Molecular and Biochemical Parasitology, 29 (1988) 159-169 159 Elsevier
MBP 00977
Metabolism of phospholipids and lysophospholipids by Trypanosoma brucei
A b d u l S a m a d 1, B e r n d L i c h t 2, M a r y E . S t a l m a c h 2 a n d A l a n M e l l o r s 2
1Department of Biomedical Sciences, University of Guelph and ZGuelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada
(Received 2 December 1987; accepted 8 February 1988)
African trypanosomes (Trypanosoma brucei brucei) rapidly metabolize exogenous 1-acyl-lysophospholipids by at least two routes: (1) hydrolysis by a phospholipase A1, (2) acylation by an acyl-CoA-dependent acyltransferase. In contrast to lysophospholipids, exogenous phospholipids are not rapidly metabolized by T. brucei. The acyltransferase (EC 2.3.1.23) converts exogenous 1-acyl lysophosphatidylcholine and exogenous acyl-CoA to phosphatidylcholine and CoA-SH. It is a membrane-bound enzyme and shows maximal activity within the first 2 min of exposure of trypanosomes to the exogenous substrates. The acyltransferase specificity for lysophospholipids is lysophosphatidylcholine > lysophosphatidylinositol > lysophosphatidylethanolamine > lysophosphati- date. Phosphatidylcholine enhances the enzyme activity towards lysophosphatidylethanolamine and lysophosphatidic acid. The preference for CoA acyl thioesters is oleoyl > palmitoyl > myristoyl > stearoyl > arachidonoyl, and this specificity distinguishes the protozoan enzyme from those of cells of mammalian hosts, which are specific for arachidonoyl-CoA. When the acyltransferase converts exogenous lysophosphatidylethanolamine to phosphatidylethanolamine, the latter is rapidly methylated to form dime- thylphosphatidylethanolamine. There is also rapid hydrolysis of exogenous oleoyl-CoA by a thioester hydrolase in living trypan- osomes, to yield free oleate and CoA-SH.
In Trypanosoma brucei, an active phospholi- pase A1 is present in high concentrations [1,2]. It can generate lysophospholipids including both 1- acyl and 2-acyl sn-glycero-3-phosphocholine. In this study we describe the presence in T. brucei of a potent acyltransferase (EC 2.3.1.23, acyl- CoA: 1-acyl-sn-glycero-3-phosphocholine O-acyl- transferase) which converts exogenous lysophos- phatides to phospholipids, which are then incor-
Correspondence address: Dr. Alan Mellors, Chemisty & Bio- chemistry, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
porated into the membranes of the organism. When the acyltransferase converts exogenous ly- sophosphatidylethanolamine (Lyso-PtdEt-NH2) to PtdEt-NH2, the product is rapidly methylated to dimethyl-PtdEt-NH2, so that the B r e m e r - Greenberg pathway appears to be important in t rypanosomes for the de novo synthesis of phos- phatidylcholine (PtdCho). We also show that there is a potent oleoyl-CoA acylhydrolase (EC 3.1.2.2) activity in these organisms, which can degrade exogenous thioesters to yield CoA-SH and free oleate.
Materials and Methods
Labelled substrates. Dimyristoyl or dipalmitoyl phosphatidyl[14C]choline was prepared by the method of Stoffel et al. [3], i.e., by demethyla- tion of PtdCho (Serdary, London, ON) using benzothiolate, followed by remethylation with
[methylJ4C]iodide (NEN-Dupont Canada; Mont- real, QU). The resulting [methylJ4C]PtdCho was isolated using silica gel G thin layer chromato- graphy with CHC13/methanol/water (65:25:4, v/v) as the mobile phase. Labelled PtdCho was hydro- lyzed by phospholipase A2 (Naja naja venom; Sigma Chemical Co., St. Louis, MO) to yield 1- acyllysophosphatidyl-[methyl-14C]choline, by the method of Colard et al. [4]. The radiolabelled Lyso-PtdCho was isolated by thin layer chroma- tography on silica G plates, and extracted by the method of Bligh and Dyer [5]. Samples of the product was assayed for phosphorus [6]. [1- 14C]Oleoyl CoA, [1-14C]myristoyl CoA and di[1- 14C]oleoyl PtdCho were obtained from NEN-Du- pont Canada (Montreal, QU). Dioleoyl-phos- phatidyl[2-14C]ethanolamine was from Amers- ham (Oakville, ON). Phosphatidyl[14C]ethanol - amine was cleaved with phospholipase A2 by the method outlined above for Lyso-PtdCho produc- tion. All other lipids were from Serdary (Lon- don, ON).
