Lu-Te Chuang

Identification and Expression of Mammalian Long-Chain PUFA Elongation Enzymes

In mammalian cells, Sprecher has proposed that the synthesis of long-chain PUFA from the 20-carbon substrates involves two consecutive elongation steps, a Δ6-desaturation step followed by retroconversion (Sprecher, H., Biochim. Biophys. Acta 1486, 219–231, 2000). We searched the database using the translated sequence of human elongase ELOVL5, whose encoded enzyme elongates monounsaturated and polyunsaturated FA, as a query to identify the enzyme(s) involved in elongation of very long chain PUFA. The database search led to the isolation of two cDNA clones from human and mouse. These clones displayed deduced amino acid sequences that had 56.4 and 58% identity, respectively, to that of ELOVL5. The open reading frame of the human clone (ELOVL2) encodes a 296-amino acid peptide, whereas the mouse clone (Elovl2) encodes a 292-amino acid peptide. Expression of these open reading frames in bakers yeast, Saccharomyces cerevisiae, demonstrated that the encoded proteins were involved in the elongation of both 20-and 22-carbon long-chain PUFA, as determined by the conversion of 20∶4n−6 to 22∶4n−6, 22∶4n−6 to 24∶4n−6, 20∶5n−3 to 22∶5n−3, and 22∶5n−3 to 24∶5n−3. The elongation activity of the mouse Elovl2 was further demonstrated in the transformed mouse L cells incubated with long-chain (C20-and C22-carbon) n−6 and n−3 PUFA substrates by the significant increase in the levels of 24∶4n−6 and 24∶5n−3, respectively. This report demonstrates the isolation and identification of two mammalian genes that encode very long chain PUFA specific elongation enzymes in the Sprecher pathway for DHA synthesis.

Identification of two novel microalgal enzymes involved in the conversion of the ω3-fatty acid, eicosapentaenoic acid, into docosahexaenoic acid

Marine microalgae such as Pavlova and Isochrysis produce abundant amounts of the omega3-PUFAs (polyunsaturated fatty acids), EPA (eicosapentaenoic acid, 20:5n-3) and DHA (docosahexaenoic acid, 22:6n-3). The pathway leading to the conversion of EPA into DHA in these lower eukaryotes is not well established although it is predicted to involve an elongation step, catalysed by an elongating enzyme complex, leading to the conversion of EPA into omega3-DPA (omega-3-docosapentaenoic acid, 22:5n-3); followed by a desaturation step, catalysed by a Delta4-desaturase, which results in the conversion of DPA into DHA. To date, the enzymes involved in the elongation of EPA have not been identified from any lower eukaryote. In the present study, we describe the identification of microalgal genes involved in the two-step conversion of EPA into DHA. By expressed sequence tag analysis, a gene (pavELO) encoding a novel elongase was identified from Pavlova, which catalysed the conversion of EPA into omega3-DPA in yeast. Unlike any previously identified elongase from higher or lower eukaryotes, this enzyme displayed unique substrate specificity for both n-6 and n-3 C20-PUFA substrates, with no activity towards any C18- or C22-PUFA substrates. In addition, a novel Delta4-desaturase gene (IgD4) was isolated from Isochrysis, which was capable of converting omega3-DPA into DHA, as well as adrenic acid (22:4n-6) into omega6-DPA. Yeast co-expression studies, with pavELO and IgD4, revealed that these genes were capable of functioning together to carry out the two-step conversion of EPA into DHA.

Effect of conjugated linoleic acid on fungal Δ6-desaturase activity in a transformed yeast system

Conjugated linoleic acid (CLA; 18∶2), a group of positional and geometric isomers of linoleic acid (LA; 18∶2n−6), has been shown to modulate immune function through its effect on eicosanoid synthesis. This effect has been attributed to a reduced production of n−6 polyunsaturated fatty acid (PUFA), the precursor of eicosanoids. Since Δ6-desaturase is the rate-limiting enzyme of the n−6 PUFA production, it is our hypothesis that CLA, which has similar chemical structure to LA, interacts directly with Δ6-desaturase. A unique and simple model, i.e., bakers yeast (Saccharomyces cerevisiae) transformed with fungal Δ6-desaturase gene, previously established, was used to investigate the direct effect of CLA on Δ6-desaturase. This model allows LA to be converted to γ-linolenic acid (GLA; 18∶3n−6) but not GLA to its metabolite(s). No metabolites of CLA were found in the lipids of the yeast transformed with Δ6-desaturase. The inability to convert CLA to conjugated GLA was not due to the failure of yeast cells to take up the CLA isomers. CLA mixture and individual isomers significantly inhibited the activity of Δ6-desaturase of the transformed yeast in vivo. Even though its uptake by the yeast was low, CLA c9, t11 isomer was found to be the most potent inhibitor of the four isomers tested, owing to its high inhibitory effect on Δ6-desaturase. Since CLA did not cause significant changes in the level of Δ6-desaturase mRNA, the inhibition of GLA production could not be attributed to suppression of Δ6-desaturate gene expression at the transcriptional level.

