Eur. J. Biochem. 263, 189±194 (1999) q FEBS 1999
`
`Biosynthesis of 1,2-dieicosapentaenoyl-sn-glycero-3-phosphocholine in
`Caenorhabditis elegans
`
`Tamotsu Tanaka1, Shinya Izuwa2, Katsuhisa Tanaka1, Daisuke Yamamoto1, Tatsunori Takimoto1, Fumito Matsuura2
`and Kiyoshi Satouchi1
`
`1Department of Food Science and Technology and 2Department of Biotechnology, Fukuyama University, Japan
`
`Previously, we showed that lowering the growth temperature increased the level of eicosapentaenoic acid (EPA)
`in the phosphatidylcholine (PtdCho) of Caenorhabditis elegans. In this study, we investigated the molecular
`species composition of PtdCho of C. elegans, with an emphasis on EPA-containing species. C. elegans contained
`a substantial amount of 1,2-dipolyunsaturated fatty acid-containing PtdCho (1,2-diPUFA-PtdCho) species, such
`as arachidonic acid/EPA and EPA/EPA, which are unusual phospholipids in higher animals. The EPA/EPA-
`PtdCho content was significantly increased in C. elegans grown at a low temperature. To examine the
`possibility that the acyltransferase activity involved in the remodeling of phospholipids accounts for the
`production of 1,2-diPUFA-PtdCho, we investigated the substrate specificity of this enzyme in C. elegans
`and found that it did not exhibit a preference for saturated fatty acid for acylation to the sn-1 position
`of PtdCho. The efficacy of the esterification of EPA to the sn-1 position was almost equal to that of stearic acid.
`The lack of preference for a saturated fatty acid for acylation to the sn-1 position of PtdCho is thought to result in
`the existence of the unusual 1,2-diEPA-PtdCho in C. elegans.
`
`Keywords: acyltransferase; Caenorhabditis elegans; eicosapentaenoic acid; phosphatidylcholine; nematode.
`
`The free-living nematode Caenorhabditis elegans is the first
`multicellular animal to have its genome sequenced [1].
`Previously, we investigated the fatty acid composition of
`C. elegans and found that the nematode contains abundant
`eicosapentaenoic acid (EPA) especially in phosphatidylcholine
`(PtdCho) fraction [2]. Our recent investigation [3] showed that
`C. elegans has the ability to synthesize polyunsaturated fatty
`acids (PUFAs) de novo, as do the other nematode species
`Turbatrix aceti [4] and Steinernema carpocapsae [5]. In this
`regard, three C. elegans genes encoding fatty acid desaturases
`have been cloned [6±8]. We also showed that growth
`temperature affected the fatty acid composition of C. elegans.
`A reduction in growth temperature from 25 8C to 15 8C caused
`the proportion of EPA to increase from 23.6% to 32.5% in the
`PtdCho fraction. Conversely, the levels of dihomo-g-linolenic
`acid and arachidonic acid in the PtdCho fraction were reduced
`at the low growth temperature [3]. On the other hand, in the
`phosphatidylethanolamine (PtdEtn) fraction, the diacyl subclass
`of PtdEtn was markedly increased at low growth temperature [3].
`In the present study, we investigated the effect of growth
`temperature on the molecular species composition of PtdCho
`of C. elegans with an emphasis on EPA-containing species.
`The results showed that the proportion of 1,2-dieicosapenta-
`enoyl-sn-glycero-3-phosphocholine (1,2-diEPA-PtdCho), which
`
`Correspondence to T. Tanaka, Department of Food Science and
`Technology, Fukuyama University, Fukuyama 729-0292, Japan.
`Fax: + 81 849 36 2459, Tel.: + 81 849 36 2111, ext. 4051,
`E-mail: tamot@fubac.fukuyama-u.ac.jp
`Abbreviations: AgTLC, argentation thin-layer chromatography; EPA,
`eicosapentaenoic acid; PtdCho, phosphatidylcholine; PtdEtn,
`phosphatidylethanolamine; PUFA, polyunsaturated fatty acid.
`Enzymes: acylCoA, lysophospholipid acyltransferase; (EC 2.3.1.23 and
`EC 2.3.1.62); acylCoA synthetase, (EC 6.2.1.10); phospholipase A2,
`(EC. 3.1.1.4); phospholipase C, (EC 3.1.4.3).
`(Received 1 December 1998, revised 17 March 1999, accepted
`16 April 1999)
`
`in higher animals, was
`is an unusual PtdCho species
`significantly increased at a low growth temperature. We also
`investigated the substrate specificity of acylCoA : lysophos-
`pholipid acyltransferase activity of C. elegans. By comparison
`with acyltransferase of rat liver, we discuss the possibility that
`the lack of a preference for saturated fatty acid for acylation to
`the sn-1 of PtdCho underlies the existence of 1,2-diEPA-
`PtdCho in C. elegans.