Trypanosomes and lymphocytes. Trypanosomes (T.b. brucei of a monomorphic strain, Shinyanga III) were isolated from infected rat blood by the method of Lanham and Godfrey [7], and stored at -70°C in phosphate-buffered saline, pH 8.0, containing 1% glucose and 10% glycerol, at a concentration of 3 x 10 9 trypanosomes m1-1. Lymphocytes were isolated from spleens of 10-13- week-old female Swiss-Webster mice by the Fi- coll-Isopaque method of Boyle [8].
Trypanosomalfractions. Approximately 3 x 10 l° trypanosomes were disrupted by sonication and centrifuged at 48 000 rpm (150 000 x g) for 1 h in a Beckman Ti50 rotor. The resulting membrane pellet was homogenized in 0.1 M sodium phos- phate buffer, pH 7.4, containing 1 mg m1-1 bo- vine serum albumin, and centrifuged at 150000 x g for 1 h. The pellet was resuspended in 10 mM sodium phosphate buffer, pH 7.4, and centri- fuged again at 150000 x g for 1 h to obtain a membrane pellet. The supernatant from such preparations was used for soluble enzyme activ- ity.
Acyltransferase assay. Each acyltransferase assay tube contained 6 nmol 1-palmitoyl-lyso-PtdCho, containing 6000-10000 cpm of radiolabel, sus- pended in 49 ~1 RPMI-1640 medium (Gibco, Burlington, ON) containing 25 mM HEPES buffer, pH 7.4, and 0.7% (w/v) glucose. This was sonicated with 1 ixl dimethylsulfoxide containing 5 nmol oleoyl-CoA, and then 150 txl trypanoso- mal suspension in the RPMI-HEPES-glucose medium described above, was added. The incu- bation was at 37°C in a shaking water bath. The reaction was stopped by the addition of 750 ~1 of chloroform/methanol (1:2), containing an unla- belled standard lipid mixture, and the phases were separated by the addition of 200 txl of chloroform and 200 ~1 of water, followed by centrifugation for 30 s at 10000 x g.
The chloroform-soluble lipids were dried un- der nitrogen, resuspended in a suitable volume of chloroform, and separated by thin-layer chro- matography on silica LK6-D plates (Whatman). The mobile phase was either chloroform/ methanol/water (65:25:4, v/v) or the solvent of Skipski et al. [9], chloroform/methanol/acetic acid/water (25:15:4:2, v/v). Lipid spots were lo- cated by iodine staining, scraped into liquid scin- tillation vials and the silica gel was deactivated with 0.5 ml water overnight. The samples were assayed for radioactivity by liquid scintillation counting [10]. Recovery of radioactivity from sil- ica gel thin layer chromatography fractions was determined by chromatographing standards of radiolabelled Lyso-PtdCho or PtdCho on the same plates as samples. Percentage recovery values for these standards were used to correct the ob- served rates of conversion of Lyso-PtdCho to PtdCho in the acyltransferase assays. The results are expressed as nmol PtdCho produced per as- say tube.
For identification of aqueous methanol-soluble products, the aqueous phase from the extraction was concentrated by evaporation under nitrogen. The residue was analyzed by chromatography on cellulose thin layers. Samples were applied to freshly prepared cellulose plates, together with unlabelled choline, phosphocholine, CDP-cho- line and glycero-3-phosphocholine (GPC) as standards (Sigma, St. Louis, MO). The mobile phase was n-butanol/acetic acid/water (5:2:3, v/v)
and spots containing choline were detected with Dragendorff 's spray reagent [11]. The 14C-radio- activity in these water-soluble compounds was determined as described earlier.
Phospholipase A1 assay. The methodology was as described for the acyltransferase activity except that Lyso-PtdCho and oleoyl-CoA were omitted and 5 nmol di[lac]oleoyl-PtdCho was added. The release of [14C]oleate was measured as described previously [1].
Acyl-CoA hydrolase assay. Acyl-CoA hydrolase activity was measured as for the acyltransferase activity except that Lyso-PtdCho was omitted and [14C]acyl-CoA (15000 cpm) was included. Prod- ucts of acyl-CoA hydrolase were identified by thin layer chromatography of the lipid fraction on sil- ica gel, with development in the following solvent system: petroleum ether/diethyl ether/acetic acid (80:20:1, v/v). Compounds were visualized, scraped and counted as above.
