Trypanosoma cruzi:Nitrogenous-Base-Containing Phosphatides in Trypomastigote Forms—Isolation and Chemical Analysis

EXPERIMENTAL PARASITOLOGY ARTICLE NO. PR974181

87, 8–19 (1997)

Trypanosoma cruzi: Nitrogenous-Base-Containing Phosphatides in Trypomastigote Forms—Isolation and Chemical Analysis Marı´a Laura Uhrig,* Alicia S. Couto,* Maria Ju´lia M. Alves,† Walter Colli,† and Rosa M. de Lederkremer* *CIHIDECAR, Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428, Buenos Aires, Argentina; and †Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil UHRIG, M. L., COUTO, A. S., ALVES, M. J. M., COLLI, W., AND DE LEDERKREMER, R. M. 1997. Trypanosoma cruzi: Nitrogenous-base-containing phosphatides in trypomastigote forms—Isolation and chemical analysis. Experimental Parasitology 87, 8–19. In trypanosomatids, little is known about the biosynthetic pathways involved in the metabolism of ethanolamine. In an attempt to clarify this point, an exhaustive analysis of the chloroform:methanol extract of T. cruzi trypomastigotes metabolically labeled with [14C]ethanolamine, in comparison with the lipids from [3H]palmitic acidincorporated parasites, was performed. In both cases, phosphatidylethanolamine and lysophosphatidylethanolamine were detected, while phosphatidylcholine and lysophosphatidylcholine were only labeled with the fatty acid precursor. However, dimethylphosphatidylethanolamine was isolated from parasites labeled with the base precursor, indicating the ability of trypanosomes to methylate phosphatidylethanolamine to dimethylphosphatidylethanolamine. Fatty acids of the labeled phospholipids were analyzed by reverse-phase thin-layer chromatography and fluorography. Interestingly, phospholipids from the trypomastigote stage show palmitic acid (C16:0) and stearic acid (C18:0) as the only labeled components. The same saturated fatty acids were found free and as components of the radioactive triglycerides. No unsaturated fatty acids were detected, in accordance with the results obtained with inositolphospholipids. Conversely, when the fatty acids of phospholipids purified from nonlabeled parasites were analyzed by gas–liquid chromatography and gas–liquid chromatography– mass spectrometry, C18:1 was also detected. A striking finding was the presence of a considerable amount of free lignoceric acid (C24:0). Also, the C24:0 fatty acid was identified in the triglyceride fraction and as a component of phosphatidylcholine. The limited capacity of trypomastigote forms to elongate fatty acids was determined. In contrast with the results reported for other noninfective forms of the parasite, the absence of unsaturated fatty acids due to a low activity of desaturases was observed. © 1997 Academic Press INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; trypomastigote; zwitterionic phospholipids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; DMPE, dimethylphosphatidylethanolamine; MMPE, monomethylphosphatidylethanolamine; GPI, glycosylphosphatidylinositol; PLC, phospholipase C; PLA2, phospholipase A2; SAPA: shed acute-phase antigen; DAG, diacylglycerol; TAG, triacylglycerol; DME, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; TLC, thin-layer chromatography; RP-TLC, reverse-phase thin-layer chromatography; GLC, gas–liquid chromatography; GLC-MS, gas–liquid chromatography–mass spectrometry.

INTRODUCTION

surface glycoprotein coat leads to parasite destruction. Thus, the specific transport system utilized for the ethanolamine uptake plays an important role in parasite survival (Rifkin et al. 1995). The Trypanosoma cruzi invasion process is very complex. Several different predominantly stage-specific molecules appear to be involved in the recognition and invasion steps. However, in all stages, the known antigenic structures ex-

Ethanolamine is found in trypanosomes as a component of the anchor moiety of surface glycoproteins. In Trypanosoma brucei, the variant surface glycoprotein contains one ethanolamine per mole, accounting for the bridge between the protein and the glycosylphosphatidylinositol (GPI) fragment (Englund 1993). It is known that the inability to synthesize or replace the 8 0014-4894/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.