`
`M A T E R I A L S A N D M E T H O D S
`
`Chemicals
`
`Agar, peptone, tryptone and yeast extract were obtained from
`Difco Laboratories (Detroit, MI, USA). Glycerol and precoated
`thin-layer plates (Silica gel 60) were from Merck (Darmstadt,
`Germany). Phospholipase A2 (EC 3.1.1.4, from Crotalus ada-
`manteus venom), phospholipase C (EC 3.1.4.3, from Bacillus
`cereus) ATP, CoA and essentially fatty acid-free BSA were
`from Sigma Chemical Co. (St Louis, MO, USA). EPA was from
`Funakoshi Co. (Tokyo, Japan). Linoleic acid was from Doosan
`Serdary Research Laboratories (London, ON, Canada). Sep-Pak
`C18 cartridges were the product of Waters Associates (Milford,
`MA, USA). [1-14C]EPA and [1-14C]stearic acid were from
`NEN Life Science Products (Boston, MA, USA). [1-14C]Lino-
`leic acid was from Amersham Life Science (Arlington Heights,
`IL, USA). All other chemicals were analytical grade.
`
`Growth and isolation of C. elegans
`
`The standard wild-type strain N2 of C. elegans was grown at
`15 8C, 20 8C or 25 8C on 90-mm nematode-growing-medium
`agar plates seeded with Esherichia coli (OP50 strain) [2]. When
`microscopic inspection showed the plates were rich with adult
`worms, they were washed off with M9 buffer (22 mm KH2PO4,
`42 mm Na2HPO4, 86 mm NaCl, 1 mm MgSO4), purified by
`sucrose flotation, and rinsed as described previously [3]. Worms
`
`000001
`
`

`
`190 T. Tanaka et al. (Eur. J. Biochem. 263)
`
`q FEBS 1999
`
`were then subjected to lipid extraction or french press for enzyme
`assay.
`
`eicosapentaenoyl chloride was prepared using EPA and oxalyl
`chloride [12].
`
`Molecular species analysis of PtdCho
`
`Preparation of lyso-phospholipid
`
`Purified live C. elegans were homogenized by grinding in a
`mortar with a pestle in a small amount of chloroform/methanol
`mixture (1 : 2, v/v). Total
`lipids were extracted from the
`homogenate according to the method of Bligh and Dyer [9].
`The PtdCho and PtdEtn fractions of C. elegans were prepared
`by thin-layer chromatography (TLC) using the solvent chloro-
`form/methanol/H2O (65 : 35 : 6, v/v) (solvent system A) [2].
`Detection was done with 0.01% primuline (in acetone/water,
`4 : 1, v/v) under ultraviolet
`light. The PtdCho fraction
`(1 mmol) from C. elegans was subjected to phospholipase C
`treatment. As a large portion of PtdCho of C. elegans is
`accounted for by the diacyl subclass (96.4%) [2], almost all of
`the resulting glyceride is diacylglycerol. The diacylglycerol
`was converted to the monoacetyldiacylglyceride derivative or
`dinitrobenzoyl derivative [10]. For acetylation, diacylglycerol
`dissolved in dehydrated pyridine (0.5 mL) was mixed with
`1 mL of acetic anhydride at 30 8C. After 15 h, 2.5 mL of
`chloroform/methanol (1 : 2, v/v) and 1.5 mL of water was
`added to the solution. The monoacetyldiglyceride was recov-
`ered from the chloroform phase and subjected to argentation
`TLC (AgTLC) on chromatoplates prepared as described
`previously [11]. The developing system of AgTLC was
`benzene/chloroform/methanol (80 : 20 : 10, v/v), and detection
`was performed with 0.2% 2,7-dichlorofluorescein (in ethanol)
`under ultraviolet light. Monoacetyldiglyceride was recovered
`from each of the zones of silica gel by the method of Bligh and
`Dyer [9] after spotting the authentic monoacetyldiglyceride
`(20 : 0/20 : 0) (10 mg as fatty acid) in each zone of silica gel.
`The recovered monoacetyldiglyceride was then dissolved in 5%
`methanolic HCl solution for methanolysis [3]. The fatty acid
`methyl esters derived from monoacetyldiglyceride were
`determined by gas chromatography (GC) using 20 : 0 as the
`internal standard as described below.
`
`Positional distribution of fatty acid in the PtdCho and PtdEtn
`fraction
`
`The PtdCho or PtdEtn of C. elegans was hydrolyzed with
`phospholipase A2, and the resulting free fatty acid and
`lysophospholipid fractions were isolated by TLC [3]. After
`methanolysis of each fraction, the fatty acid composition was
`determined by GC.
`
`GC and HPLC
`
`Fatty acid methyl esters were analyzed by GC (Shimadzu
`GC-14 A, Kyoto, Japan) equipped with a capillary column
`coated with a 0.25-mm film of polar CBP 20 (0.22 (cid:2) 50 m;
`Shimadzu). The temperature of both the injector and the flame
`ionization detector was 250 8C. The initial column temperature
`was set at 170 8C and then raised to 225 8C at 58 C´min21.
`The dinitrobenzoyl derivative of diacylglycerol was analyzed
`by high-performance liquid chromatography (HPLC) (Tosoh
`CCPD, Tokyo, Japan) equipped with a 0.45 (cid:2) 15-cm TSK-gel
`ODS-80 TM column (Tosoh) using acetonitrile-isopropanol
`(90 : 10, v/v) as eluent. The flow rate was 0.7 mL´min21.
