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J. Biol. Chem., Vol. 278, Issue 37, 35115-35126, September 12, 2003
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¶
From the
Institut für Allgemeine Botanik,
Universität Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany and the
||Division of Immunochemistry, Research Center
Borstel, Parkallee 22, 23845, Borstel, Germany
Received for publication, June 6, 2003 , and in revised form, June 27, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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6-elongase and front-end desaturases from different organisms, we have
reconstituted in Saccharomyces cerevisiae the biosynthesis of
arachidonic acid from exogenously supplied linoleic acid in order to identify
these acyl carriers. Acyl-CoA measurements strongly suggest that the
elongation step involved in polyunsaturated fatty acids biosynthesis is taking
place within the acyl-CoA pool. In contrast, detailed analyses of lipids
revealed that the two desaturation steps (5 and 6) occur
predominantly at the sn-2 position of phosphatidylcholine when using
5- and 6-desaturases from lower plants, fungi, worms, and algae.
The specificity of these 6-desaturases for the fatty acid acylated at
this particular position as well as a limiting re-equilibration with the
acyl-CoA pool result in the accumulation of 
-linolenic acid at the
sn-2 position of phosphatidylcholine and prevent efficient
arachidonic acid biosynthesis in yeast. We confirm by using a similar
experimental approach that, in contrast, the human 6-desaturase uses
linoleoyl-CoA as substrate, which results in high efficiency of the subsequent
elongation step. In addition, we report that 12-desaturases have no
specificity toward the lipid polar headgroup or the sn-position. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5,8,11,14),
eicosapentaenoic acid (20:55,8,11,14,17), and
docosahexaenoic acid (22:64,7,10,13,16,19) are
important constituents of membranes (particularly in the retina and the
central nervous system) as well as precursors of several biologically active
eicosanoids (1). The presence
of VLC-PUFAs in the human diet affects diverse physiological processes
involved in cardiovascular, immune, neuronal, and visual functions
(2). Many clinical studies have
linked PUFA intake with normal health and development, particularly in the
case of newborns and infants
(3). VLC-PUFAs are mainly found
in fish, in some fungi and lower plants, as well as in a variety of
microorganisms of the phytoplankton. With the exception of the anaerobically
operating polyketide synthase-like systems found in some marine bacteria and
primitive eukaryotes (4),
VLC-PUFAs are synthesized by elongation and desaturation of linoleic acid (LA,
18:29,12)or 
-linolenic acid (ALA,
18:39,12,15) in the endoplasmic reticulum. Most
algae, fungi, and lower plants producing VLC-PUFAs possess the entire
biosynthetic pathway to synthesize these fatty acids from acetate, whereas
mammalia, which lack 12- and 15-desaturases, use as precursors
LA and ALA that have to be supplied in their diet and thus are essential fatty
acids.
The numerous health benefits attributed to VLC-PUFAs as well as the absence of sustainable and low cost sources has led to the long-term goal of producing such fatty acids in transgenic oilseed crops (5). Using organisms producing VLC-PUFAs such as the fungus Mortierella alpina, the moss Physcomitrella patens, the worm Caenorhaditis elegans, and the diatom Phaeo-dactylum tricornutum as gene sources, a large collection of sequences coding for elongases and desaturases was created in the last 10 years (reviewed in Ref. 6). Each coding sequence was separately expressed in yeast or plant and the substrate specificity of the encoded enzyme verified so that cDNAs encoding all the enzymatic activities required for DHA synthesis are available.
The fatty acid desaturases involved in VLC-PUFA biosynthesis can be divided
into two groups, the 
6-/3-desaturases and the so-called
front-end desaturases (7),
which contain a cytochrome b5-domain fused to their N
terminus (8). Whereas the
latter group of desaturases inserts the new double bond between the fatty acid
carboxyl group and a pre-existing double bond, the
6-/3-desaturases insert it between a pre-existing double bond
and the fatty acid methyl end. Using alkenylether glycerolipids and tomato
cell cultures, it was unambiguously proven that plant 6- and
3-desaturases are acting on lipid-linked substrates
(9). In addition, biochemical
studies with plants and fungi strongly suggest that 6-desaturases are
acting on both positions (sn-1 and sn-2) of
phosphatidylcholine (PC), whereas 6-desaturases are confined to the
sn-2 position of PC
(10–12).
On the other hand, fatty acid desaturases from vertebrates are referred to as
acyl-CoA desaturases (13,
14).
In contrast to the fatty acid desaturases involved in VLC-PUFA
biosynthesis, the elongation activities remain to be biochemically
characterized. Elongation of PUFAs has been linked to ELO sequences. ELO-type
proteins were first characterized in yeast, where ELO1 is involved in the
elongation of medium-chain saturated and monounsaturated fatty acids, whereas
ELO2 and ELO3 catalyze the subsequent elongation yielding the
C24–26 saturated fatty acids present in sphingolipids
(15,
16). Related sequences were
then identified in M. alpina
(17), C. elegans
(18), man
(19), and P. patens
(20), and their involvement in
the elongation of PUFAs was demonstrated by expression in yeast. Nevertheless,
so far there is no unequivocal evidence that any polypeptide encoded by an ELO
sequence catalyzes the actual condensation reaction involved in fatty acid
elongation. Compared with the poorly described elongation of PUFAs, the
elongation of saturated and monounsaturated fatty acids has been extensively
characterized biochemically in both plant and rat liver microsomes. Each
C2 elongation is a four-step process
(condensation-reduction-dehydration-reduction), which involves four different
enzymes, most probably organized in a multifunctional complex. The
rate-limiting step of this process is the condensation of the acyl primer with
malonyl-CoA catalyzed by the 
-ketoacyl-CoA synthase (KCS or FAE for
fatty acid elongation), which also confers substrate specificity to the whole
elongase complex. Expression of the KCS/FAE condensing enzyme or of an
ELO-type protein alone is sufficient to restore elongation activity in yeast,
suggesting that the three other enzymes are ubiquitously expressed. Recently,
using an Arabidopsis thaliana KCS/FAE condensing enzyme purified
nearly to homogeneity, Ghanevati and Jaworski
(21) measured high in
vitro activities with various acyl-CoAs, indicating that acyl-CoAs are
most probably the substrates of this type of elongase. Since the elongation
process condenses an acyl-CoA primer with malonyl-CoA and finally results in
the production of a C2-elongated acyl-CoA, the entire elongation process most
probably takes place within the acyl-CoA pool.