R e s u l t s
Fate of exogenous PtdCho and Lyso-PtdCho. Living trypanosomes (T. b. brucei) do not metab- olize exogenous micellar PtdCho. Table I shows that, in the absence of detergent, PtdCho was a poor substrate for trypanosomal phospholipase A1. Upon addition of Tri ton X-100, living try- panosomes cleaved 20% of the exogenous di[14C]oleoyl-PtdCho to Lyso-PtdCho and free [14C]oleate. Very little of the PtdCho or resulting Lyso-PtdCho was further converted to water-sol- uble cleavage products. Our previous studies have characterized this detergent-activated activity as phospholipase A1, a membrane-bound trypano- somal enzyme [1], and we have shown that the isolated enzyme is only active against PtdCho when detergents are added.
In contrast to PtdCho metabolism, exogenous Lyso-PtdCho can be metabolized at high rates, as shown in Fig. 1. In the absence of detergent or exogenous fatty acyl-CoA, the product of Lyso- PtdCho metabolism is principally GPC which re- sults from phospholipase A1 action [12]. How- ever, in the presence of exogenous oleoyl-CoA, the major metabolite of Lyso-PtdCho is PtdCho.
161
TABLE I
Cleavage of exogenous PtdCho to Lyso-PtdCho and oleic acid by trypanosomes
I × 1 0 7 trypanosomes were incubated for 20 min at 37°C, in the presence of 25 ~M di[14C]oleoyl-PtdCho or 25 txM Ptd[14C]Cho with and without 0,125% Triton X-100. Mean --- S.E.M., n = 2.
At the concentrations of lysophosphatides and acyl-CoA used in this study there was no loss of viability, as shown by trypanosome motility, and no trypanosomal lysis even after 30 min incuba- tion with substrates. The PtdCho product was found to be associated with the trypanosomes, whereas the water-soluble GPC was found exclu- sively in the medium, which suggests that the phospholipase A1 action is extracellular (data not shown). At a concentration of trypanosomes of 5 × 107 per ml, conversion of Lyso-PtdCho to PtdCho is very rapid with 40% converted within 2 min, while 15% of the Lyso-PtdCho is hydro- lyzed to GPC in this time. The water-soluble product, GPC, was characterized by thin layer chromatography where its Rf corresponded to that of an authentic standard. At high trypanosomal concentrations, the phospholipase A1 activity ap-
5 --A :J. GPC (-oleoylCoA) T
4 I v
? % E 2
0 I I I I 5x10 s 5x10 e 5x10 7 5x10 B
Tryp~nosome ¢oncrl. mL -1
Fig. 1. Exogenous oleoyl-CoA promotes conversion of ex- ogenous Lyso-PtdCho to PtdCho. Trypanosomes were incu- bated with 30 ~,M Lyso-Ptd[14C]Cho and 25 p,M oleoyl-CoA for 2 min at 37°C in 0.2 ml media. Mean -+ S.E.M., n = 3.
162
pears to be more significant than the production of PtdCho.
Several enzymes are known to synthesize PtdCho from Lyso-PtdCho in eucaryotes. An en- zyme [13] that converts two molecules of Lyso- PtdCho to one molecule each of PtdCho and GPC, in the absence of acyl-CoA, is not involved here. In the absence of acyl-CoA, t rypanosomes produced little PtdCho and much GPC, in a ratio of 0.05 mol PtdCho/1.0 mol GPC (data not shown). Similarly the direct acylation of GPC, produced by phospholipase A1 action on Lyso- PtdCho, was ruled out by a pulse-chase experi- ment in which unlabelled G P C was added at var- ious times to the incubation mixture and did not inhibit PtdCho synthesis (Fig. 2). Hydrolysis of Lyso-PtdCho to GPC was significantly inhibited by the unlabelled G P C pulse-chase, which sug- gests that product inhibition of the phospholipase A1 by GPC can occur.