`Several EPA-containing PtdCho species were chemically
`synthesized as standard PtdCho for HPLC analysis. They
`were prepared by condensation of 1-acyl-2-lyso-PtdCho with
`eicosapentaenoyl chloride. 1-Acyl-2-lyso-PtdCho was obtained
`by hydrolysis of egg yolk PtdCho with phospholipase A2, and
`
`First, 1,2-diEPA-PtdCho was prepared by condensation of
`l-a-glycerophosphorylcholine with EPA anhydride [13] and
`purified by silicic acid chromatography and TLC with solvent
`system A. 1-EPA-2-lyso-PtdCho was prepared by hydrolysis of
`1,2-diEPA-PtdCho with phospholipase A2 [3], and purified by
`TLC with solvent system A. 1-Alkenyl(alkyl)-2-EPA-PtdCho
`was prepared by the condensation of 1-alkenyl(alkyl)-2-lyso-
`PtdCho with EPA anhydride [14]. 1-Alkenyl(alkyl)-2-EPA-
`PtdCho was subjected to mild acid hydrolysis and resulting
`1-lyso-2-EPA-PtdCho was purified by TLC with solvent
`system A. 1-Lyso-2-acyl-PtdEtn was obtained by acid
`hydrolysis of 1-alkenyl-2-acyl-PtdEtn of bovine heart, and
`1-acyl-2-lyso-PtdEtn was prepared from egg yolk PtdEtn with
`phospholipase A2.
`
`Determination of the acyl migration of 1-lyso-2-EPA-PtdCho
`
`It has been pointed out that the acyl group of the sn-2 position
`of lysophospholipid is liable to migrate to the sn-1 position to
`form 1-acyl-2-lyso-PtdCho [15]. Therefore, we checked the
`extent of the migration of EPA residue in 1-lyso-2-EPA-
`PtdCho. The lyso-PtdCho was acetylated, treated with phos-
`pholipase C and converted to tert-butyldimethylsilil derivative
`for determination of isomer ratio by GC [14]. During 24 h
`storage at 220 8C after preparation of 1-lyso-2-EPA-PtdCho,
`the
`formation of 1-EPA-2-lyso-PtdCho was about 10%.
`Lysophospholipids were used within 24 h. We also checked
`the extent of migration under our acyltransferase assay
`conditions. The migration of EPA residue from sn-2 to sn-1
`during the 10 min incubation was estimated to be 20% at pH 7
`at 20 8C. At higher pHs, the migration rate was pronounced
`(50% at pH 8). On the other hand, it was reduced at acidic pH
`(10% at pH 6), so the assay was conducted at a slightly acidic
`pH (pH 6.8) at 20 8C for 10 min
`
`Preparation of membrane fraction of C. elegans
`
`The purified C. elegans (2 g, wet weight) was suspended in
`8 mL of 50 mm potassium phosphate buffer (pH 7.0) contain-
`ing 1.5 mm glutathione, 0.15 m KCl, 1 mm EDTA, 1 mm
`dithiothreitol, 1 mm phenylmethanesulfonyl
`fluoride
`and
`0.25 m sucrose (homogenizing buffer), and subjected to the
`french press. Because the enzyme activity was impaired above
`200 kg´cm22 in preliminary experiments, the french press was
`conducted at a pressure lower than 200 kg´cm22. The C.
`elegans homogenate was centrifuged at 11 000 g for 30 min.
`The resulting supernatant was further centrifuged at 105 000 g
`for 60 min. The pellet was suspended in the homogenizing
`buffer (omitting EDTA, dithiothreitol and phenylmethanesul-
`fonyl fluoride) and immediately used for the enzyme assay
`described below. The protein content was estimated by the
`method of Lowry et al. [16] using BSA as a standard.
`
`Preparation of microsome fraction of rat liver
`
`Male Sprague±Dawley rats (250±300 g) were killed, and their
`livers were perfused with 0.9% NaCl to remove contaminating
`hemoglobin. The tissue was homogenized in 50 mm potassium
`(pH 7.0) containing 1.5 mm glutathione,
`phosphate buffer
`0.15 m KCl, 1 mm EDTA, and 0.25 m sucrose with a Potter±
`Elvehjem glass-Teflon homogenizer. The microsomal fraction
`
`000002
`
`

`
`q FEBS 1999
`
`Molecular species composition of PtdCho in C. elegans (Eur. J. Biochem. 263) 191
`
`Table 1. Typical results of molecular species analysis of PtdCho of
`C. elegans grown at 15 8C by AgTLC coupled with GC. PtdCho from
`C. elegans was converted to monoacetyldiacylglyceride derivative and
`fractionated by AgTLC. Fatty acids in each fractions were determined by
`GC using 20 : 0 as internal standard.
`
`Fraction
`No.
`
`Degree of
`unsaturation
`
`Fatty acid
`detected
`
`Possible
`molecular species
`
`(nmol)
`
`(nmol)
`
`1
`2
`
`3
`
`4
`
`5
`
`6
`
`10
`9
`
`8
`
`7
`
`6
`
`5
`
`was prepared by sequential centrifugation [17]. The final
`microsomal pellet was suspended in the homogenizing buffer
`(omitting EDTA) and immediately used for the enzyme assay.