T. cruzi: PHOSPHATIDES IN TRYPOMASTIGOTE FORMS hibit a GPI-anchor moiety: mucins (Previato et al. 1995) and GIPLs (Lederkremer et al. 1990, 1991, 1993; Previato et al. 1990) from the epimastigote stage; F2/3 mucins (Almeida et al. 1994), Tc-85 (Couto et al. 1993) and transsialidase (SAPA) (Pollevick et al. 1991; Agusti et al., 1997) from the trypomastigote stage; Ssp4 in amastigotes (Bertello et al. 1996); and 1G7 antigen (Gu¨ther et al. 1992; Heise et al. 1995) and mucins (Schenkman et al. 1993; Acosta Serrano et al. 1995) in metacyclic forms. Although ethanolamine has been determined to be a component of the anchor moiety of some of the T. cruzi antigens mentioned above, the radioactive precursor seems not to be easily incorporated into T. cruzi glycoproteins (Rifkin and Fairlamb 1985) or GPIs (Heise et al. 1996). We were interested in characterizing the molecules labeled by this precursor. Phospholipids constitute a major proportion of the lipids of trypanosomatids. In T. cruzi epimastigotes about 30% of the total lipid extract is composed of phospholipids, with phosphatidylethanolamine (PE) and phosphatidylcholine (PC) being the predominant species (Da Silveira and Colli 1981). In blood and culture forms of T. lewisi and T. rhodesiense they account for 73–77% of the total lipids (Dixon and Williamson 1970). In Leishmania donovani promastigote phospholipids comprise 58% of total lipid (Ghosh 1963) and in other flagellates it varies from 65 to 79% (Haughan and Goad 1991). The most prominent classes in both bloodstream and procyclic forms of Trypanosoma brucei brucei are PC, PE, and sphingomyelin, accounting for about 80% of the total phospholipids (Patnaik et al. 1993). PE is the precursor for the introduction of ethanolamine in GPI anchors (Menon et al. 1993). Provided that exogenous ethanolamine is available, the majority of PE is formed via the CDP– ethanolamine pathway in mammalian cells (‘‘Kennedy pathway’’) (Weiss et al. 1958). However, in tissue culture, PE is also biosynthesized via decarboxylation of phosphatidylserine (Voelker 1984). A third pathway involves direct incorporation of ethanolamine into phospholipids by a base exchange mechanism (Kanfer 1980). Recently, it has been reported

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that ethanolamine is continuously released from PE and recycled back into PE, suggesting that an active ethanolamine cycle exists in CHO-K1 cells (Shiao and Vance 1995). On the other hand, the transport system in T. brucei brucei is distinct from that of mammalians and yeast. It has been recently reported that the Kennedy pathway is mainly used for most of the PE biosynthesis, but, in addition, other minor metabolites such as dCDP–ethanolamine and glycerophosphoryl–ethanolamine, which may be important in other biosynthetic pathways, were found (Rifkin et al. 1995). Phosphatidylcholine is apparently essential for mammalian life and is the major phospholipid found in most eukaryotic cells (Vance 1990). In the present work, we characterized and quantified nitrogenous-base-containing metabolites of the trypomastigote stage of T. cruzi. Metabolic labeling with [14C]ethanolamine or [3H]palmitic acid was performed and chloroform:methanol extracts were analyzed. Also, the lipids from nonlabeled parasites were analyzed by GLC and GLC-MS. MATERIALS AND METHODS Parasites. Trypomastigotes of T. cruzi (Y strain) were obtained from infected LLC-MK2 epitelial cell monolayers maintained in DME containing 2% FCS. Parasites were collected on the fifth day after infection (Andrews and Colli, 1982). Parasite labeling. Parasites (1.5 × 109) were resuspended at a density of 8 × 107 cells/ml in DME and metabolically labeled with [9,10(n)-3H]palmitic acid (Amersham, 54 Ci/ mmole, 41 mCi/ml) for 7 hr at 37°C. Incorporation of [1,214 C]ethanolamine (NEN, 3 mCi/mmole) was performed with 1 × 109 trypomastigotes. Parasites were resuspended at a density of 10 × 107 cells/ml in DME, 2% FCS, 20 mM Hepes and were incubated with [1,2-14C]ethanolamine, 5 mCi/ml, for 6 hr at 37°C. After incubation, microscopic observation showed that all parasites remained viable and did not suffer morphological modifications. Trypomastigotes were harvested and washed with medium 199, and the final pellet was freezedried. Lipid extraction. The labeled parasites were extracted with chloroform:methanol 2:1 (2 × 1 ml) and 1:1 (2 × 1 ml). The combined extracts were fractionated on DEAESephadex A-25 (acetate form) as previously described (Couto et al. 1985). Briefly, lipids which do not interact with the resin were eluted with chloroform:methanol:water