Because the format ion of PtdCho is stimulated by oleoyl-CoA, it appeared likely that an acyl- transferase enzyme was involved, and this was confirmed by using Lyso-PtdCho radiolabelled in the 1-acyl fatty acid residue, and by using [14C]oleoyl-coenzyme A (Table II). The similar rates of conversion for all three types of labelled substrate show that an acyl group is transferred
lO]r- A i GPC
E
0 ~' l I I I l 0 30 60 90 120 150
Time of pulse-chase (secondsi
Fig. 2. A pulse-chase of exogenous unlabelled GPC inhibits glycero-3-phospho[14C]choline production without decreasing PC formation. For assay of GPC production, 1 × 10 7 trypan- osomes were incubated with 30 ixM Lyso-Ptd[14C]Cho in 0.2 ml media for 2 min at 37°C. Unlabelled GPC was added, at a final concentration of 1.5 mM, at various times during the in- cubation and the reaction was stopped as described in Mate- rials and Methods. For PtdCho production, the assay was as above except that 1 x 10 6 trypanosomes and 25 IxM oleoyl-
CoA were used. Mean -+ S.E.M., n = 3.
TABLE II
Incorporation of label from acyltransferase substrates into PtdCho, by T. b. brucei
5 × 105 trypanosomes were incubated with 251xM oleoyl-CoA and 125 txM Lyso-PtdCho in 0.2 ml media for 4 rain at 37°C. Mean -+ S.E.M., n = 2.
to the 2-hydroxyl position of Lyso-PtdCho. These results also eliminate the possibility that PtdCho is synthesized by base exchange between Lyso- PtdCho and another membrane phospholipid.
Within a t rypanosome concentration rfinge of 5 x 105 - 5 x 106 per ml, the oleoyl-CoA-depend- ent acyltransferase activity is proport ional to cell numbers, with little interference f rom phospho- lipase A1, and this concentration range was used for further investigation of the acyltransferase. The acyltransferase activity of t rypanosomes is maximal within the first 2 min of exposure of the organisms to substrates, the rate of production of PtdCho declines rapidly thereafter , and accumu- lated radiolabelled PtdCho declines after a few minutes (data not shown). The transient high in- itial activity is characteristic also of membrane fractions from mammals and yeasts [14-17]. The decline in accumulated PtdCho is due in part to phospholipase A1 hydrolysis of PtdCho to Lyso- PtdCho, and subsequent acyl group migration, followed by phospholipase A1 hydrolysis to give GPC as described earlier. This is not inconsistent with the finding that exogenous PtdCho is not converted to Lyso-PtdCho or GPC in the absence of detergents, since we find that most of the PtdCho formed is associated with t rypanosomal membrane , where it is in a lipid environment that may render it susceptible to phospholipase A1 hydrolysis.
The t rypanosomal membrane acyltransferase was assayed over a p H range of p H 6.0-9.2 in 0.2 M phosphate buffer (pH 6.0-8.0) and 0.04 M barbital buffer (pH 8.0-9.2). Opt imum activity was determined at pH 7.2-7.4. In experiments not shown, it was determined that the enzyme activ- ity was heat-labile, in that organisms killed by
TABLE III
Acyl donor specificity for Lyso-PtdCho:acyl CoA acyltransferase of T. b. brucei
163
CoA thioester PtdCho produced (pmol +-- S.E.M.)
1:1, Lyso-PtdCho:acyl CoA 1:4, Lyso-PtdCho:acyl CoA
10 7 trypanosomes or equivalent membrane fraction were incubated with 50 txM or 12.5 IzM Lyso-PtdCho and 50 IzM acyl CoA in 0.1 ml media for 20 in at 37°C. Mean ± S.E.M., n = 2.
heating at 60°C for 2 min did not show enzyme activity towards exogenous Lyso-PtdCho.
The specificities for acyl-CoA thioesters of the trypanosomal acyltransferase are shown in Table III. Under the conditions used throughout this study, namely at an equimolar ratio of Lyso- PtdCho/acyl-CoA, oleoyl-CoA is the preferred acyl donor, and the preference order is oleoyl > palmitoyl = myristoyl > stearoyl > arachidon- oyl. When lower concentrations of Lyso-PtdCho are used, so that the Lyso-PtdCho/acyl-CoA ra- tio is 1:4, the preference changes to: myristoyl > oleoyl > stearoyl > palmitoyl > arachidonoyl. However, under these condi t ions the isolated membrane fraction shows preference for oleoyl- CoA over myristoyl-CoA. There may be several acyltransferases active in living trypanosomes which can act on exogenous substrates. An acyl- transferase which is specific to myristoyl-CoA may be inhibited at the higher Lyso-PtdCho concen- trations usually used here. Lower Lyso-PtdCho concentrations may avoid substrate inhibition of a myristoyl-CoA:Lyso-PtdCho acyltransferase, and such substrate inhibition has been observed for mammalian membrane-bound acyltransfer- ases [18,19]. In trypanosomes, however, it ap- pears from Table III that the membrane-bound acyltransferase prefers oleoyl-CoA whereas the intact organism shows maximal transfer from myristoyl-CoA at these substrate concentrations. There is no obvious correlation of the acyl-CoA specificities with fatty acyl chain length, critical micellar concentrations [20] or degree of unsatu- ration. The lack of utilization of arachidonoyl-
CoA as a substrate is in marked contrast to mam- malian acyltransferases where many tissues and organelles possess arachidonoyl-CoA-specific aCyltransferases [16,18,19,21-23].