`
`Acyltransferase assay
`
`lysoPtdCho or
`Each incubation consisted of 32 nmol of
`lysoPtdEtn, 0.5 mm nicotinamide, 1.5 mm glutathione, 0.15 m
`KCl, 5 mm MgCl2, 0.25 m sucrose, 7.5 mm ATP, 0.4 mm CoA,
`50 mm potassium phosphate buffer (pH 6.8), 0.8 mg protein of
`the membrane fraction of C. elegans, and the desired fatty acid
`in a total volume of 2.0 mL. The fatty acids were added as a
`fatty acid/albumin complex [18], and the specific activity was
`0.05 mCi/50 nmol. The incubation was conducted at 20 8C for
`10 min, and the reaction was stopped by mixing with 7.5 mL of
`chloroform/methanol (1 : 2, v/v). The lipids were extracted by
`the method of Bligh and Dyer [9], and PtdCho or PtdEtn was
`isolated by TLC with solvent system A. The isolated phos-
`pholipids were hydrolyzed with phospholipase A2, and the
`resulting lysophospholipids and free fatty acid were separated
`by TLC [14]. Each zone of silica gel was scraped off the plate
`and radioactivity was determined. The assay for acyltransferase
`activity of the rat liver microsomes was conducted similarly,
`except for the temperature of the incubation (37 8C), micro-
`somal protein (0.1 mg), and the total volume of the incubation
`(1 mL).
`
`AcylCoA synthetase assay
`
`The incubation was conducted in the same manner as the assay
`for acyltransferase except for the omission of lysophospholipid
`from the reaction mixture. After
`incubation, 10 mL of
`isopropanol/n-heptane/1 m sulfuric acid (40 : 10 : 1, v/v) was
`added. Then, 2 mL of water and 10 mL of n-heptane were
`added for phase separation. The lower phase was washed twice
`with 10 mL of n-heptane, and radioactivity of the lower phase
`was measured to determine acylCoA formation.
`
`R E S U L T S
`
`Effect of growth temperature on the EPA-containing
`molecular species composition of PtdCho
`
`The monoacetyldiglyceride derivative obtained from the
`PtdCho of C. elegans was separated on AgTLC by degree of
`unsaturation (total unsaturation 10±0). Typical results of fatty
`acid analyses of fractions 1 (total unsaturation 10) through 6
`(total unsaturation 5) are shown in Table 1. Because of the
`tailing of monoacetyldiglycerides on AgTLC plate, the streaked
`monoacetyldiglycerides contaminated to the next
`fraction.
`Therefore, the number of nmol of EPA detected as fatty acid
`was slightly higher than the sum of the number of nmol of
`counterpart fatty acids in each fraction except for fraction 1.
`The PtdCho of C. elegans contained substantial amounts of 1,2-
`diPUFA-PtdChos, such as 20 : 4(n-6)/20 : 5 and 20 : 5/20 : 5
`which are unusual phospholipids in higher animals. Among
`them, 20 : 5/20 : 5-PtdCho was significantly increased in
`C. elegans grown at 15 8C (Fig. 1).
`We also investigated the molecular species composition of
`PtdCho as dinitrobenzoyl derivative by HPLC. Many peaks
`were detected, and peaks corresponding to dinitrobenzoyl
`derivative of 18 : 0/20 : 5, 18 : 1/20 : 5, 20 : 4/20 : 5 and
`20 : 5/20 : 5 were identified by comparison with synthetic
`standards. The increase in 20 : 5/20 : 5-PtdCho species at low
`growth temperature was confirmed. The proportions of this
`
`20 : 5(n-3)/20 : 5(n-3) 82
`20 : 4(n-3)/20 : 5(n-3) 30
`20 : 4(n-6)/20 : 5(n-3) 23
`
`20 : 3(n-6)/20 : 5(n-3)
`20 : 4/20 : 4
`
`22
`
`18 : 2(n-6)/20 : 5(n-3)
`20 : 3(n-6)/20 : 4(n-3)
`
`32
`
`18 : 1(n-7)/20 : 5(n-3) 71
`18 : 1(n-9)/20 : 5(n-3)
`13
`18 : 2(n-6)/20 : 4(n-3)
`
`16 : 0/20 : 5(n-3)
`18 : 0/20 : 5(n-3)
`19 : D/20 : 5(n-3)
`17 : 0iso/20 : 5(n-3)
`16 : 0iso/20 : 5(n-3)
`17 : 0/20 : 5(n-3)
`17 : D/20 : 5(n-3)
`18 : 2(n-6)/20 : 3(n-6)
`18 : 1/20 : 4
`
`11
`76
`12
`13
`7
`5
`7
`
`20 : 5(n-3) 163
`20 : 5(n-3)
`54
`20 : 4(n-3)
`30
`20 : 4(n-6)
`23
`20 : 5(n-3)
`30
`20 : 4(n-3)
`10
`20 : 4(n-6)
`8
`20 : 3(n-6)
`22
`20 : 5(n-3)
`55
`20 : 4(n-3)
`20 : 3(n-6)
`32
`18 : 2(n-6)
`20 : 5(n-3) 104
`20 : 4(n-3)
`18 : 2(n-6)
`71
`18 : 1(n-7)
`13
`18 : 1(n-9)
`20 : 5(n-3) 167
`20 : 4(n-3)
`15
`20 : 4(n-6)
`5
`20 : 3(n-6)
`8
`18 : 2(n-6)
`7
`18 : 1(n-7)
`12
`18 : 1(n-9)
`5
`19 : Da
`12
`18 : 0
`76
`17 : Db
`17 : 0
`17 : 0iso
`16 : 0
`16 : 0iso
`
`6 3
`
`9 8
`
`7 5
`
`13
`11
`7
`
`a 19:D, cis-11, 12-methyleneoctadecanoic acid. b 17:D, cis-9, 10-methylene-
`hexadecanoic acid.