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UHRIG ET AL.

(15:30:4, 100 ml), and strongly acidic lipids were eluted with chloroform:methanol:0.8 M NaAcO (15:30:4, 100 ml). Nonbound lipids labeled with [14C]ethanolamine were resuspended in water and passed through a C18 clean-up cartridge (Worldwide Monitoring, PA, U.S.A.). Salts and free radioactive precursor were eluted with water and lipids were recovered with methanol. Nonradioactive lipids were extracted from 1 × 1010 parasites and purified by the same procedure. Materials. Phospholipids and lipid standards were purchased from Sigma. Thin-layer chromatography (TLC) was performed on silica gel 60 precoated plates (Merck) using the following solvent systems: (A) chloroform:methanol: water (65:25:4, by vol); (B) hexane:ethyl ether:acetic acid (70:35:1, by vol); and (C) hexane:isopropyl alcohol (93:7, v/v). In order to resolve PC and LPE, the components running as these standards in solvent system A were eluted from the plate and analyzed by two-dimensional TLC using chloroform:methanol:13.3 M NH4OH (65:25:5, by vol) in the first direction and chloroform:acetone:methanol:acetic acid:water (30:40:10:10:1, by vol) in the second direction. In this case, standard lipids were run in the same plate and located with iodine vapor. Reverse-phase TLC was performed on RP-18 F-254 precoated plates (Merck) using acetonitrile:acetic acid (1:1, v/v) (solvent system D). Analysis of the nitrogenous hydrolysis products of lipids labeled with [14C]ethanolamine was performed on cellulose TLC sheets (Merck) using the following as developing solvents: (E) n-butanol:acetic acid:water (5:2:3, by vol) or (F) phenol:n-butanol:formic acid:water (47:50:3:10, by vol), which was shaken with solid KCl and decanted. Plates were sprayed with 1 N KCl and dried before spotting (Dittmer and Wells 1969). In all cases, radioactive samples were located by fluorography at −70°C using EN3HANCE (NEN) and Kodak XOmat AR films. Quantification of the labeled spots was performed after fluorography by extracting the silica gel scrapings with chloroform:methanol:water (5:5:1, by vol) and counting radioactivity. Radioactivity was determined in a 1214 RackBeta Wallac liquid scintillation counter. PLC digestion. Samples were resuspended in 20 mM Tris/HCl, pH 7.4 (100 ml), containing 0.01% Triton X-100, 1 mM CaCl2, and incubated overnight with PLC (5 U) of Bacillus cereus (Sigma, Type IV, 139 U/mg, 1260 U/mg prot.) at 37°C. A control sample in the absence of enzyme was performed under the same conditions. The lipids were extracted with chloroform:methanol (2:1, v/v) and analyzed by TLC. PLA2 digestion. Samples were resuspended in 50 mM Tris/HCl, pH 7.4 (200 ml), containing 0.1% of deoxycholate, 2 mM CaCl2 and incubated with 50 U of PLA2 from Crotalus adamanteus venom (Sigma, 270 U/mg, 400 U/mg prot.). The mixture was shaken vigorously in a water bath at 37°C for 4 hr. When the reaction was stopped the lipids were extracted with chloroform:methanol (2:1, v/v) and

