Pradip Mukerji

Recent advances in the study of fatty acid desaturases from animals and lower eukaryotes.

The biosynthesis of polyunsaturated fatty acids (PUFAs) in different organisms can involve a variety of pathways, catalyzed by a complex series of desaturation and elongation steps. A range of different desaturases have been identified to date, capable of introducing double bonds at various locations on the fatty acyl chain. Some recently identified novel desaturases include a delta4 desaturase from marine fungi, and a bi-functional delta5/delta6 desaturase from zebrafish. Using molecular genetics approaches, these desaturase genes have been isolated, identified, and expressed in variety of heterologous hosts. Results from these studies will help increase our understanding of the biochemistry of desaturases and the regulation of PUFA biosynthesis. This is of significance because PUFAs play critical roles in multiple aspects of membrane physiology and signaling mechanisms which impact human health and development.

Cloning of Δ12- and Δ6-desaturases from Mortierella alpina and recombinant production of γ-linolenic acid in Saccharomyces cerevisiae

Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited Δ12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid. The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited Δ6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the Δ12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the Δ6-desaturase activity, the level of γ-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the Δ6- and Δ12-desaturase cDNA resulted in the endogenous production of γ-linolenic acid. The yields of γ-linolenic acid reached as high as 8% of total fatty acids in yeast.

Identification of delta5-desaturase from Mortierella alpina by heterologous expression in bakers' yeast and canola

A DNA fragment with homology to Δ6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known Δ6-desaturase genes. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M. alpina library. Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had Δ5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant Δ5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina Δ5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Δ5,9–18:2) and pinolenic acid (Δ5,9,12–18:3), which are the Δ5-desaturation products of oleic and linoleic acids, respectively.

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.

A novel omega3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid.

Long-chain n-3 PUFAs (polyunsaturated fatty acids) such as EPA (eicosapentaenoic acid; 20:5 n-3) have important therapeutic and nutritional benefits in humans. In plants, cyanobacteria and nematodes, omega3-desaturases catalyse the formation of these n-3 fatty acids from n-6 fatty acid precursors. Here we describe the isolation and characterization of a gene ( sdd17 ) derived from an EPA-rich fungus, Saprolegnia diclina, that encodes a novel omega3-desaturase. This gene was isolated by PCR amplification of an S. diclina cDNA library using oligonucleotide primers corresponding to conserved regions of known omega3-desaturases. Expression of this gene in Saccharomyces cerevisiae, in the presence of various fatty acid substrates, revealed that the recombinant protein could exclusively desaturate 20-carbon n-6 fatty acid substrates with a distinct preference for ARA (arachidonic acid; 20:4 n-6), converting it into EPA. This activity differs from that of the known omega3-desaturases from any organism. Plant and cyanobacterial omega3-desaturases exclusively desaturate 18-carbon n-6 PUFAs, and a Caenorhabditis elegans omega3-desaturase preferentially desaturated 18-carbon PUFAs over 20-carbon substrates, and could not convert ARA into EPA when expressed in yeast. The sdd17 -encoded desaturase was also functional in transgenic somatic soya bean embryos, resulting in the production of EPA from exogenously supplied ARA, thus demonstrating its potential for use in the production of EPA in transgenic oilseed crops.

Green tea polyphenols modulate secretion of urokinase plasminogen activator (uPA) and inhibit invasive behavior of breast cancer cells.

Many epidemiological studies have suggested that consumption of green tea may decrease the risk of cancer. The chemopreventive effect of green tea polyphenols (GTP) has been demonstrated through the inhibition of cell proliferation and angiogenesis in cell culture and animal models of breast cancer. Metastasis of breast cancer is the major reason for the high mortality of breast cancer patients and is directly linked to the invasive behavior of breast cancer cells. Cancer metastasis consists of several interdependent processes including cancer cell adhesion, cancer cell migration, and invasion of cancer cells. In this study, we evaluated the effect of GTP on human breast cancer cells, and we show that in addition to inhibiting cell growth, GTP also suppressed the invasive behavior of MDA-MB-231 cells. These anti-invasive effects of GTP were the result of the inhibition of constitutively active transcription factors AP-1 and NF-κB, which further suppressed secretion of urokinase plasminogen activator (uPA) from breast cancer cells. Based on these results, it can be hypothesized that GTP treatment resulted in the inhibition of formation of signaling complexes responsible for cell adhesion and migration (uPA, uPA receptor, vitronectin, integrin receptor) and cell invasion (uPA, uPA receptor). Our results indicate that GTP may contribute to the anticancer effects of green tea by inhibiting the invasive behavior of cancer cells.

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.

ANALYSIS OF BETA-CASEIN AND ITS PHOSPHOFORMS IN HUMAN MILK

Abstract Quantitation of β-casein was achieved through the use of urea-polyacrylamide gel electrophoresis of whole milk and scanning densitometry. This method also provided electrophorectic separation of the different phosphoforms of β-casein which were also quantitated. Fifty-eight human milk samples collected in 4 different countries were analyzed for β-casein and β-casein phosphoform concentrations. The average β-casein concentration obtained using the whole milk methodology was 4.72 ± 1.44 mg/ml. We found that β-casein is found in all the fractions of milk that has been centrifuged to remove the lipid or acid precipitated to collect the caseins. This study used whole human milk and therefore all the β-casein present was included in the analysis. The whole milk analysis of β-casein indicated that on average the phosphoforms are present in the following order ranked by concentration: tetra- > di- > non- > mono- > tri- > penta-phosphorylated β-casein. However, the phosphoform distribution of individual donors varied widely. Four different methods were used to determine the concentration of total protein in human milk samples. UV absorbance-based and colorimetric methods produced higher values of protein concentration than the Kjeldahl method.