPA synthesis with increasing EPA concentration (Fig. 3a). For Elovl2 there was also a proportional increase in the product of the first reaction, DPA, but not the product of the second reaction, 24:5n-3 (Fig. 3b). This may suggest that the second reaction of endogenously generated DPA conversion to 24:5n-3 is saturated at concentrations of EPA that are not saturating for the first reaction, EPA conversion to DPA. When DPA was used as an exogenous substrate, apparent saturation of conversion to 24:5n-3 was also observed (Fig. 3c). The elongation products of EPA and DPA were not detected in the empty vector dose response experiments (Fig. 3). Expression of Elovl2 and Elovl5 in rat liver and heart. The dose responses indicate that both elongase enzymes have similar affinities for EPA and therefore, both could produce DPA. However, expression levels in vivo are quite different with Elovl5 being present at much higher levels than Elovl2 in liver and heart (Fig. 4). The expression of both Elovl5 and Elovl2 was higher in liver compared to heart (Fig. 4). D6-Desaturase (Fads2) Sequence analysis of Fads2. Amplification of the 1335 bp Fads2 ORF and subsequent alignment with the GenBank sequence NM_031344 revealed one nucleotide difference. The Fads2 sequence was deposited in GenBank as accession number HQ909027. The predicted Fads2 protein of 444 amino acids contained one residue difference compared to NP_112634. The residue change was not within the regions characteristic of fatty Fgure 1. Comparison of the rat Elovl5 and Elovl2 substrate specificities. Recombinant S. cerevisiae expressing Elovl5 (A) or Elovl2 (B) were grown in the presence of 100 mM of various C18, C20 and C22 PUFA substrates. Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to longer chain fatty acid product(s) was calculated as [product(s)/(product(s) + substrate)]6100. The product was 2 carbons longer than the substrate. 1 denotes that the conversion includes the 4 carbon elongation product. The results are the means 6 S.D. of triplicate incubations. Values with different symbols are significantly different from each other. ND, not detected. doi:10.1371/journal.pone.0029662.g001 PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29662