Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids.

The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.

Cholesterol supplementation attenuates the hypocholesterolemic effect of rice bran oil in rats.

Rice bran oil (RBO), when blended with safflower oil (SFO) at the ratio of 7 to 3, has been shown to lower serum cholesterol in humans consuming cholesterol. The mechanism as to how this oil blend exerts its effect is not yet clear. This study examined the effect of cholesterol supplementation on the cholesterol-lowering ability of different RBO/SFO blends. Male Sprague Dawley rats (4 wk old) were fed purified diets containing 10% fat with or without the addition of 0.5% cholesterol for 3 wk. The fat was either SFO or RBO alone, or the mixture of these two oils at the ratio of 7: 3 (7S/3R), 5:5 (5S/5R), or 3:7 (3S/7R). Without cholesterol supplementation, there were no significant differences in the serum and liver total cholesterol levels among different dietary fats. However, the HDL cholesterol level of rats fed the RBO-containing diets (especially in rats fed the 3S/7R diet) was higher than that of rats fed the diet containing SFO alone. This resulted in an increase in the ratio of HDL/total cholesterol-a desirable outcome. Supplementation of the diets with 0.5% cholesterol significantly increased the cholesterol level in both the serum and the liver. Increasing the proportion of RBO in the diet further raised the total cholesterol level in the serum whereas it reduced liver cholesterol. Then, the specific effect of the 3S/7R mixture on the ratio of HDL/total cholesterol disappeared. These findings suggest that cholesterol supplemented at the level of 0.5% in this study masked the cholesterol-lowering effect of RBO. Smaller percentages of polyunsaturated fatty acid (i.e., 18:2n-6) in the RBO-containing diets than in the SFO diet might have reduced their ability to dispose the circulating serum cholesterol into the liver.

Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids.

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of gamma-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

cDNA cloning and characterization of human Delta5-desaturase involved in the biosynthesis of arachidonic acid.

Two human expressed sequence tag (EST) cDNA sequences with identity with Delta(5)- and Delta(6)-desaturases from a filamentous fungus, Mortierella alpina, were identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. (Palo Alto, CA, U.S.A.). An oligonucleotide complementary to the 3' EST cDNA sequences was used to screen human liver cDNA using rapid amplification of cDNA ends (RACE)-PCR. The amplified DNA fragment had 98% identity with a putative open reading frame (ORF) predicted from a human genomic sequence, and encoded 444 amino acids. Expression of this ORF in mouse fibroblast cells demonstrated that the encoded protein was a Delta(5)-desaturase, as determined by the conversion of dihomo-gamma-linolenic acid (C(20:3,n-6)) into arachidonic acid (C(20:4,n-6)). The human Delta(5)-desaturase contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. A tissue expression profile revealed that this gene is highly expressed in fetal liver, fetal brain, adult brain and adrenal gland. A search of the existing databases led to localization of this ORF within a 14 kb interval flanked by the flap endonuclease-1 (FEN1) and vitelliform macular dystrophy (Best's disease; VMD2) loci of chromosome 11q12.

Polyunsaturated fatty acid-specific elongation enzymes.

We have isolated a novel gene (GLELO) from Mortierella alpina and its homologue (CEELO1) from Caenorhabditis elegans and demonstrate the involvement of their encoded proteins in the elongation of C(18) polyunsaturated fatty acids.

Modulation of cholesterol concentration in Caco-2 cells by incubation with different n-6 fatty acids.

Incorporation of exogenous cholesterol was compared in human adenocarcinoma colon cells (Caco-2) after incubation with 100 microM of either linoleic acid (LA, 18:2n-6), gamma-linolenic acid (GLA, 18:3n-6), arachidonic acid (AA, 20:4n-6) or adrenic acid (or n-6 docosatetraenoic acid, DTA, 22:4n-6). In both cells 7 days after seeding and 14 days after confluency, incubation with LA significantly raised the proportion of 18:2n-6 but not its long-chain metabolites in cellular phospholipid. Incubation with GLA increased the levels of 18:3n-6, 20:3n-6, and 20:4n-6. Incubation with AA increased the levels of 20:4n-6 and 22:4n-6, and incubation with DTA increased the levels of 22:4n-6 as well as its retro-conversion metabolite, 20:4n-6. A subsequent addition of cholesterol (180 microM) to the medium significantly raised the cellular cholesterol level but less so in the cells 7 days after seeding incubated with GLA. The increase in cellular cholesterol level was generally greater in the cells of 7 days after seeding, particularly those incubated with long-chain highly unsaturated n-6 fatty acids, than in those of 14 days after confluency. These findings suggest that the cell growth and the extent of unsaturation in cell membrane phospholipid fatty acids modulate the incorporation of the exogenous cholesterol into the Caco-2 cells.

Effects of gamma-linolenic acid and docosahexaenoic acid in formulae on brain fatty acid composition in artificially reared rats.