When the biosynthesis of VLC-PUFAs was reconstituted in yeast by
co-expressing the 6-desaturase from Borago officinalis, the
6-elongase from C. elegans and the 5-desaturase from
M. alpina in the presence of LA or ALA, small but significant amounts
of ARA and EPA, respectively, were detected
(18). We obtained similar
results by co-expressing the 5- and 6-desaturases from P.
tricornutum together with the 6-elongase from P. patens
(22). Despite this success,
these reconstitution experiments were rather inefficient compared with the
situation in the genuine organisms and regarding the relatively high
activities measured with the separately expressed enzymes. A closer look at
the activities of the different enzymes expressed to reconstitute VLC-PUFAs
biosynthesis in yeast showed that the elongation of endogenously produced
6-fatty acids was less than half that observed with exogenously
supplied 6-fatty acids. These results suggest a great difference in the
availability of exogenously added or in situ produced 6-fatty
acids for elongation: in contrast to exogenously supplied fatty acids, those
produced endogenously by 6-desaturation may remain in a pool that is
not available for elongation, which consequently limits VLC-PUFAs
biosynthesis.
In the present work we sought to identify which acyl carriers could be used
as substrate by the different enzymes involved in the biosynthesis of ARA
reconstituted in S. cerevisiae. In view of the data presented above,
special attention was paid to phosphatidylcholine and the acyl-CoA pool. Using
cDNAs from various organisms, the four activities leading to the synthesis of
arachidonic acid from oleic acid (12-desaturase, 6-desaturase,
6-elongase, and 5-desaturase) were expressed in yeast separately
or in combination. After short or long incubation times, the fatty acid
profiles of various lipid pools were determined in order to evaluate which
acyl carriers are preferentially used by the different desaturases and the
elongase.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Construction of Vectors—The different fatty acid desaturases and yeast expression constructs used in this study are listed in Table I. Usually, complete open reading frames (ORF) were modified by PCR to create appropriate restriction sites adjacent to the start and stop codons. The amplified DNAs were cloned into the pGEM-T vector (Promega, Madison, WI) before being released and cloned into a yeast expression vector (pVT102-U, pYES2 or pESC-LEU) using the restriction sites inserted by PCR.
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Expression in S. cerevisiae—The S. cerevisiae strain C13ABYS86 (leu2, ura3, his, pra1, prb1, prc1, cps) (22) was used in all the expressions described in this study. Transformation, selection and growth of the transgenic yeast cells have already been described (23). When the cultures had reached an OD600 of about 0.2, expressions were induced by supplementing galactose (2%, w/v) and the appropriate fatty acids to a final concentration of 500 µM. All cultures were then grown for another 24 or 48 h at 20 or 30 °C, as indicated, and harvested by centrifugation. For short time pulses, cultures were grown for 24 h at 30 °C, reaching an OD600 of about 1.5, before the exogenous fatty acid was added. After 1 min, 2.2-ml aliquots were harvested and the cells sedimented by short centrifugation (20 s). After removal of the supernatant, the cell pellets were frozen in liquid nitrogen and stored at –80 °C until needed.
Lipid Analysis—Lipid analysis of transgenic yeast cells were made from 150-ml cultures grown for 24 h at 20 °C unless otherwise indicated. Cells were harvested by centrifugation, washed with 30 ml of 0.1 M NaHCO3 and the lipids were extracted on a shaker for 4 h with 15 ml of chloroform/methanol (1:1) and then for 20 h with 15 ml of chloroform/methanol (2:1). The resulting organic phase was extracted with 9 ml of 0.45% NaCl, dried with Na2SO4, and evaporated under vacuum. The residue was dissolved in 2 ml of chloroform and corresponded to the total lipid extract. The major lipid classes PC (phosphatidylcholine), PI+PS (phosphatidylinositol and -serine), PE (phosphatidylethanolamine) and NL (neutral lipids) were purified by thin layer chromatography using chloroform/methanol/acetic acid (65:35:8; v/v/v) as solvent mixture. The different spots were scraped off the plate and the lipids were extracted from silica by adding successively 400 µl of water, 2 ml of methanol, and 2 ml of chloroform and vigorously shaking. After adding 2 ml of 0.2 M H3PO4/1 M KCl, the organic phase was extracted and the resulting aqueous phase re-extracted with 2 ml of chloroform. Both organic phases were combined, dried with Na2SO4 and evaporated under argon. The residues dissolved in 2 ml of chloroform corresponded to the different lipid fractions. For quantification, an aliquot of the total lipid extract was resolved by thin layer chromatography using chloroform/methanol/acetic acid (65:35:8; v/v/v) as solvent mixture. After drying, the plate was dipped in 10% CuSO4 in 8% (v/v) phosphoric acid, dried at 100 °C and heated to 178 °C until the spots appeared (24). The different spots were then quantified by scanning densitometry using a CAMAG TLC Scanner 3 (CAMAG, Muttenz, Switzerland) as already described (25).