The ability of the T. brucei acyltransferase to use different lyso-phospholipid acyl acceptors was tested in a different type of assay. Table IV sum- marizes the incorporation of [14C]oleate from [14C]oleoyl-CoA in the corresponding phospho- lipids when different unlabelled exogenous lyso- phospholipid acyl acceptors are incubated with intact trypanosomes. Lyso-PtdCho is the best acyl acceptor and all other activities are compared to it. The ability of the acyltransferase to transfer [14C]oleate from oleoyl-CoA to lysophosphatidyl- inositol (Lyso-PtdIns), lysophosphatidylserine (Lyso-PtdSer), or Lyso-PtdEt-NH 2 was 69%, 11% and 27%, of the Lyso-PtdCho acyltransferase ac- tivity, respectively. Lysophosphatidic acid (Lyso- Ptd), a precursor for phospholipids, was con- verted to phosphatidate at 19% of the conversion of Lyso-PtdCho to PtdCho. Glycero-3-phos- phate, a substrate for glycero-3-phosphate acyl- transferase, was not acylated by the trypanoso- mal enzyme. These data suggest T. brucei has no glycero-3-phosphate acyltransferase, or no up- take of the substrate, in contrast to Tetrahymena which has an active enzyme system [24]. Plate- lets, rat liver, mouse brain and Tetrahymena all possess multiple lysophosphatide: acyl-CoA transferases, specific for various acyl donors and acceptors [16,19,21,25].
Table IV also shows the influence of unlabelled exogenous micellar dipalmitoyl-PtdCho (25 IxM)
164
T A B L E IV
Conversion of exogenous lysophosphat ides to phospholipids by t rypanosomes
Reaction Phospholipid formed Change (pmol rain -1 (5 × 105 cells) - l )
5 x 105 t rypanosomes were incubated in the presence of 120 ixM lysophosphatide, 25 IxM oleoyl-CoA and 29000 cpm [14C]oleoyl -CoA in dimethylsulfoxide with and without 25 IzM PtdCho, for 4 min at 37°C. Means ± S.E.M. are shown for triplicates. *P < 0.01, **P < 0.005.
when added with lysophosphatide substrates at a molar ratio of 1:5 [26]. The physical state of the substrates is known to be critical for many phos- pholipid-metabolizing enzymes and some phos- pholipase A2 enzymes show much greater activ- ities against PtdEt-NH 2 when the substrate is dispersed in mixed micelles with PtdCho [26]. Ptd[laC]Cho was used in control replicates to show that PtdCho is not metabolized by intact trypan- osomes under the conditions of these experi- ments. The addition of PtdCho to Lyso-Ptdlns had little effect on the acyltransferase activity. The presence of PtdCho caused a 1.5 fold increase in PtdEt-NH 2 production from Lyso-PtdEt-NH 2 and Ptd production from Lyso-Ptd was enhanced 3.4 fold, as compared to controls in the absence of PtdCho carrier. None of the lysophosphatides, in the presence or absence of PtdCho carrier, was as effective a substrate as was Lyso-PtdCho for the trypanosomal acyltransferase. The acylations of Lyso-PtdCho and Lyso-PtdSer were slightly inhibited (20%) when PtdCho was present. For the acylation of Lyso-PtdCho this inhibition may be due to product inhibition. The inhibition of Lyso-PtdSer conversion to PdtSer may be due to some physical interaction between the substrate
and PtdCho which decreases its ability to act as an effective substrate.