analyzed by TLC. A control was performed in the absence of the enzyme under the same conditions. Authentic dipalmitoylphosphatidylcholine (Sigma) was treated under the same conditions as an enzyme control and to obtain a standard of 1-O-palmitoyl-glycero-3-phosphocholine. Acid hydrolysis. Samples of lipids metabolically labeled with [14C]ethanolamine were hydrolyzed with 4 N HCl overnight at 100°C. The acid was eliminated by several evaporations with the addition of water and then in vacuo over NaOH pellets. The residue was redissolved in water and analyzed on cellulose TLC plates as described previously. Analysis of fatty acids. Samples were treated with 0.1 M NaOH in methanol for 1 hr at room temperature. The solution was neutralized, dried, and resuspended in water. Free fatty acids were extracted with chloroform and the corresponding methyl esters were obtained by treatment with BF3/methanol (20% in methanol, 1 ml, Merck) at 80°C for 1 hr in a screw-cap test tube (Manku 1983). Labeled fatty acid methyl esters were analyzed by RP-TLC in solvent D. Hydrogenation. Labeled fatty acid methyl esters were subjected to hydrogenation with palladium on activated carbon (palladium content, 10%; Aldrich), using a hydrogen pressure of 3 atm. The reaction was performed for 4 to 5 hr with shaking at room temperature. A sample of linoleic acid methyl ester (C18:2) was treated under the same conditions as a reaction control. Analysis by GLC and GLC-MS of nonlabeled fatty acid methyl esters. Capillary GLC was carried out with a Hewlett–Packard 5890 gas chromatograph with nitrogen as the carrier gas. An HP-5 column (0.32 mm × 50 m) was used. The oven temperature program was 180°C (1 min) to 210°C at 5°C/min, 210°C (7 min) to 270°C at 15°C/min, 270°C (17 min); injector temperature, 230°C; detector temperature, 290°C. GLC-MS was performed on a TRIO-2VS MASSLAB at 70 eV, using an SPB-5 column (0.25 mm × 30 m). A similar temperature program was used, with an initial oven temperature of 150°C. Plasmalogen assay. Samples were dissolved in 90% acetic acid and incubated under N2 at 37°C for 18 hr (Gray 1969). The reaction mixture was dried in a speed-vac concentrator (Savant), redissolved in chloroform:methanol (1: 1), and analyzed by TLC.

RESULTS Nitrogenous-base-containing phosphatides from T. cruzi trypomastigotes (Y strain) metabolically labeled with [3H]palmitic acid were obtained by extraction with chloroform:methanol. Further separation from acidic lipids was achieved by DEAE-Sephadex A-25. Analysis by TLC in solvent A (Fig. 1, lane 1) showed a complex mixture of compounds. Among others, three spots coincident with authentic standards of PE, PC, and LPC were obtained. As PC and

T. cruzi: PHOSPHATIDES IN TRYPOMASTIGOTE FORMS

FIG. 1. TLC followed by fluorography of metabolically labeled neutral lipids from T. cruzi trypomastigotes. Lane 1, lipids extracted from [3H]palmitic acid-labeled trypomastigotes were separated on DEAE-Sephadex and the nonbound fraction was analyzed. Lane 2, the same fraction obtained from [14C]ethanolamine-labeled trypomastigotes. I, II, and III were further analyzed as in Fig. 3. The solvent system was chloroform:methanol:water (65:25:4, by vol). Standards: TAG, triacylglycerols; FA, fatty acids; PE, phosphatidylethanolamine; DMPE, dimethylphosphatidylethanolamine; PC, phosphatidylcholine; LPE, lysophosphatidylethanolamine; SM, sphingomyelin; LPC, lysophosphatidylcholine. O, origin.

lysophosphatidylethanolamine (LPE) overlap in the neutral solvent used, two-dimensional TLC in basic and acidic solvent systems was necessary to confirm their identity and to purify each component (Fig. 2). Quantification of the labeled spots showed a 5:1 ratio between these components. Analysis by TLC in solvent B of the strong bands in the front of the plate (Fig. 1, lane 1) showed them to be mainly triacylglycerols (TAG) and free fatty acids. When free fatty acids were extracted from the plate, methylated, and analyzed by RP-TLC, C16:0 and