Positional Analysis—Positional analyses were conducted using the Rhizopus arrhizus lipase (26) from Sigma. Samples containing PC or PE in chloroform were dried under argon and resuspended in 1 ml of 0.03% Triton X-100, 2 mM CaCl2, 50 mM HEPES, pH 7.2 by sonication. 10,000 units of lipase were added and the digestions were conducted for 2 h at 37 °C. The aqueous phase was extracted with 2.5 ml of chloroform/methanol (2:1) and then 2 ml of chloroform. The resulting organic phase was dried under argon and separated by thin layer chromatography using chloroform/methanol/aqueous ammonia (65:25:0.7; v/v/v) as solvent mix. The spots corresponding to free fatty acids and lysophospholipids were scraped and directly transmethylated for gas-liquid chromatography analysis.
Total and Esterified Fatty Acid Analysis—For the analysis of total fatty acids, fatty acid methyl esters (FAMEs) were prepared from sedimented cell pellets or lipid extracts by direct transmethylation with 0.5 M sulfuric acid in methanol containing 2% (v/v) dimethoxypropane. After 1 h at 80 °C, 0.2 ml of 5 M NaCl were added and FAMEs were extracted with 1 ml of petroleum-ether. For the analysis of esterified fatty acids, 1.35 ml of toluene/methanol (1:2, v/v), and 0.5 ml of 0.5 M NaOCH3 in methanol were successively added to sedimented cell pellets. After homogenization, samples were shaken for 1 h at room temperature before adding 2 ml of petroleum-ether and 0.4 ml of 5 M NaCl and extracting FAMEs. FAMEs were then analyzed by gas-liquid chromatography as previously described (23).
Acyl-CoA Analysis—For acyl-CoA analysis, the highly sensitive method relying on the fluorescence of etheno derivatives developed by Larson and Graham for plant tissues (27) was used. Frozen yeast cell pellets equivalent to 2.2 ml of yeast culture (OD600 of 1.5) were used as starting material and the extraction of acyl-CoAs was performed exactly as described (27). After drying the extracted acyl-CoAs under argon at 50 °C, derivatization was carried out in 300 µl of chloracetaldehyde derivatization reagent (27) at 85 °C for 20 min. HPLC analysis was made using a Thermoquest HPLC system (Thermoquest, Egels-bach, Germany) equipped with a LUNA 150 x 2.0 mm column with phenylhexyl-coated 5 µm silica particles (Phenomenex, Torrance, CA) under the same conditions as described by Larson et al. (28).
Acyl-CoA were identified using saturated and mono-unsaturated acyl-CoAs
from Sigma or enzymatically synthesized polyunsaturated acyl-CoAs
(18:29,12-,
18:36,9,12-,
20:211,14-,
20:38,11,14-, and
20:45,8,11,14-CoA) as standards. Synthesis was
achieved in 200 µl reaction mixture containing 100 mM Tris-HCl,
pH 8.1, 10 mM MgCl2, 5 mM CoASH, 2
mM dithiothreitol, 25 µM free fatty acid, and 2.5
units of acyl-coenzyme A synthetase from Pseudomanas sp. (Sigma).
After2hof incubation at 37 °C, the reaction was stopped with 50 µl of
glacial acetic acid/ethanol 1:1 (v/v), and the samples were washed with
petroleum ether to extract unreacted fatty acids. Acyl-CoAs were purified on a
short reverse phase column (Strata C18-E, Phenomenex, Torrance, CA) using
acetonitrile as eluent, dried under argon, and dissolved in 50 mM
MES, pH 5.0.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Elongation of GLA Takes Place in the Acyl-CoA Pool—We then
used the same approach with a yeast expressing an ELO-type elongase in order
to see whether the elongation of GLA takes place within the acyl-CoA pool. For
this experiment, a yeast culture transformed with a construct carrying the
gene of the 6-elongase from P. patens was grown in the
presence of galactose for 24 h before adding GLA so that the elongase was
present within the cells before supplying its substrate. The fatty acid
composition of the three different fractions defined above were then
determined before and 1 min after the addition of GLA. Before the pulse, the
fatty acid profiles were similar to those reported in
Fig. 1A (data not
shown). After 1 min in the presence of GLA
(Fig. 1C), GLA
represented about 40% of the total, but only 2.5% of the esterified fatty
acids, confirming the data obtained with LA. In these two fractions, the
elongation product of GLA, i.e.
20:38,11,14, could not be detected. On the other
hand, both GLA and 20:38,11,14 were present in
high proportions in the acyl-CoA pool. Besides the predominant 16:1, each
represented more than 25% of the acyl-CoA species only 1 min after the
addition of GLA, reflecting an elongation of about 50% in that pool. These
data strongly suggest that upon entrance into yeast cells exogenously supplied
GLA is converted into GLA-CoA and thus becomes immediately available for
6-elongation.