Table V shows data for acyltransferase activity in the soluble (150000 x g supernatant) and membrane (150000 x g pellet) fractions of soni- cated trypanosomes. Under conditions where in- tact trypanosomes convert 20% of exogenous Lyso-PtdCho to PtdCho, membrane fractions from an equivalent number of organisms have twice the activity of intact cells, whereas the su- pernatant fraction has half the activity of intact cells. The supernatant activity may be due in part to small membrane fragments from sonicated or- ganisms, which are not sedimented under these conditions. The 1-acyl-glycerophosphocholine acyltransferases that have been characterized from a variety of species have shown to be membrane- bound enzyme systems [14-21,24,27-29]. The re- quirement for oleoyl-CoA for enzymatic activity is clearly seen in Table V. An acyl derivative of coenzyme A is absolutely essential for activity since coenzyme A alone cannot substitute for oleoyl-CoA (Table V, legend).
In Table V, the mouse spleen lymphocyte, a mammalian cell from a susceptible host, is com- pared for acyltransferase activity, with trypano-
TABLE V
GPC and PtdCho formation in trypanosomes and trypanosomal fractions, compared with mouse spleen lymphocytes
165
nmol min -1 (10 7 cells) -1 (nmol min -1 (mg protein) 1)
A control experiment in which CoA was added in place of oleoyl-CoA, showed 0.06 nmol PtdCho produced min -1 per 5 x 105 trypanosomes and 0.13 nmol GPC produced min -1 per 5 x 105 trypanosomes. Values in brackets are nmol min -1 (rag protein) -1 based on 0.04 mg protein per 107 trypanosomes (2) and 0.61 mg protein per 107 small murine spleen lymphocytes. Cell numbers represent either 107 cells or isolated cell fractions from 107 cells. Cells were incubated with 30 p,M Lyso-Ptd[14C] --- 25~M oleoyl- CoA or CoA for 2 min at 37°C in 0.2 ml media.
somes. U n d e r these condit ions, the acyltransfer- ase in intact t r ypanosomes converts Lyso -P tdCho to P tdCho at a rate of 1.05 --- 0.11 nmol min -1 (107 cells) -1. This observat ion suggests that the t rypanosome's capacity to utilize exogenous Lyso- P tdCho is greater than that of the mouse lym- phocyte for which the rate is 0.59 --- 0.01 nmol min -1 (107 cells) -1. While t rypanosomes and lymphocytes differ great ly in cell shape, size and prote in content , the two cell types do have simi- lar surface areas [30,31]. Thus acyltransferase content may be related to the area of the plasma membrane . Despi te the high prote in content of t rypanosomal p lasma m e m b r a n e s due to the presence of V S G coat prote in [32], specific activ- ities for acyltransferase, in t rypanosomal mem- brane fract ions or intact t rypanosomes , are not lower than for lymphocytes .
LPE-Acyltransferase and the rnethylation of eth- anolamine phospholipids. Fig. 3 shows that when the L y s o - P t d E t - N H 2 : o l e o y l - C o A acyltransferase of intact t rypanosomes , descr ibed above, was measured directly by incubat ion of t rypanosomes with exogenous Lyso-Ptd[14C]Et-NH2 and oleoyl- C o A , there was convers ion of about 10% of the
Lyso-PtdEt -NH2 to P tdEt -NH2 within 20 min. In the absence of o leoy l -CoA, no P tdEt -NH2 was formed (data not shown). However , ano ther ma- jor p roduc t of Lyso-Ptd[14C]Et-NH2 metabol ism accumulates , especially at lower t rypanosome concentrat ions. This was tentat ively identified as d imethyl -PtdEt-NH2 f rom its co -ch roma tog raphy with standards, and this c o m p o u n d appears to arise via rapid methyla t ion of the acyltransferase product , P t d E t - N H 2. The methyla t ion react ion was not seen in the absence of o l eoy l -CoA so presumably the d imethyl -PtdEt-NH2 arises by methylat ion of PtdEt-NH2. The extent of conver- sion of d ime thy l -P tdEt -NH 2 to P tdCho by fur ther methylat ion was not de te rmined in these experi- ments.
Acyl-CoA hydrolase activity. While s tudying the product ion of phospholipids f rom various acyl ac- ceptors in the presence of [14C]oleoyl-CoA, it be- came apparen t that free fat ty acid was being re- leased f rom exogenous o l eoy l -CoA by living t rypanosomes (Table VI) . In most cases about 10% of the 14C-products appeared in the free fatty acid region of the chromatogram. The produc- tion of the free fat ty acids was decreased by the
166
3O
Dimethyf PtdEt-NH2 25 " ~ H PtdEt- NH2
qc_ 15 . g 1 0
Q.