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C18:0 in a 5:1 ratio were obtained. The same result was obtained when fatty acids from TAG were analyzed (not shown). The spot with Rf coincident with sphingomielin was extracted, treated with sphingomielinase, and analyzed by TLC, but no ceramide was detected. At this point, an analysis of the zwitterionic lipids obtained from trypomastigotes metabolically labeled with [14C]ethanolamine (Fig. 1, lane 2) became necessary. Strong bands corresponding to PE (I), as well as LPE (III), were observed. A minor component (II) coincident with a standard of dimethylphosphatidylethanolamine (DMPE) was also present. To rule out the possibility that the radiolabel found in PE, LPE, and DMPE appeared in fatty acids due to the long time of incorporation of the radioactive precursor, each spot was eluted from the plate and hydrolyzed with HCl. After extraction with chloroform more than 90% of the radioactivity was recovered in the aqueous phase. This fact indicated that radioactive ethanolamine was incorporated into the polar component of these lipids. The identity of the water-soluble radioactive moieties of these three compounds was confirmed by a TLC analysis on cellulose using solvent system E. In every case one spot of Rf 0.59 coincident with standards of ethanolamine, dimethylethanolamine, and choline (which are not resolved under these conditions) was observed (not shown). No radiolabeled spots were detected elsewhere on the plate, excluding the presence of other related species such as serine. In order to distinguish between the different bases, the three labeled spots of Rf 0.59 were individually eluted from the chromatogram and rerun in solvent system F (Fig. 3). As expected, ethanolamine was detected in the two main components (I and III, Fig. 1, lane 2) while dimethylethanolamine was mainly observed in the minor one (II). Bands migrating above I and below III were also hydrolyzed under the same conditions and analyzed by TLC. Faint bands corresponding to ethanolamine were detected in both cases. The low amount of these components isolated from the mixture precluded further investigation. Interestingly, no radioactive PC was detected

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UHRIG ET AL.

FIG. 2. Purification of PC and LPE. Two-dimensional TLC of the compound migrating as PC and LPE from Fig. 1, lane 1, using chloroform:methanol:13.3 M NH4OH(65:25:5) in the first direction and chloroform:acetone:methanol:acetic acid:water (30:40:10:10:1) in the second direction. Circles correspond to standard PC and LPE that were run in the same plate.

in the [14C]ethanolamine-labeled lipids although PC is the major phospholipid present in T. cruzi epimastigotes (Da Silveira and Colli 1981) and in the [3H]palmitic acid-labeled trypomastigotes (Table I). This fact suggests that PC is not biosynthesized to any appreciable extent by methylation of PE. In contrast, DMPE labeled with the base is present. This result is in accordance with the findings of Rifkin et al. (1995) for T. brucei. Analysis of the lipidic moiety of [3H]palmitic acid-labeled PE, PC, LPE, and LPC of trypomastigotes of T. cruzi. PE, PC, and LPC were purified by TLC from trypomastigotes metabolically labeled with [3H]palmitic acid. These components were hydrolyzed by PLC and the lipids were analyzed by TLC in solvent system C. Lipids migrating as authentic samples of dipalmitoylglycerols were obtained for PC and PE (Fig. 4A, lanes 2 and 3). The presence of the 1,3-diacylglycerol is explained by the wellknown intramolecular acyl migration of the 1,2-

diacyl isomer in organic solvents (Sjursnes and Anthonsen 1994). Only one spot coincident with a standard of 1-palmitoylglycerol was detected in the case of LPC (Fig. 4A, lane 1). When these lipids were recovered from the plate and subjected to alkaline treatment only labeled fatty acids were obtained. This fact confirmed the acyl nature of these compounds. The corresponding fatty acids obtained from each phospholipid were methylated with BF3/ methanol and analyzed by RP-TLC in solvent system D. PC showed palmitic acid (C16:0) together with stearic acid in a 4:1 ratio (Fig. 4B, lane 1); the same fatty acids were found as components of PE, but in a 3:1 ratio (Fig. 4B, lane 3). The only fatty acid found in LPC was C16:0; no C18:0 was detected as component of this lysophospholipid even when the plate was overexposed for a longer period of time. However, LPE showed the presence of both C16:0 and C18:0, in a 6:1 ratio (not shown). Catalytic hydrogenation did not change the pattern obtained,