Next we analyzed the distribution of GLA and
20:38,11,14 in the different lipids of a yeast
culture that had expressed the 6-elongase in the presence of GLA for 24
h. After extraction and separation of the major lipid classes, the fatty acid
pattern of PC, phosphatidylethanolamine (PE), the neutral lipids (NL
fraction), and a fraction comprising phosphatidylinositol and
phosphatidylserine (PI + PS fraction) were analyzed. In addition, the fatty
acid profiles of the sn-1 and sn-2 positions of PE and PC
were determined. In the total lipid extract, LA and GLA represented each about
20% of the total fatty acids, indicating that the elongase had converted 50%
of GLA (Fig. 2). In the various
lipid fractions and sn-positions, GLA and
20:38,11,14 were always found in roughly similar
proportions. Both fatty acids were present in equimolar proportions in PC and
in the PI + PS and NL fractions, while in PE, GLA was slightly more abundant
than 20:38,11,14. Despite a nearly constant GLA
to 20:38,11,14 ratio, the content of GLA and
20:38,11,14 in the various lipid fractions and
sn-positions clearly differed. Both fatty acids were enriched in PC
and the NL fraction (each about 23–24%), but significantly reduced in
both PE and the PI + PS fraction (each about 12%). Most importantly, the
proportions of GLA and 20:38,11,14 were twice as
high at the sn-2 position than at the sn-1 position in both
PC and PE. These data suggest that after 6-elongation in the acyl-CoA
pool, various acyltransferases transfer indiscriminately both GLA and
20:38,11,14 into the different lipids, and that
the sn-2 positions of PC and PE as well as the NL fraction represent
the major acceptors.
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Lipid Pools Involved in Arachidonic Acid Biosynthesis—If the
elongation step involved in ARA biosynthesis most probably takes place in the
acyl-CoA pool, the low elongation yields measured with endogenously produced
6-fatty acids suggest that the 6-desaturase responsible for
their synthesis operates within another lipid pool. To identify this pool, the
ARA biosynthetic pathway was reconstituted in yeast by co-expressing the
5- and 6-desaturases from P. tricornutum together with
the 6-elongase from P. patens in the presence of exogenously
supplied LA. After 48 h of expression, the fatty acid profiles of the total
lipid extract, the acyl-CoA pool and the putative desaturase substrate PC were
examined (Fig. 3). In the total
lipid extract (Fig.
3A), LA and GLA represented about 50 and 7% of the total
fatty acids, respectively, whereas the major C20-PUFA was
20:211,14 (about 3%), resulting from the
elongation of LA. Due to the very low elongation of endogenously formed GLA,
only traces of ARA were detected (0.4%). In the acyl-CoA pool
(Fig. 3B), the
endogenous 16:0, 16:1, 18:0, and 18:1 were abundant, but the exogenously
supplied LA still represented the major fatty acid specie. Its elongation
product, 20:211,14, was present in considerable
proportions, whereas both GLA and 20:38,11,14
were minor components. In the acyl-CoA pool, about 50 and 10% of GLA and LA
were elongated, respectively, which is similar to the elongation activities
measured with exogenously supplied fatty acids
(Fig. 2). In PC
(Fig. 3C), LA was
predominating, but the content of GLA was more than doubled in comparison to
the lipid extract. As the 5-desaturated products were also enriched
(Fig. 3C,
insert), these data suggest that the two desaturation steps involved
in the biosynthesis of ARA may take place in PC.
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5- and 6-Desaturases from P. tricornutum Are
Specific for the sn-2 Position of PC—In order to confirm that the
6-desaturation of LA occurs in PC, the main lipid fractions of the
transgenic yeast culture that had expressed the 6-desaturase in the
presence of LA were isolated and their fatty acid profiles determined
(Table II). In the
unfractionated lipid extract, LA and GLA represented 48.0 and 10.5% of the
total fatty acids, respectively, indicating that about 18% of LA had been
desaturated. The NL fraction had a fatty acid profile similar to that of the
lipid extract, but it contained slightly more LA and less GLA. In agreement
with the general fatty acid composition of yeast phospholipids
(29), the PI + PS fraction was
enriched in saturated fatty acids (16:0 and 18:0), whereas PE was enriched in
16:1 and poor in 18:0 (Table
II). Although these two fractions had proportions of LA similar to
that of the lipid extract, GLA was much less abundant, suggesting that none of
these phospholipids was a major site for 6-desaturation. In contrast,
GLA represented about 22% of the total fatty acids in PC, which is more than
two times the proportion in the lipid extract
(Table II).
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The positional analysis of PE and PC showed that the sn-1
positions of both phospholipids were enriched in 16:0, 16:1, and 18:0, whereas
the sn-2 positions were enriched in 18:1, LA and GLA
(Table II). In both
phospholipids, the LA plus GLA content accounted for about 38 and 78% of the
total fatty acids at sn-1 and sn-2 positions, respectively,
in line with the sn-2 position of these two phospholipids being a
major acyl acceptor. In PE, the GLA level was equally low at both positions
(2–6%), whereas in PC GLA represented 3.7% of the total fatty acids at
the sn-1 position, but as much as 46% at the sn-2 position.
In contrast to the nearly constant educt/product ratio in all fractions
reported for the elongase in Fig.
2, the data presented in Table
II demonstrate a significant variation of this ratio between the
different lipids and the two sn-positions. After 24 h of expression
in the presence of LA, the neutral lipid fraction represented about 50% of the
total lipids, while PC, PE and the PI + PS fraction accounted for 26, 15 and
8%, respectively (Table II).
Assuming that the neutral lipid fraction was equally composed of triglycerides
and sterol esters (30), these
data indicated that although the sn-2 position of PC represented only
13% of all acyl groups, it contained about half (56%) of the total GLA. This
strongly suggests that the sn-2 position of PC served as the major
site for 6-desaturation.