`
`molecular species of C. elegans grown at 25 8C and 15 8C were
`6.3 ^ 1.2% and 10.0 ^ 1.9%, respectively.
`
`Positional distribution of fatty acid in PtdCho and PtdEtn of
`C. elegans
`
`In C. elegans, PtdEtn had a preponderance of PUFAs at the sn-
`2 position (Table 2). In contrast, substantial amounts of PUFAs
`were found at the sn-1 position of PtdCho (Table 2). This
`indicates that the positional distribution of fatty acid in PtdCho
`molecule of C. elegans is not so strictly regulated as the phos-
`pholipids of higher animals, which show a strict PUFA-
`distribution toward the sn-2 position. Therefore, the mechanism
`that regulates the positional distribution of PUFA of PtdCho
`may be different from that of PtdEtn in C. elegans.
`
`Substrate specificity of acylCoA: lysophospholipid
`acyltransferase activity
`
`To examine the possibility that the acylCoA:lysophospholipid
`acyltransferase involved in the deacylation/reacylation of phos-
`pholipid accounts for the biosynthesis of 1,2-diEPA-PtdCho
`which increases at
`the lower growth temperature, we
`
`000003
`
`

`
`192 T. Tanaka et al. (Eur. J. Biochem. 263)
`
`q FEBS 1999
`
`Fig. 1. Effect of growth temperature on the
`proportion of EPA-containing PtdCho from
`C. elegans analyzed by AgTLC coupled with
`GC. The PtdCho from C. elegans grown at
`25 8C and 15 8C was converted to
`monoacetyldiacylglyceride derivative and
`fractionated by AgTLC. The fatty acid analysis
`of each fraction was conducted by GC. Values
`are percentage of total molecular species of
`PtdCho and means ^ SD (three harvests of
`nematodes grown at 15 8C and 25 8C,
`respectively). * Indicates significantly different
`from corresponding molecular species grown at
`25 8C using Student's t-test (P , 0.05).
`
`this enzyme of
`investigated the substrate specificity of
`C. elegans. We also compared the enzyme activity to that in
`rat liver. Because EPA is an abundant fatty acid at both sn-1 and
`sn-2 of PtdCho of C. elegans (Table 2),
`the acyl acceptor
`used in this study was 1-lyso-2-EPA-PtdCho or 1-EPA-2-
`lyso-PtdCho. When 1-lyso-2-EPA-PtdCho was used as the
`acyl acceptor, stearic acid and EPA were incorporated into
`the sn-1 position of PtdCho in a dose-dependent manner. The
`saturated level was around 2 nmol per 10 min´mg21 protein in
`both fatty acids (Fig. 2A, Table 3).
`In contrast,
`the level
`of acylation of 1-EPA-2-lyso-PtdCho with EPA was over 10
`times that with stearic acid at any concentration of the fatty acid
`(Fig. 2B). The substrate specificity of acyltransferase activity
`
`Table 2. Positional distribution of fatty acid in PtdCho and PtdEtn of
`C. elegans. PtdCho or PtdEtn of C. elegans grown at 20 8C was hydrolyzed
`with phospholipase A2, and fatty acid composition of resulting free fatty
`acid and lysophospholipid fractions was analyzed by GC. ND, not detected.
`
`PC (%)
`
`PE (%)
`
`of the C. elegans membrane fraction was compared with that of
`rat liver microsomes using fixed concentrations of stearic acid,
`linoleic acid and EPA. When the 1-EPA-2-lyso-PtdCho was
`used as acyl acceptor, the acyltransferase activity of rat liver
`microsomes acylated linoleic acid and EPA to the sn-2 position
`of PtdCho more effectively than stearic acid (Table 4).
`Likewise,
`the acyltransferase activity of
`the C. elegans
`membrane fraction preferred PUFA to stearic acid for acylation
`
`Table 3. Incorporation of various fatty acids into exogenously added
`lysoPtdCho and lysoPtdEtn in C. elegans membrane fraction. The
`incubation was conducted at 20 8C for 10 min with 0.8 mg protein of
`C. elegans membrane fraction and 50 nmol of fatty acid. The acyl acceptors
`used were 32 nmol (a)1-lyso-2-EPA-PtdCho, (b)1-EPA-2-lyso-PtdCho, (c)
`1-lyso-2-acyl-PtdEtn, and (d)1-acyl-2-lyso-PtdEtn. Values are means ^ SD
`(three harvests of nematodes grown at 20 8C).