T. cruzi: PHOSPHATIDES IN TRYPOMASTIGOTE FORMS

FIG. 3. Analysis of the nitrogenous-containing moieties of compounds I, II, and III from Fig. 1, lane 2. Compounds I, II, and III were extracted from the plate (Fig. 1, lane 2), subjected to acid hydrolysis, and analyzed on cellulose TLC sheets using phenol:n-butanol:formic acid:water (47:50:3:10, by vol), saturated with KCl.

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UHRIG ET AL. TABLE I [3H]Palmitic Acid-Labeled Lipids of Trypanosoma cruzi Trypomastigotes

Component

Percentagea

Triacylglycerol PE LPE PC LPC Nondetermined neutral lipids

10.8 3.1 2.6 13.5 5.1 39.0

IPL1 IPL2 Lyso-IPL2 PA Other acidic lipids

6.9 4.9 1.1 2.5 10.5

Note. IPL1, inositolphosphoceramides; IPL2, phosphatidylinositol (diacyl and alkylacyl forms) (Uhrig et al. 1996). a Lipid composition is expressed in percentages of the metabolically incorporated [3H]palmitic acid in a chloroform:methanol extract. Quantification was performed by counting radioacativity after TLC (Figs. 1 and 2) and extraction of the silica gel scrapings corresponding to each compound. Radioactivity corresponding to free fatty acids was subtracted in order to discard unincorporated precursor.

showing the absence of unsaturation (Fig. 4B, lanes 2 and 4). Table II summarizes the results obtained. In order to investigate the presence of plasmalogens in labeled PC and PE, a mild acid hydrolysis with acetic acid was performed (Gray 1969). Analysis by TLC in solvent system A showed only minor degradation to free fatty acids, but neither long-chain aldehydes nor lysophospholipids were detected. PLA2 treatment of PC and PE. Purified [3H]palmitic acid-labeled PC and PE obtained from trypomastigotes of T. cruzi were treated with PLA2 of C. adamanteus venom and the products were analyzed by TLC in solvent system A (Fig. 5). Products coincident with LPC and LPE of T. cruzi, respectively, were obtained. Free fatty acids released by the enzyme appeared at the top of the plate. Analysis of fatty acids by GLC and GLC-MS. In order to compare the fatty acids in nonlabeled samples with those metabolized from [3H]palmitic acid, an analysis by GLC and GLC-MS was performed. The lipids from trypomastigotes

(1 × 1010) were extracted and purified as described above. The fatty acids obtained by saponification of PE, PC, and TAG and the free fatty acids were identified as methyl esters by their retention times in GLC and by GLC-MS (Table III). Although C16:0 and C18:0 were the major fatty acids observed in all cases, C18:1 was also detected. An interesting observation was the presence of C24:0 in a relatively high ratio in the free fatty acid fraction. DISCUSSION In the present work we extend our analysis of lipids from T. cruzi trypomastigotes to the zwitterionic components. We have recently characterized two classes of inositolphospholipids (Uhrig et al. 1996). In an attempt to relate nitrogenous-base-containing phospholipids with putative anchor precursors, [14C]ethanolamine metabolically labeled components were analyzed. While a chloroform:methanol parasite extract was strongly labeled (1 × 106 cpm/109 cells), the more polar extract obtained with chloroform:methanol:water, where glycosylphosphatidylinositol precursors are usually found (Menon et al. 1993), was practically nonlabeled (2 × 103 cpm/109 cells). This result is in accordance with the rapid turnover recently described, for the only ethanolamine-labeled ‘‘glycolipid-A like 1’’ precursor, obtained from metacyclic forms (Heise et al. 1996). An exhaustive analysis of the chloroform: methanol extract in comparison with a palmitic acid-labeled extract was performed. In both cases, PE and LPE were detected. Base incorporation percentages (57.7 and 32.0%, respectively) were in accordance with those described for T. brucei (Rifkin et al. 1995). Accordingly, no PC or LPC was metabolically labeled with the radioactive base although PC is the major phospholipid, as indicated by [3H]palmitic acid incorporation. However, DMPE in an amount of 4.3% was found. Methylation of PE to biosynthesize PC appears to be quantitatively significant in liver. In this case, the phosphatidylethanolamine methyltransferase, which has been isolated and purified, is able to methylate PE, monomethylphosphatidylethanolamine