To demonstrate that GLA accumulation at the sn-2 position of PC
was not resulting from the selectivity of yeast acyltransferase activities but
from the 6-desaturase activity, a wild type yeast was supplemented with
a 3:1 mixture of LA/GLA and grown for 24 h before determining the fatty acid
composition of different lipid fractions (data not shown). After this
incubation, LA and GLA represented about 46 and 11%, respectively, of the
total fatty acids. These percentages mimic the fatty acid profile resulting
from the expression of a 6-desaturase. In PC and particularly at the
sn-2 position of PC, both LA and GLA were enriched. Nevertheless, the
LA/GLA ratio was about the same at the sn-2 position of PC, in intact
PC as well as in the total lipid extract. Accordingly, the enrichment of GLA
at the sn-2 position of PC compared with the lipid extract as
presented in Table II is due to
the fact that the 6-desaturase from P. tricornutum mainly uses
the acyl chain at the sn-2 position of PC as substrate, rather than
to lipid remodeling.
We then analyzed the lipid extract of a transgenic yeast producing ARA by
coexpressing the 6-desaturase, the 6-elongase and the
5-desaturase in the presence of LA. In this case, not only GLA but also
the two products of the 5-desaturase
(20:35,11,14 and ARA) were enriched in PC and
predominantly found at the sn-2 position of PC. Although the
substrates of the 5-desaturase 20:211,14
and 20:38,11,14 were present in low proportions
due to the poor activity of the 6-elongase with LA and the
inaccessibility of the endogenously synthesized GLA,
20:35,11,14 and ARA represented up to 1.2 and 1%,
respectively, of the total fatty acids at the sn-2 position of PC
(data not shown). In contrast, no 5-desaturated fatty acids could be
detected in PE, in the PI + PS fraction and only trace amounts were present at
the sn-1 position of PC. When the desaturase activities were
calculated as (product x 100)/(educt + product) using values
corresponding to percent of total fatty acids, 5-desaturation was
rather low at the sn-1 position of PC, but very high at the
sn-2 position, where 44 and 69% of
20:211,14 and
20:38,11,14, respectively, were desaturated
(Fig. 4). These analyses
clearly demonstrate that the activities of both the 5- and
6-desaturases from P. tricornutum are mainly confined to the
acyl substrate esterified at the sn-2 position of PC. In addition,
they suggest that the fatty acids modified by these desaturases stay at the
sn-2 position of PC because of a low level of lipid remodeling in
yeast. Since the 6-elongation may take place in the acyl-CoA pool, we
are forced to conclude that an inefficient release of GLA from the
sn-2 position of PC into the acyl-CoA pool may cause the poor
elongation activity measured with in situ produced GLA.
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Acyl Substrate Used by Front-end Desaturases from Other
Organisms—To see whether the conclusions drawn above can be
extended to front-end desaturases from organisms other than algae, we
expressed 5- and 6-desaturases from fungi, mosses, higher
plants, worms and mammals in yeast and looked at the desaturation in the
different lipid fractions, particularly in PC and at its sn-2
position. The different 6-desaturases were expressed in the presence of
LA, whereas 5-desaturases were co-expressed with the 6-elongase
from P. patens in the presence of GLA, as it results in high
incorporation of 20:38,11,14 into the
sn-2 position of PC (see Fig.
2). As an example, Fig.
5 presents the results obtained with the 5-desaturase from
the fungus M. alpina. After 24 h, about 50% of GLA had been elongated
and 28% of the resulting 20:38,11,14 had been
desaturated to ARA, which represented as much as 5% of the total fatty acids
in the lipid extract (Fig.
5A). The proportion of ARA was more than doubled in PC
(12% of the total fatty acids; Fig.
5B), whereas it was lower in all the other individually
analyzed lipid fractions (not shown). On the other hand, ARA accounted for
less than 4% of the total fatty acids at the sn-1 position of PC
(Fig. 5C), but as much
as 25% at the sn-2 position (Fig.
5D). The desaturation of
20:38,11,14 at the sn-2 position of PC
was so efficient (82% conversion) that hardly any substrate for the
5-desaturase was left at this position after 24 h. As clearly shown in
Fig. 5, the enzyme from M.
alpina resulted in two and three times higher 5-desaturations in
PC and at its sn-2 position, respectively, than in the lipid extract.
These results are similar to those obtained with the 6-desaturase from
P. tricornutum, where the activity was also two and three times
better in PC and at its sn-2 position, respectively, than in the
lipid extract (Table II).
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The results obtained with 5- and 6-desaturases from all the
organisms tested are summarized in Fig.
6. The 5-desaturases from the moss P. patens, the
fungus Phytophthora megaspema, the worm C. elegans, and the
diatom P. tricornutum gave results similar to those presented in
Fig. 5 for the enzyme from
M. alpina. Although these desaturases differed in their level of
activity in yeast, desaturation was always two and three times higher in PC
and at its sn-2 position, respectively, than in the lipid extract.
The same correlation was found for the 6-desaturases from the mosses
P. patens and C. purpureus, the fungus M. alpina
and the alga P. tricornutum, suggesting that the 5- and
6-desaturases from fungi, lower plants, and diatoms are specific for
the sn-2 position of PC. In contrast to theses enzymes, the
expression of the 6-desaturases from man and the higher plant B.
officinalis led to different patterns. Desaturation was not higher in PC
than in the lipid extract. The proportions of GLA were about the same in the
lipid extract, PC and at its sn-2 position after expression of the
human 6-desaturase, whereas expression of the 6-desaturase from
B. officinalis resulted in about 50% higher GLA proportions at the
sn-2 position of PC as compared with PC or the lipid extract.