5
0 I I I O 5 10 15
Trypanosomes x 107 mL -1
Fig. 3. Production of PtdEt-NH 2 and dimethyl-PtdEt-NH2 from exogenous Lyso-PtdEt-NH2 and oleoyl-CoA. Trypano- somes were incubated with 10.7 ~xM Lyso-Ptd[14C]Et-NH2 and 33.3 ~M oleoyl-CoA for 20 min at 37°C in 0.15 ml media.
Mean -+ S.E.M. , n = 3.
addition of exogenous lysophospholipids (Table VII). To confirm that the product was free fatty acid, trypanosomes were incubated with 25 p~M [14C]oleoyl-CoA or [14C]myristoyl-CoA for 4 min. The chloroform-soluble extract was chromato- graphed on thin layers of silica gel in a system that separates diglycerides, monoglycerides, acyl-CoA and free fatty acid. The radioactive product was identified as free fatty acid by its relative mobil- ity. The acyl-CoA hydrolase activity is more ac- tive against oleoyl-CoA than against myristoyl- CoA. When exogenous lysophospholipids are added along with the oleoyl-CoA, the acyltrans- ferase activity apparently competes with the acyl- CoA hydrolase for the fatty acyl-CoA substrates and reduces markedly the oleoyl-CoA hydrolysis. Unlike acyltransferase activity, which is higher in membrane fractions than in equivalent numbers of intact organisms (Table V), the acyl-CoA hy-
drolase of membrane fractions is less active than the living cells against exogenous substrate.
Discussion
In the blood stream and tissue spaces of the mammalian host, trypanosomes are exposed to a variety of metabolites that may be beneficial, or detrimental, to the survival of the parasites. Slen- der blood stream forms of T. b. brucei do not synthesize fatty acids but incorporate host fatty acids into their lipids and membrane glycopro- teins [33,34]. A potent trypanosomal phospholi- pase A1 can release free fatty acids from Lyso- PtdCho, thereby removing a membrane-active amphiphile from the external environment, and at the same time releasing free fatty acid which the parasites can utilize. The excessive release of free fatty acid may contribute to the hemolysis seen in some host species [35]. As shown in this study, the phospholipase A1 appears to be inac- tive against PtdCho, at least under the in vitro culture conditions used here, and requires deter- gents for action on diacylphosphoglycerides.
If exogenous fatty acyl-CoA is provided to the trypanosomes along with exogenous lysophos- pholipids, then a rapid transacylation occurs and the resultant phospholipid is incorporated into the trypanosomal membrane lipids. It is not clear un- der what circumstances trypanosomes may en- counter significant concentrations of exogenous lysophospholipids and fatty acyl-CoA, though in vivo hemolysis or host cell plasma membrane lysis may expose the parasite to these metabolites. This study shows that there is a rapid utilization of exo- genous oleoyl-CoA and exogenous Lyso-PtdCho
T A B L E VI
Compar ison of phosphol ipase A1, acyltransferase and oleoyl-CoA hydrolase in t rypanosomes and t rypanosomal membranes
Enzyme activity (nmol min ~ m l 1)
Phospholipase AI" Lyso-PtdCho Oleoyl-CoA acyltransferase b hydrolase c
a 1 X 107 t rypanosomes were incubated with 30 p,M Lyso-Ptd[14C]Cho for 2 min at 37°C in 0.2 ml media (n = 3). b 1 × 107 t rypanosomes were incubated as in (a) with the addition of 25 ~M oleoyl-CoA (n = 3). c 5 × 106 t rypanosomes were incubated with 25 jxM oleoyl-CoA and 50000 cpm [14C]oleoyl-CoA for 4 min at 37°C in 0.2 ml media
(n = 2; mean +- S.E.M:) .