T. cruzi: PHOSPHATIDES IN TRYPOMASTIGOTE FORMS

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FIG. 4. (A) Analysis by TLC of the lipid moiety of LPC, PC, and PE purified from trypomastigotes metabolically labeled with [3H]palmitic acid. LPC (lane 1), PC (lane 2), and PE (lane 3) were hydrolyzed with phospholipase C and the products were analyzed by TLC using hexane:ethyl ether:acetic acid (70:35:1, by vol) as the developing solvent system. Standards: 1,3-DAG, 1,3-di-O-palmitoylglycerol; 1,2-DAG, 1,2-di-O-palmitoylglycerol; MAG, monoacylglycerol. O, origin. (B) Reverse-phase TLC of the labeled fatty acid methyl esters obtained from PE and PC. Lane 1, fatty acid methyl esters obtained from PC; lane 2, a sample from lane 1 subjected to hydrogenation; lane 3, fatty acid methyl esters obtained from PE; lane 4, idem 3 subjected to hydrogenation. The solvent system was acetonitrile:acetic acid (1:1). Standards: C12:0, C14:0, C16:0, C18:0, and C20:0, methyl esters of dodecanoic acid, myristic acid, palmitic acid, stearic acid, and arachidic acid, respectively. O, origin.

(MMPE), and DMPE. In yeast, two methyltransferase activities have been described by molecular cloning of the genes. One gene codes for the enzyme that catalyzes only the methylTABLE II Fatty Acid Composition of Phospholipids of Trypanosoma cruzi, Trypomastigote Forms Incorporated with [3H]Palmitic Acida Fatty acid 16:0 18:0

PE

LPE

PC

LPC

75% 25%

86% 14%

80% 20%

100% —

a Fatty acid percentages were calculated by counting radioactivity of each eluted spot from the plates shown in Fig. 4B.

ation of PE to MMPE and the second one has preference for the methylation of MMPE and DMPE (Vance 1990). The results obtained would suggest the absence or inhibition of this second activity in trypanosomes, precluding the appearance of labeled PC. Thus, the predominant route of PC biosynthesis in T. cruzi trypomastigotes would be the Kennedy pathway: choline → choline-P → CDP-choline → PC (Weiss et al. 1958). Interestingly, high percentages of LPE and LPC were found when labeling with the fatty acid. It is known that cell manipulation, sonication, or lyophilization may lead to the appearance of lysophosphatides. But the higher per-

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UHRIG ET AL. TABLE III Comparison of Fatty Acid Composition Appearing Free or as Components of PE, PC, and TAG from T. cruzi Trypomastigotesa Fatty acid

Free fatty acids

TAG

PE

PC

14:0 16:1 16:0 18:2 18:1 18:0 20:0 22:0 24:0

0.04 0.01 0.93 + 0.02 1.00 0.02 0.04 0.34

+ + 0.83 − 0.12 1.00 0.10 0.08 0.11

0.28 − 0.77 − 0.05 1.00 − − +

− + 1.05 + 0.65 1.00 0.11 + 0.12

a Fatty acid methyl esters were identified by GLC and GLC-MS. Relative ratios refer to C18:0. +, indicates trace component only.