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Mammalian desaturases like the human 6-desaturase have been
biochemically characterized as being acyl-CoA desaturases
(13,
14). In order to confirm this
acyl-substrate specificity, the human 6-desaturase (FADS2) and for
comparison the 6-desaturase of P. tricornutum (PtD6p) were
expressed in yeast in the presence of LA, and the fatty acids profiles of the
lipid extract, the sn-2 position of PC and the acyl-CoA pool were
determined (Fig. 7). After
expression of the algal 6-desaturase, GLA as well as the
"side-products" 16:26,9 and
18:26,9 were highly enriched at the sn-2
position of PC, but GLA was barely detectable in the acyl-CoA pool. In
contrast, after expression of FADS2, GLA was clearly visible in the acyl-CoA
pool, but present in similar proportions in both the total fatty acids and at
the sn-2 position of PC. This strongly suggests that the human
6-desaturase uses CoA-thioesters as acyl substrates. Such acyl
substrate specificity could be responsible for the even distribution of both
substrate and product of the desaturase shown in Figs.
6 and
7, similar to the situation
reported for the elongase in Fig.
2. Further confirmation that the human 6-desaturase uses
acyl-CoAs as substrates was obtained by its coexpression with the
6-elongase from P. patens
(Fig. 8). When the human
6-desaturase was expressed in the presence of LA,
18:29,12 and
18:36,9,12 represented 49.5 and 7.5% of the total
fatty acids (Fig. 8A).
When the 6-elongase was expressed in addition
(Fig. 8B), nearly all GLA (93%)
was elongated to 20:38,11,14. This result
suggests that both enzymes use substrates from the acyl-CoA pool and that the
retention of the different intermediates within this pool led to a highly
efficient cooperation between the acyl-CoA-desaturase and elongase.
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|
Acyl Substrate Used by 12/6-Fatty Acid
Desaturase—As mentioned above, incubations of tomato cell cultures
with alkenylether glycerolipids resulted in 12-desaturated products
found at both position of PC and monogalactosyldiacylglycerol
(9). Therefore,
12/6-desaturases may not be specific for the sn-2
position of PC or glycolipids, in contrast to the front-end desaturases from
fungi, diatoms, worms and lower plants. When we expressed the
12-desaturase from P. tricornutum
(31) for 24 h in the presence
of LA, all the glycerolipid fractions were similarly desaturated (data not
shown). In order to get a closer look at the substrate specificity of
12/6-desaturase, a transgenic yeast producing
diglucosyldiacylglycerol (DGD) by expressing the processive
glucosyltransferase from Staphylococcus aureus
(32) was transformed with a
vector containing the gene coding for the 12-desaturase from sunflower
(Helianthus annuus L.). After 72 h of expression, the different lipid
fractions were isolated to determine the conversion of
16:19 and 18:19 to
16:29,12 and LA, respectively. As clearly shown
in Fig. 9, the
12-desaturase from sunflower was not specific for any lipid. In
contrast, all the lipids and both positions of PC, PE and DGD displayed
similar levels of desaturation for both 16:19 and
18:19. Since similar results were also obtained
with the 12-desaturase from A. thaliana (data not shown), we
conclude that plant 12/6-desaturases use lipid-linked acyl
chains as substrates and are most probably not specific for the polar head
group of the lipid and the sn-position of the acyl group on the
glycerol backbone.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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12-, 6-, 5-desaturase and
6-elongase) involved in VLC-PUFAs biosynthesis. Using the model
organism S. cerevisiae as heterologous expression system, we have
expressed the genes coding for the activities responsible for the conversion
of oleic to arachidonic acid and analyzed the fatty acid composition of
different lipid fractions and the acyl-CoA pool after short and long
incubation times. We made use of the fact that feeding of exogenous fatty
acids results in immediate labeling of the acyl-CoA pool, since uptake is
coupled to acyl-thioester formation. These experiments allowed the
identification of the acyl carriers used by both the desaturases and the
elongase involved in ARA biosynthesis.
Transport of exogenous long-chain fatty acids into S. cerevisiae
was shown to rely on the activities of the fatty acid transport protein Fat1p
and long-chain fatty acyl-CoA synthetases (primarily Faa1p)
(33,
34).
Figs. 1 shows that an
exogenously supplied fatty acid (LA or GLA) represents the dominating acyl
group in the acyl-CoA pool already only 1 min after addition to the medium.
These data demonstrate for the first time the efficiency of labeling the
acyl-CoA pool by exogenous fatty acids, regarding both timing and extent.
Wagner and Paltauf (29) have
shown that exogenous, radiolabeled 16:0 and 18:1 fatty acids were
predominantly incorporated into the phospholipids after 2 min of incubation
and that most of the label in PC and PE was found at the sn-2
position. Interestingly, the exogenously supplied fatty acid and its
downstream product were still highest at the sn-2 position of PC and
PE after 24 h of incubation (Table
II and Fig. 2). In
addition, the fact that the substrate to product ratio was similar in all the
different lipid fractions and positions when expressing enzymes using
acyl-CoAs as substrates (the 6-elongase in
Fig. 2 or the human
6-desaturase in Fig. 6) suggests that the fatty acids present within the acyl-CoA pool are transferred
indiscriminately into the different lipids by various acyltransferases. The
extent to which the different phospholipids are acylated appears to depend
mainly on their metabolic involvement in yeast.
The data presented in Fig.