This study evaluated the effects of dietary supplementation with gamma-linolenic acid (GLA, 18:3n-6) and docosahexaenoic acid (DHA, 22:6n-3) on the fatty acid composition of the neonatal brain in gastrostomized rat pups reared artificially from days 5-18. These pups were fed rat milk substitutes containing fats that provided 10% linoleic acid and 1% alpha-linolenic acid (% fatty acids) and, using a 2x3 factorial design, one of two levels of DHA (0.5 and 2.5%), and one of three levels of GLA (0.5, 1.0, and 3.0%). A seventh artificially reared group served as a reference group and was fed 0.5% DHA and 0.5% arachidonic acid (AA, 20:4n-6); these levels are within the range of those found in rat milk. The eighth group, the suckled control group, was reared by nursing dams fed a standard American Institute of Nutrition 93M chow. The fatty acid composition of the phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine/phosphatidylinositol membrane fractions of the forebrain on day 18 reflected the dietary composition in that high levels of dietary DHA resulted in increases in DHA but decreases in 22:4n-6 and 22:5n-6 in brain. High levels of GLA increased 22:4n-6 but, in contrast to previous findings with high levels of AA, did not decrease levels of DHA. These results suggest that dietary GLA, during development, differs from high dietary levels of AA in that it does not lead to reductions in brain DHA.

Cloning of delta12- and delta6-desaturases from Mortierella alpina and recombinant production of gamma-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 delta12-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 delta6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the delta12-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 delta6-desaturase activity, the level of gamma-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the delta6- and delta12-desaturase cDNA resulted in the endogenous production of gamma-linolenic acid. The yields of gamma-linolenic acid reached as high as 8% of total fatty acids in yeast.

Water maze performance is unaffected in artificially reared rats fed diets supplemented with arachidonic acid and docosahexaenoic acid.

Four groups of male Long-Evans rats were reared artificially from postnatal d 5 to 18 by being fed through a gastrostomy tube with rat milk substitutes containing oils providing 10% linoleic acid and 1% alpha-linolenic acid (g/100 g fat); with the use of a 2 x 2 design, they were fed one of two levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) (0.0 and 2.5 g/100 g of fatty acids). A fifth artificially reared group was fed a diet high in saturated fat, and a sixth group was reared by dams fed a standard AIN-93M diet. The pups were weaned onto modified AIN-93G diets, with a fat composition similar to that fed during the artificial rearing period. Behavioral testing was conducted between 6 and 9 wk of age; brain lipid composition was then assessed. Relative to the unsupplemented group (0.0 g/100 g AA and DHA), dietary supplementation resulted in a wide range of AA (84-103%) and particularly DHA (86-119%) levels in forebrain membrane phospholipids. AA supplementation increased AA levels and decreased DHA levels, and DHA supplementation increased DHA levels and decreased AA levels, with the magnitude of these effects dependent on the level of the other fatty acid. DHA levels were very low in the saturated fat group. The groups did not differ on the place or cued version of the Morris water-maze, but on a test of working memory, the saturated fat group was impaired relative to the suckled control group. Further correlational analyses in the artificially reared animals did not support a relationship between the wide range of DHA and AA levels in the forebrain and working-memory performance.

Long-chain polyunsaturated fatty acid levels in formulae influence deposition of docosahexaenoic acid and arachidonic acid in brain and red blood cells of artificially reared neonatal rats.

We studied the effects of dietary long-chain polyunsaturated fatty acids (PUFA) on the fatty acid composition of the brain and red blood cells in gastrostomized rat pups reared artificially from postnatal Days 5-18. These pups were fed rat milk substitutes in which the fat comprised 10% linoleic acid and 1% alpha-linolenic acid and, using a 3 x 3 factorial design, one of three levels of both arachidonic acid (AA) and docosahexaenoic acid (DHA) supplied as single cell microbial oils (0.0, 0.4 and 2.4% fatty acids). A tenth group was reared by nursing dams. The fatty acid composition of the phosphatidylethanolamine (PE) and phosphatidylserine/phosphatidylinositol (PS/PI) phospholipids in the brain and red blood cells on Day 18 reflected the dietary composition in that pups receiving long-chain supplementation of each had higher levels of the supplemented PUFA, but lower levels of the other, relative to unsupplemented groups. In contrast to these results, there were few changes in the brain in phosphatidylcholine (PC) phospholipids whereas, in the red blood cells, changes in PC were similar to those in PE and PS/PI. Regression analyses showed that DHA levels in the brain correlated more closely with those of the red blood cells than did AA levels. The results of this study indicate that, although supplementation of formula with AA or DHA during the period of rapid brain development in rats increases deposition of the long-chain PUFA in the developing tissues, each also affects the levels of the other.

Identification of Delta5-desaturase from Mortierella alpina by heterologous expression in Bakers' yeast and canola.

A DNA fragment with homology to Delta6-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 Delta6-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 Delta5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant Delta5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina Delta5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Delta5,9-18:2) and pinolenic acid (Delta5,9,12-18:3), which are the Delta5-desaturation products of oleic and linoleic acids, respectively.