Inhibitory effect of conjugated linoleic acid on linoleic acid elongation in transformed yeast with human elongase

Conjugated linoleic acid (CLA; 18∶2) refers to a group of positional and geometric isomers derived from linoleic acid (LA; Δ9, 12–18∶2). Using a growing bakers yeast (Saccharomyces cerevisiae) transformed with human elongase gene, we examined the inhibitory effect of CLA at various concentrations (10, 25, 50, and 100 μM) on elongation of LA (25 μM) to eicosadienoic acid (EDA; Δ11,14–20∶2). Among four available individual CLA isomers, only c9,t11- and t10,c12-isomers inhibited elongation of LA to EDA. The extent of inhibition (ranging from 20 to 60%) was related to the concentration of CLA added to the medium. In the meantime, only these two isomers, when added at 50 μM to the media, were elongated to conjugated EDA (c11,t13- and t12,c14–20∶2) by the same recombinant elongase at the rate of 28 and 24%, respectively. The inhibitory effect of CLA on LA elongation is possibly due to competition between CLA isomers and LA for the recombinant elongase. Thus, results from this study and a previous study suggest that the biological effect of CLA is exerted through its inhibitory effect on Δ6-desaturation as well as elongation of LA which results in a decrease in long-chain n−6 fatty acids and consequently the eicosanoid synthesis.

Effects of high-γ-linolenic acid canola oil compared with borage oil on reproduction, growth, and brain and behavioral development in mice

Previous research in rats and mice has suggested that γ-linolenic acid (GLA) derived from borage oil (BO: 23% GLA) may be an appropriate source for increasing levels of long-chain n−6 FA in the developing brain. Recently, transgenic technology has made available a highly enriched GLA seed oil from the canola plant (HGCO: 36% GLA). The first objective of this study was to compare the effects of diets containing equal levels of GLA (23%) from either BO or HGCO on reproduction, pup development, and pup brain FA composition in mice. The second objective was to compare the effects of the HGCO diluted to 23% GLA (GLA-23) with those of undiluted HGCO containing 36% GLA (GLA-36). The diets were fed to the dams prior to conception and throughout pregnancy and lactation, as well as to the pups after weaning. The behavioral development of the pups was measured 12 d after birth, and anxiety in the adult male offspring was assessed using the plus maze. The findings show that despite equivalent levels of GLA, GLA-23 differed from BO in that it reduced pup body weight and was associated with a slight increase in neonatal pup attrition. However, there were no significant effects on pup behavioral development or on performance in the plus maze. An increase in dietary GLA resulted in an increase in brain 20∶4n−6 and 22∶4n−6, with a corresponding decrease in 22∶6n−3. Again, despite their similar levels of GLA, these effects tended to be larger in GLA-23 than in BO. In comparison with GLA-23, GLA-36 had larger effects on growth and brain FA composition but no differences with respect to effects on reproduction and behavioral development. These findings suggest that the HGCO can be used as an alternative source of GLA.

Effect of conjugated linoleic acid on Delta-5 desaturase activity in yeast transformed with fungal Delta-5 desaturase gene

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers derived from linoleic acid (LA:Δ9, 12-18:2), has been shown to exhibit various biological functions based on studies using cell culture and animal models. It was postulated that the beneficial effects of CLA were exerted through suppression of production of arachidonic acid (AA;Δ5,8,11,14-20:4) and consequently, production of pro-inflammatory eicosanoids. In this study, we used the bakers yeast, Saccharomyces cerevisiae, transformed with fungal Δ5-desaturase gene as a model, to study whether CLA affects the activity of Δ5-desaturase, a rate-limiting step which converts dihomo-γ-linolenic acid (DGLA;Δ8,11, 14-20:3) to AA. The activity of Δ5-desaturase was examined in the transformed yeast incubated in a medium supplemented with DGLA and one of four different CLA isomers (c9, t11−, t10, c12−, c9, c11− and t9, t11). Results show that all four isomers were taken up readily by the yeast, and all of them suppressed the conversion of DGLA to AA. The degree of suppression, which varied significantly among four isomers was modulated by the level of CLA isomers added in the medium. Since portions of these CLA isomers could be converted to form Δ5-CLA metabolites (Δ5, c9, t11−, Δ5, t10, c12−, Δ5, c9, c11− and Δ5, t9, t11-18:3), it is suggested that CLA suppressed the Δ5-desaturation of DGLA to AA through substrate competition between DGLA and CLA isomers. (Mol Cell Biochem 265: 11–18, 2004)