1C show that only 1 min after addition of GLA to the
culture medium, this fatty acid as well as its elongation product
20:38,11,14 account in nearly equal proportions
for more than 50% of the acyl species of the acyl-CoA pool. In contrast, GLA
represents only a minor peak in the esterified fatty acid profile, whereas
20:38,11,14 is not detected at all. In view of
the size of the different pools of acyl groups, i.e. acyl-CoA
thioesters versus lipid-bound oxygen esters, this result is not
surprising. Quantitative acyl-CoA measurements with yeast have shown that
lipid-bound acyl groups exceed acyl-CoA thioesters by a factor of about 2000
(35). With the method used in
the present study and in the presence of 500 µM exogenous fatty
acid, we found even larger factors (data not shown). Throughout the present
study, the activity of the elongase deduced from educt/product ratios in the
acyl-CoA pool always reflected the total activity measured with exogenously
supplied fatty acids (about 10 and 50% conversion of LA and GLA,
respectively). These data cannot prove that the ELO-enzyme from P.
patens catalyzes the condensation reaction, but they strongly suggest
that the elongation of GLA takes place within the acyl-CoA pool. It seems
accordingly that similar to the situation encountered in the elongation of
monounsaturated fatty acids catalyzed by KCS/FAE condensing enzymes
(21), the elongation steps
involved in VLC-PUFAs biosynthesis most probably utilize acyl-CoAs as
substrates and produce acyl-CoAs as products.
Our experimental procedures also allowed the identification and/or
confirmation of the different acyl carriers used as substrate by the various
desaturases involved in VLC-PUFAs biosynthesis. Biochemical studies have shown
that the first desaturase, the 12-desaturase responsible for the
synthesis of LA from oleic acid, acts on both sn-positions of PC
(10,
11,
36). The data presented in
Fig. 9 confirm these results,
but also show that the activity of 12-desaturases is not restricted to
PC. Since it was unambiguously proven that such enzymes can act on
lipid-linked substrates (9),
and because of the low level of lipid remodeling in yeast observed in this
study, we are forced to conclude that 12-desaturases are not specific
for any polar head group and display activity on both sn-positions of
all glycerolipids. In contrast to this very wide acyl carrier specificity, the
6- and 5-desaturases involved in VLC-PUFAs biosynthesis were
shown to be very specific for the acyl chain esterified at the sn-2
position of PC in most cases (Fig.
6). Front-end desaturases from algae, fungi, lower plants, and
worms were mainly active on this particular position, which resulted in the
accumulation of the desaturation products at the sn-2 position of PC
(Table II). The results
obtained with the human 6-desaturase FADS2 confirm that the front-end
desaturases from mammalia in contrast most probably use CoA-thioesters as acyl
carriers. Whereas the desaturases discussed above resulted in significant
variation of the educt/product ratio between the different lipids and the
sn-positions of PC (Table
II and Fig. 6), the
educt/product ratio resulting from the expression of the human
6-desaturase was roughly constant in the different lipids and
sn-positions. This situation reflected the pattern resulting from the
expression of the acyl-CoA elongase (Fig.
2). Although we carried out several experiments with the human
6-desaturase, we could not detect desaturation products in the acyl-CoA
pool 1 min after adding the fatty acid substrate to the medium. This could
either be due to the existence of different pools of CoA-thioesters or to
different localizations of the desaturases and elongases expressed in yeast.
Nevertheless, coexpression with the elongase strongly suggests that mammalian
front-end desaturases use CoA-thioesters as substrate. Finally, the results
presented in Fig. 6 indicate
that the 6-desaturase from B. officinalis may have yet another
acyl carrier specificity. The rather low elongation of GLA obtained upon
coexpression with the C. elegans 6-elongase suggests that this
desaturase also uses lipid-linked substrates
(18). In microsomes prepared
from B. officinalis seeds, GLA was found in both PC and PE, but
in vitro assays with [14C]-18:1 have shown that the
sn-2 position of PC was the preferred site for desaturation
(10,
11). When we expressed the
B. officinalis 6-desaturase in yeast in the presence of LA,
this desaturase was not specific for PC, since significant proportions of GLA
were also found in PE and in the PI + PS and neutral lipid fractions, but in
both PC and PE, desaturated fatty acids were nearly exclusively found at the
sn-2 position (data not shown). The B. officinalis
6-desaturase appears to be highly specific for the sn-2
position, but its specificity toward the polar head of lipids differs from the
other front-end desaturases.
Although our experimental approach could differentiate all these
desaturases according to the acyl carriers used as substrate, we could not
demonstrate that the activity of any of these enzymes was absolutely
restricted to an unique acyl substrate. For example, the data presented in
Fig. 9 could be compatible, if
considered at their own, with the operation of the 12-desaturase with
acyl-CoA substrates. Similarly, the presence of minor proportions of
desaturated fatty acids at the sn-1 position of PC and in most of the
other lipids resulting from the expression of front-end desaturases specific
for the sn-2 position of PC raises the question as whether this was
due to lipid remodeling or to substrate unspecificity. On the other hand, the
distribution of the desaturated products varied considerably between the
different desaturases assayed, whereas lipid remodeling should be considered
as being always the same in the various expressions carried out with the same
yeast strain. Therefore, it seems more probable that these desaturases are
preferentially, but not absolutely specific for the sn-2 position of
PC. As shown in Table II for
the 6-desaturase from P. tricornutum, about 96% of the total
GLA was found at the sn-2 position of PC and in the neutral lipids,
which represented half of all lipids. These data indicate that after 24 h in
the presence of 500 µM LA, all the different pathways involved
in storage lipid biosynthesis in yeast were highly active and that lipid
remodeling mainly transferred GLA from PC into the neutral lipids. Three main
pathways responsible for storage lipid synthesis have been described in yeast
(37). TAG are mainly
synthesized from DAG and acyl-CoAs by the acyl-CoA:DAG-acyltransferase
(encoded by DGA1)
(38), whereas sterols ester
are exclusively made from acyl-CoAs and sterols by a set of two
acyl-CoA:sterol-acyltransferases (encoded by ARE1 and ARE2)
(39). The third route involves
the phospholipid:DAG-acyltransferase activity (encoded by LRO1),
which in yeast specifically transfers the acyl chain from the sn-2
position of PC to DAG, yielding TAG and sn-2-lyso-PC
(40). If we consider that the
low elongation of endogenously produced GLA indicates that GLA is present in
very low level in the acyl-CoA pool (see next), then most of the GLA made in
PC is transferred to the NL fraction without passing through the acyl-CoA
pool. Accordingly, the phospholipid:DAG-acyltransferase as well as activities
synthesizing DAG from PC (phospholipase C or D, cholinephospho-transferase)
(41) may account for most of
the GLA synthesized in PC, but found in the NL fraction.