FIG. 5. [3H]Palmitic acid-labeled components obtained after PLA2 hydrolysis. Lane 1, PC obtained from [3H]palmitic acid-labeled trypomastigotes; lane 2, PLA2 digestion of PC from lane 1; lane 3, LPC obtained from [3H]palmitic acid-labeled trypomastigotes; lane 4, PE obtained from [3H]palmitic acid-labeled trypomastigotes; lane 5, PE from lane 4 after PLA2 digestion. Standards: FA, fatty acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine. O, origin.

centages obtained when compared with epimastigote forms (Da Silveira and Colli 1981), and the fact that the lipid moieties of anchors from two trypomastigote glycoproteins, namely, Tc85 (Couto et al. 1993) and trans-sialidase (Agusti et al., 1997), have 2-lysoglycerol structures, would suggest that trypomastigote forms contain an active phospholipase A2 that hydrolyzes the fatty acid of the 2 position. The ratio of TAG to PC in trypomastigotes is markedly higher than that in epimastigote forms. In fact, for epimastigote whole cells, it

has been reported that 3.6% of TAG and 13.9% of PC can be calculated as percentages of the total lipid extract dry weight (cf. Da Silveira and Colli 1981). As TAGs are thought to be used by protozoal parasites as energy depots and as storage of fatty acyl groups (Dixon and Williamson 1970), the results obtained would be due to the long time of incorporation (in a cell-free medium) without a ready supply of TAG from host cells. Fatty acid composition of labeled phospholipids from the trypomastigote stage shows C16:0 and C18:0 as the only components (Table II). No unsaturated fatty acids were detected. Accordingly, no unsaturated fatty acids were detected, neither free nor as components of inositolphospholipids (Uhrig et al. 1996). On the other hand, C18:1 and traces of other unsaturated fatty acids were detected as components of PC, TAG, and also free, when cold trypomastigote forms were analyzed (Table III). A striking finding was the presence of C24:0 in a relative high proportion, either free or as a component of TAG, PC, and PE. From the comparison of Tables II and III, it follows that while the ratio of C16:0 and C18:0 in PE is 3:1 (when labeled with palmitic acid; Table II), it is 3:4 when not labeled (Table III). The same is true for PC, the ratio of C16:0 and C18:0 being 4:1 (when la-

T. cruzi: PHOSPHATIDES IN TRYPOMASTIGOTE FORMS beled with palmitic acid) but 1:1 when not labeled. Thus, a limited biosynthetic capacity for fatty acid transformation can be observed. Trypomastigote forms were not efficient in elongating C16:0 to C18:0. Furthermore, the absence of unsaturated labeled fatty acids may be ascribed to a low activity of desaturases. Lignoceric acid has been reported as component of the ceramide moiety of LPPG (Lederkremer et al. 1990, 1991, 1993; Previato et al. 1990), and in neutral glycosphingolipids obtained from epimastigote forms (BarretoBergter et al. 1992). On the other hand, C18:2 and C18:1 are the major components of phospholipids of T. cruzi epimastigotes (Timm et al. 1982). It has been suggested that the lipid composition of trypanosomes might reflect the fatty acid content of their environment (Dixon and Williamson 1970). The extent of their absorption varies over the stages of the life cycle, with different fatty acids being absorbed at different points (Dixon et al. 1971). However, for T. lewisi and T. rhodesiense the fatty acid pattern does not mimic that of their medium (Dixon and Williamson 1970). Furthermore, T. cruzi epimastigotes are able to transform palmitate and stearate exogeneously supplied to C18:2 and C18:1 (Lema and Aeberhard 1986). The fact that the lipid composition of metabolically labeled compounds of T. cruzi trypomastigotes is different from that obtained from cold parasites may be explained by a limited biosynthetic capacity of trypomastigotes as nondividing forms and, thus, it must reflect the fatty acid composition of the medium. Phospholipid biosynthesis and regulation may constitute a future area of attack for novel chemotherapeutic agents. ACKNOWLEDGMENTS This investigation received financial support from UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), and Universidad de Buenos Aires to R. M. de Lederkremer; Fundacio´n Antorchas/Vitae to R. M. de Lederkremer and W. Colli; and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP Tema´tico, 95/ 4562-3) and Conselho Nacional do Desenvolvimento Cien-

17

tı´fico e Tecnolo´gico (CNPq62.0251/94.8 PADCT II) to W. Colli and M. J. M. Alves. R.M.L. and A.S.C. are research members of CONICET.

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Received 18 November 1996; accepted with revision 30 April 1997