`
`Acylation (nmol´10 min21´mg21 protein)
`
`To lysoPtdCho
`
`To lysoPtdEtn
`
`Fatty acid
`
`sn-1
`
`sn-2
`
`sn-1
`
`sn-2
`
`Fatty acid
`
`sn-1a
`
`sn-2b
`
`sn-1c
`
`sn-2d
`
`14 : 0
`15 : 0iso
`15 : 0ante
`16 : 0iso
`16 : 0
`16 : 1(n-7)
`17 : 0iso
`17 : 0
`17 : Da
`18 : 0DMAb
`18 : 0
`18 : 1(n-9)
`18 : 1(n-7)
`18 : 2(n-6)
`19 : Dc
`20 : 3(n-6)
`20 : 4(n-6)
`20 : 4(n-3)
`20 : 5(n-3)
`
`1.1 (cid:138)^ 0.2
`1.5 (cid:138)^ 0.2
`0.6 (cid:138)^ 0.1
`2.3 (cid:138)^ 0.3
`5.1 (cid:138)^ 1.1
`1.0 (cid:138)^ 0.4
`2.9 (cid:138)^ 0.3
`1.2 (cid:138)^ 0.1
`6.2 (cid:138)^ 2.4
`Trace
`5.4 (cid:138)^ 0.3
`3.9 (cid:138)^ 0.8
`16.4 (cid:138)^ 0.4
`7.8 (cid:138)^ 0.6
`5.3 (cid:138)^ 1.1
`1.5 (cid:138)^ 0.5
`3.7 (cid:138)^ 1.3
`4.0 (cid:138)^ 0.9
`13.8 (cid:138)^ 5.2
`
`0.6 (cid:138)^ 0.2
`Trace
`Trace
`0.5 (cid:138)^ 0.2
`2.0 (cid:138)^ 0.5
`1.0 (cid:138)^ 0.4
`0.7 (cid:138)^ 0.1
`Trace
`1.2 (cid:138)^ 0.2
`ND
`1.4 (cid:138)^ 0.2
`3.8 (cid:138)^ 0.5
`5.0 (cid:138)^ 0.8
`10.3 (cid:138)^ 0.9
`1.3 (cid:138)^ 0.2
`6.7 (cid:138)^ 0.5
`7.6 (cid:138)^ 0.5
`7.8 (cid:138)^ 0.9
`42.9 (cid:138)^ 4.0
`
`0.9 (cid:138)^ 0.2
`0.9 (cid:138)^ 0.3
`Trace
`1.2 (cid:138)^ 0.1
`9.1 (cid:138)^ 1.7
`1.9 (cid:138)^ 0.9
`6.3 (cid:138)^ 1.4
`3.3 (cid:138)^ 0.1
`4.7 (cid:138)^ 1.0
`14.6 (cid:138)^ 3.1
`20.4 (cid:138)^ 1.7
`2.4 (cid:138)^ 0.8
`11.7 (cid:138)^ 0.6
`1.9 (cid:138)^ 0.8
`1.6 (cid:138)^ 0.5
`1.1 (cid:138)^ 0.1
`0.2 (cid:138)^ 0.4
`0.4 (cid:138)^ 0.5
`0.6 (cid:138)^ 0.2
`
`1.0 (cid:138)^ 0.1
`Trace
`Trace
`1.1 (cid:138)^ 0.1
`3.9 (cid:138)^ 1.8
`1.2 (cid:138)^ 0.9
`1.1 (cid:138)^ 0.3
`Trace
`2.3 (cid:138)^ 0.6
`ND
`1.6 (cid:138)^ 0.6
`3.8 (cid:138)^ 0.8
`24.4 (cid:138)^ 4.6
`14.1 (cid:138)^ 1.5
`1.6 (cid:138)^ 0.1
`3.8 (cid:138)^ 0.6
`2.0 (cid:138)^ 1.7
`3.9 (cid:138)^ 1.0
`17.5 (cid:138)^ 2.5
`
`a 17:D, cis-11, 12-methylenehexadecanoic acid; b DMA, dimethylace-
`tal; c cis-9, 10-methyleneoctadecanoic acid.
`
`Stearic acid
`Linoleic acid
`EPA
`
`1.8 (cid:138)^ 0.2
`3.4 (cid:138)^ 0.5
`2.1 (cid:138)^ 0.5
`
`0.9 (cid:138)^ 0.4
`11.6 (cid:138)^ 1.5
`18.3 (cid:138)^ 3.8
`
`2.3 (cid:138)^ 0.7
`2.2 (cid:138)^ 0.4
`0.9 (cid:138)^ 0.4
`
`0.1 (cid:138)^ 0.1
`0.5 (cid:138)^ 0.2
`0.4 (cid:138)^ 0.2
`
`Table 4. Incorporation of various fatty acids into exogenously added
`lysoPtdCho in rat liver microsomal fraction. The incubation was con-
`ducted at 37 8C for 10 min with 0.1 mg protein of rat liver microsome and
`50 nmol fatty acid. The acyl acceptors used were 32 nmol: (a) 1-lyso-2-EPA-
`PtdCho, (b) 1-EPA-2-lyso-PtdCho, (c) 1-lyso-2-acyl-PtdEtn, and (d) 1-acyl-2-
`lyso-PtdEtn. Values are means ^ SD (three microsome preparations from
`different rats).