Despite the fact that we could not demonstrate an absolute acyl carrier
specificity of any desaturase, our results clearly show the existence of
different groups of desaturases regarding the acyl carriers used as substrate.
Many indirect approaches have already led to a recognition and assignment of
these specificities, but our data represent an independent and more direct
approach since all the different enzymes, i.e. lipid- or CoA-linked,
have been expressed in the same system for direct comparison and the analyses
included both putative lipid substrates and acyl-CoAs. In a phylogenetic tree
with various desaturases (42),
the different regiospecificities (5, 6, 9, or 12)
define separate branches, although front-end desaturases with 4-,
5-, 6-, or 8-regiospecificity do not form clear-cut
groups so that the exact regiospecificity of an unknown frontend desaturase
must always be confirmed by heterologous expression. In such phylogenetic
trees, the B. officinalis 6-desaturase groups together with
the sphingolipid long-chain base 8-desaturases, rather than with the
other front-end desaturases. This was interpreted as indicating that the
6-desaturases from higher plants have arisen by gene duplication from
sphingolipid 8-desaturases
(42). This particular
phylogenetic origin may in turn explain that the 6-desaturases from
higher plants have an acyl carrier specificity different from that of the
other front-end desaturases, as clearly shown in the
Fig. 6. In addition, we would
like to point out that in phylogenetic alignments the front-end 5- and
6-desaturases from mammalia and fish always form a deeply separated
branch (42). On the basis of
the few presently known sequences, this grouping was attributed to the general
separation of vertebrates from the other organisms. On the other hand, a
criterion never considered as influencing these alignments is desaturase
similarity based on substrate preference, i.e. using acyl-CoAs or
lipid-linked acyl groups. A high priority of this difference could also result
in the separation of these desaturases, from which at least the mammalian ones
use acyl-CoA substrates. Furthermore, as there is desaturase bifunctionality
with regard to reaction outcome (for example desaturation and hydroxylation),
stereochemistry (cis and trans double bonds) and regiochemistry (5- and
6-desaturation), there may also be bifunctionality with regard to the
acyl group position (sn-1 or sn-2), the lipid headgroup or
CoA-versus lipid-linked substrate. At present it is not possible to
recognize these alternatives, which could interfere with the interpretation of
our data, from the amino acid sequences of the various desaturases.
The involvement of different acyl carriers as demonstrated in this study
explains the poor yields obtained when reconstituting ARA biosynthesis in
yeast (18,
22). The 6-desaturase
from P. tricornutum mainly uses the LA in the sn-2 position
of PC, while the 6-elongase from P. patens requires GLA in the
acyl-CoA pool. Therefore, an inefficient transfer of the 6-desaturated
products from PC into the acyl-CoA pool most probably represents a bottleneck.
As shown in Fig. 3B,
S. cerevisiae appears to possess some enzyme(s) capable of releasing
GLA from the sn-2 position of PC. Several activities can be
responsible for this transfer in yeast, such as phospholipase A2-mediated
deacylation followed by resynthesis of acyl-CoA as well as the reverse
activity of the acyl-CoA:lysophosphatidyl-choline acyltransferase. This latter
enzyme has been described in yeast, but not studied in the presence of PUFAs
(43). Using microsomal
preparations from developing safflower cotyledons and rat liver, it was shown
that the reverse reaction catalyzed by the acyl-CoA:lysophosphatidylcholine
acyltransferase represented less than 5% of the forward reaction
(44). In our study, fatty
acids exogenously supplied or endogenously produced in the acyl-CoA pool were
found enriched in PC and in particular at its sn-2 position. Wagner
and Paltauf (29) also found
most of the exogenously supplied, labeled fatty acids at this particular
position even after 2 min, suggesting that transfer from the acyl-CoA pool
into the sn-2 position of PC is very efficient in yeast. As is
clearly shown in Fig. 7 the
reverse reaction is significantly lower and results in the accumulation of GLA
at the sn-2 position of PC, which leads to low elongation activity.
These data suggest that a bottleneck, which limits VLC-PUFA production in
yeast, may be an insufficient supply of acyl-CoA substrates to the
elongase.
Recently, we obtained the first transgenic linseed producing ARA or EPA by
expressing the 5- and 6-desaturases from P. tricornutum
together with the 6-elongase from P. patens in their
seeds.2 Similar to the
results obtained in yeast, 6-desaturated fatty acids were present in
high proportion in total fatty acids, but nearly absent from the acyl-CoA
pool, suggesting that the same problem exists in the seeds of higher plants.
In order to produce high levels of VLC-PUFAs in oil seed crops, particularly
docosahexaenoic acid (22:64,7,10,13,16,19) whose
synthesis requires another elongation step after the lipid-linked
5-desaturation, this bottleneck has to be circumvented.
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FOOTNOTES |
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