TABLE VII Acyl-CoA hydrolase activity of trypanosomes and its reduction by acyltransferase substrates
167
Free fatty acid formed (nmol min -1 m1-1)
Live trypanosomes Membrane
Oleoyl-CoA 3.38 --- 0.33 0.21 --- 0.09 Myristoyl-CoA 0.26 - 0.01 not done Oleoyl-CoA + Lyso-PtdCho 0.93 - 0.08 0.00 Oleoyl-CoA + Lyso-Ptdlns 0.50 +-- 0.07 0.056 ± 0.004 5 x 105 Trypanosomes were incubated for 4 min at 37°C with 25 I~M 14C-labelled acyl-CoA substrates, and with 125 ~M lyso- phosphatides where indicated. Mean -+ S.E.M., n = 3.
by trypanosomes, which quantitatively can ex- ceed their capacity to remove Lyso-PtdCho by phospholipase A1 action. Maximal acyltransfer- ase activity is seen in the first 2 min of exposure of the living trypanosomes to the substrates, and resembles that seen for microsomal membrane fractions from rat liver [16]. The rapid initial me- tabolism may be an artefact caused by sudden ex- posure of the organisms to amphiphilic sub- strates, or it may be a defensive mechanism for the detoxification of exogenous membrane-active lysophospholipids.
The properties of the Lyso-PtdCho:acyl-CoA acyltransferase differ from those of the mammal- ian host cells in some important respects. Mam- malian Lyso-PtdCho acyltransferases are com- monly specific for arachidonoyl-CoA, though the fatty-acyl specificity can vary with the ratio of ly- sophospholipid to acyl-CoA [19]. The acyl-CoA specificity of the trypanosomal enzyme is in de- creasing order: oleoyl, palmitoyl, myristoyl, stea- royl, arachidonoyl, when the Lyso-PtdCho is equimolar with the acyl-CoA, but at a higher mo- lar ratio (4:1) of acyl-CoA to lysophospholipid the relative order of specificity changes to favor myr- istoyl-CoA. Apparent fatty acyl-CoA specificities at the plasma membrane of living cells may not correlate with specificities for isolated enzymes or membrane fractions. It is notable, however, that arachidonoyl-CoA appears to be a poor acyl do- nor, while myristoyl-CoA can under some cir- cumstances be an excellent acyl donor for the try- panosome acyltransferase. Myristate is readily incorporated into trypanosomes and is rapidly transferred to lipid precusors in the synthesis of membrane-variant-specific glycoprotein [36]. The apparent fatty acyl-CoA specificity may be corn-
plicated by the presence of acyl-CoA hydrolase and phospholipase A1 activites in the trypano- somal plasma membrane. Nevertheless the fatty acyl-CoA specificity of T. b. brucei acyltransfer- ase does resemble that seen for membrane frac- tions from the protozoan, Tetrahymena pyrifor- mis [24]. The acyl-CoA specificities for both protozoan species also resemble those for a mam- malian membrane enzyme, the Lyso-Ptd acyl- transferase of rat liver microsomes, which, unlike the corresponding Lyso-PtdCho acyltransferase, shows marked preference for oleoyl-CoA over arachidonoyl-CoA [16]. However, the trypano- somal enzyme is clearly not involved in de novo synthesis of phospholipids, since it does not cause significant acylation of either glycerol-3-phos- phate or 1-acyl-lysophosphatidate (Table IV). When unlabelled GPC is added to trypanosomes, following exposure to Lyso-Ptd[14C]Cho and oleoyl-CoA, there is no inhibition of Ptd[laC]Cho formation (Fig. 2), so that no significant acylation of GPC occurs. Other evidence confirms that the PtdCho arises via an acyltransferase specific for Lyso-PtdCho and acyl-CoA. There is no evi- dence of transacylation between two Lyso-PtdCho substrates to form GPC and PtdCho [13]. There is some indication from the studies on the fate of Lyso-Ptd[laC]Et-NHz that methylation of PtdEt- NH2 is a route for PtdCho synthesis in trypano- somes, and this deserves further attention.
The metabolic routes for the utilization of ex- ogenous Lyso-PtdCho, and related lipids, by T. b. brucei can be summarized in Fig. 4. The rela- tive flux of metabolites will depend on the con- centration of parasites and on the relative ratio of lysophospholipid to acyl-CoA, but it is clear that these organisms have a high capacity to remove
168
Glycero-P Cho FFA / ~
'x~/~Phospholil ase A1 H 2 0 7
Lyso - Pt dCho Acy[CoA
~, , ,~ H 20 "~Acyl t ransferase
..., FFA PtdCho
Fig. 4. Metabolism of exogenous Lyso-PtdCho by T. brucei.
lysophospholipids from their environment, and to incorporate the resultant metabolites.
Acknowledgements
We would like to thank Dr. P.N. Hambrey and Dr. C.M. Forsberg for their assistance in these studies. This work was supported by the Medical Research Council of Canada and by the W.H.O. Parasitic Diseases Programme.
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