`
`Acylation (nmol´10 min21´mg21 protein)
`
`To lysoPtdCho
`
`To lysoPtdEtn
`
`Fatty acid
`
`sn-1a
`
`sn-2b
`
`sn-1c
`
`sn-2d
`
`Stearic acid
`Linoleic acid
`EPA
`
`182.3 (cid:138)^ 17.7
`19.1 (cid:138)^ 1.4
`14.3 (cid:138)^ 1.8
`
`8.6 (cid:138)^ 2.1
`135.7 (cid:138)^ 7.8
`64.0 (cid:138)^ 12.9
`
`111.5 (cid:138)^ 1.8
`10.3 (cid:138)^ 3.6
`6.0 (cid:138)^ 1.8
`
`1.6 (cid:138)^ 0.6
`7.9 (cid:138)^ 0.7
`1.4 (cid:138)^ 0.9
`
`000004
`
`

`
`q FEBS 1999
`
`Molecular species composition of PtdCho in C. elegans (Eur. J. Biochem. 263) 193
`
`Fig. 2. Substrate concentration-dependent
`incorporation of stearic acid and EPA into
`1-lyso-2-EPA-PtdCho (A) and
`1-EPA-2-lyso-PtdCho (B). The incubation was
`conducted at 20 8C for 10 min with 0.8 mg
`protein of C. elegans membrane fraction,
`32 nmol of lysoPtdCho and increasing amounts
`of stearic acid or EPA. The PtdCho extracted
`from reaction mixture was hydrolyzed with
`phospholipase A2 and radioactivity of
`lysoPtdCho (1-lyso-2-EPA-PtdCho as acyl
`acceptor) or free fatty acid
`(1-EPA-2-lyso-PtdCho as acyl acceptor) was
`determined.
`
`to the sn-2 position of PtdCho (Table 3). When 1-lyso-2-EPA-
`PtdCho was used as the acyl acceptor, a quite distinct
`preference for stearic acid over linoleic acid and EPA for
`acylation to sn-1 of PtdCho was observed with rat
`liver
`microsomes (Table 4). In contrast, the acyltransferase activity
`of C. elegans did not exhibit such a strict preference to stearic
`acid for acylation to the sn-1 position of PtdCho. The efficacy
`of the acylation was highest with linoleic acid, and that of
`stearic acid and EPA was almost equal (Table 3).
`When 1-lyso-2-acyl-PtdEtn was used as acyl acceptor, the
`level of incorporation of fatty acid into the sn-1 position of
`PtdEtn was higher with stearic acid and linoleic acid, and EPA
`was a poor acyl donor in C. elegans preparation (Table 3).
`These results with 1-lyso-2-acyl-PtdEtn differed from those
`obtained with 1-lyso-2-EPA-PtdCho as the acyl acceptor in
`C. elegans. Both in C. elegans and rat
`liver preparations,
`acylation of the sn-2 of PtdEtn did not occur to a significant
`extent compared to that of the sn-2 of PtdCho. These results
`with rat liver microsomes were consistent with the results using
`rat liver hepatocytes [19,20].
`
`Substrate specificity of acylCoA synthetase of C. elegans
`
`the added
`In the acyltransferase assay used in this study,
`fatty acid was first converted to acylCoA by acylCoA
`synthetase, and then was transferred to lysophospholipid.
`Therefore,
`the substrate specificity of acylCoA synthetase
`affects the acyltransferase activity. The conversion rates of
`EPA and stearic acid to its CoA form were 11.1 ^ 2.9 and
`8.0 ^ 3.3 nmol´10 min21 mg21 protein,
`respectively. The
`difference was statistically insignificant.
`
`D I S C U S S I O N
`
`Previously, we showed that the nematode C. elegans contained
`abundant EPA [2]. Our recent
`investigation revealed that
`palmitic, oleic, dihomo-g-linolenic and arachidonic acid were
`decreased and linoleic acid and EPA were increased in the
`PtdCho fraction when C. elegans was grown at a low
`temperature [3]. In this study, we investigated the effect of
`growth temperature on the molecular species composition of
`PtdCho of C. elegans. The AgTLC system and subsequent
`GC analysis
`showed the existence of as many as 16
`molecular species of EPA-containing PtdCho in C. elegans.
`Consistent with our previous report, proportions of EPA-
`containing PtdCho molecules with palmitic, oleic, dihomo-g-
`linolenic and arachidonic acid all
`tended to decrease, and
`1,2-diEPA-PtdCho increased significantly at
`low growth
`temperature. The 1,2-diPUFA-PtdCho such as 1,2-diEPA-
`PtdCho in C. elegans is an unusual phospholipid species in
`higher animals.
`
`the
`it has been reported that
`In the fish species carp,
`proportion of oleic acid at position sn-1 of PtdEtn was
`increased at
`low temperature and 18 : 1(n-9)/22 : 6(n-3)-
`PtdEtn exerted a membrane disordering effect
`[21].
`In
`C. elegans,
`the extent of
`the change in the fatty acid
`composition due to growth temperature was greater in PtdCho
`than that in PtdEtn [3], and 1,2-diEPA molecular species were
`only detected in the PtdCho fraction. Therefore, 1,2-diEPA-
`PtdCho might be one of the molecules that plays a role in the
`regulation of physiological properties of C. elegans mem-
`branes.
`The asymmetrical distribution of fatty acids in mammalian
`phospholipid is due to the substrate specificity of acyltrans-
`ferases involved in the phospholipid biosynthesis, by either the
`de-novo or the remodeling route. For example, it has been
`shown that
`linoleic acid and docosahexaenoic acid are
`considered to be utilized primarily for de novo synthesis and
`arachidonic acid is reported to enter through the remodeling
`pathway for acylation at sn-2 [22±25]. In C. elegans, PUFAs in
`PtdCho (but not PtdEtn) do not show a strict asymmetrical
`distribution. In the other free-living nematode species Tubatrix
`aceti, PUFAs such as linoleic acid and dihomo-g-linolenic acid
`are found in sn-1 position of PtdCho, whereas fatty acid of
`PtdEtn shows strict asymmetrical distribution like mammalian
`phospholipid [26]. The difference in the pattern of PUFA
`distribution between C. elegans PtdCho and mammalian
`PtdCho or C. elegans PtdEtn could be partially explained by
`the nature of acyltransferase involved in the remodeling of the
`respective phospholipid at sn-1. The most notable difference in
`the enzyme activity between the C. elegans and rat liver was
`that the acyltransferase of C. elegans did not show the strict
`preference for stearic acid for acylation to the sn-1 position of
`PtdCho. In C. elegans, the efficacy of utilization of EPA for
`acylation to sn-1 of PtdCho was almost equal
`to that of
`stearic acid. In contrast, the acylation of sn-1 of PtdEtn tended
`to occur with less unsaturated fatty acid in C. elegans
`preparations. These results are consistent with the fact that
`EPA was found abundantly in sn-1 of PtdCho and stearic acid
`was the predominant fatty acid in sn-1 of PtdEtn (Table 2).
`In experiments using rat hepatocytes, it has been reported
`that phospholipids with PUFAs both in the sn-1 and sn-2
`position were formed by the de novo pathway when the
`cells were incubated in relatively higher concentrations of
`PUFA [27±29]. However, the PUFA residue in sn-1 of the
`1,2-diPUFA-phospholipid was
`then replaced mainly with
`stearic
`acid
`by
`the
`remodeling
`pathway
`during
`the
`subsequent
`incubation without exogenous PUFA [30±32].
`This indicates that even in higher animals the de-novo
`pathway can synthesize 1,2-diPUFA-PtdCho in certain condi-
`tions and that
`the final molecular species composition of
`
`000005
`
`

`
`194 T. Tanaka et al. (Eur. J. Biochem. 263)
`
`q FEBS 1999
`
`phospholipid is a result of the modification of phospholipids by
`deacylation/reacylation. We currently do not know the relative
`contribution to the final molecular species composition by the
`de-novo route versus the remodeling route in C. elegans.
`However, even if the 1,2-diPUFA-PtdCho is formed by de-novo
`pathway,
`the PUFA residue at sn-1 of PtdCho would not
`always be replaced by a saturated acid during the subsequent
`remodeling because the acyltransferase lacks a preference for a
`saturated acid.
`As shown in our previous study, the increase in EPA content
`in the total
`lipid fraction of C. elegans grown at
`low
`temperature was compensated for by decreases in dihomo-g-
`linolenic acid and arachidonic acid [3]. These changes might be
`explained by the v3 desaturase which acts on dihomo-g-
`linolenic acid and arachidonic acid [6]. Under such a condition,
`the proportion of EPA-CoA in the acylCoA pool in the cell
`would increase, and as a result, the utilization of EPA-CoA by
`the acyltransferase would thus form a substantial amount of
`1,2-diEPA-PtdCho. It should be emphasized that
`the lack
`of preference for saturated fatty acid for acylation to sn-1 of
`PtdCho results in the existence of the unusual 1,2-diPUFA-
`phospholipid.
`
`R E F E R E N C E S
`
`1. The C. elegans sequencing consortium (1998) Genome sequence of the
`nematode C. elegans: a platform for invest biology. Science 282,
`2012±2018.
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`Satouchi, K. (1996) Effects of growth temperature on the fatty acid
`composition of the free-living nematode Caenorhabditis elegans.
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`8. Michaelson, L.V., Napier, J.A., Lewis, M., Griffiths, G., Lazarus, C.M.
`& Stobart, A.K.
`(1998) Functional
`identification of a fatty
`acid D5 desaturase gene from Caenorhabditis elegans. FEBS Lett.
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`9. Bligh, E.G. & Dyer, W.J. (1959) A rapid method