©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Specificity of Ethanolamine Transport and Its Further Metabolism in Trypanosoma brucei(*)

Mary R. Rifkin (§) , Carolyn A. M. Strobos (¶) , Alan H. Fairlamb (¶)

From the (1)Laboratory of Medical Biochemistry, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ethanolamine is found in trypanosomes as an integral component of the variant surface glycoprotein (VSG) and the membrane phospholipid phosphatidylethanolamine (PE). Steps in the utilization of ethanolamine could represent novel targets for the development of chemotherapeutic drugs and were therefore investigated in detail. Transport of [H]ethanolamine was studied using structural analogs of ethanolamine. Compounds with substitutions in the amino group or of one of the methylene hydrogens of ethanolamine were the most effective inhibitors. Those analogs studied in detail with respect to their kinetic properties were all found to be competitive inhibitors of ethanolamine transport. Following uptake, ethanolamine is rapidly phosphorylated by an ethanolamine-specific kinase to form phosphoethanolamine. Other acid-soluble intermediates identified by thin layer chromatography were CDP-ethanolamine, dCDP-ethanolamine, and glycerophosphorylethanolamine. The relative amounts of these metabolites varied between slender (dividing) and stumpy (non-dividing) trypanosomes and may reflect special biosynthetic needs of the different morphological forms. Pulse-chase experiments indicated that the acid-soluble metabolites served as precursors for chloroform/methanol-soluble lipids. Radioactive lipids included PE, mono-methyl and dimethyl PE, and lysoPE. Further methylation of dimethylPE to phosphatidylcholine was not observed under the experimental conditions described. These results are consistent with the conclusion that trypanosomes are able to synthesize phospholipids via the Kennedy pathway.


INTRODUCTION

Ethanolamine may play a pivotal role in the survival of the parasitic protozoan Trypanosoma brucei brucei in the bloodstream of the mammalian host. Ethanolamine is taken up by T. b. brucei by a specific transport system and is utilized directly in the synthesis of the glycolipid anchor of the variant surface glycoprotein (VSG)()of this parasite(1) . The surface glycoprotein is important in protecting the parasite from immune lysis, mediated by antibodies to plasma membrane antigens. Thus, a trypanosome which can no longer synthesize or replace its surface glycoprotein coat would be destroyed. The uptake and utilization of ethanolamine becomes of interest as a possible target for chemotherapy of the diseases caused by these protozoa.

Ethanolamine is also a major component of trypanosome membrane phospholipids. Phosphatidylethanolamine (PE) constitutes 25-30% of the total phospholipid of T. b. brucei(2) . To satisfy their biosynthetic requirements for ethanolamine, trypanosomes must either efficiently scavenge ethanolamine from the external milieu or derive it via unusual metabolic pathways from other metabolites. We have previously described a specific ethanolamine transport system with a K that corresponds to the plasma concentration of ethanolamine(1) . Uptake of ethanolamine via this transporter can satisfy the ethanolamine requirements of the cell for phospholipid and VSG biosynthesis; however, there is little knowledge of any of the biosynthetic pathways involved in the further metabolism of ethanolamine.

Because of the major role PE plays not only in maintaining membrane homeostasis but also in serving as the donor for phosphoethanolamine in the biosynthesis of VSG(3) , it was of interest to study the pathway for PE biosynthesis in trypanosomes. In mammalian cells, if exogenous ethanolamine is readily available, the majority of PE is formed via the CDP-ethanolamine pathway (``Kennedy pathway'')(4) ; however, in tissue culture medium lacking ethanolamine, the decarboxylation of phosphatidylserine (PS) becomes the major pathway for biosynthesis of PE(5) . In bacteria and yeast, decarboxylation of PS is the major biosynthetic pathway, although the Kennedy pathway enzymes exist in these cells(6) . A third pathway for the direct incorporation of ethanolamine into phospholipids is by the base exchange pathway(7) . In trypanosomes, the contributions of these various pathways for PE biosynthesis have not been studied.

In this paper we describe the structural properties of ethanolamine that are important for uptake by the ethanolamine transporter. Inhibition by ethanolamine analogs of both the transport system and ethanolamine kinase, the first enzyme in the ethanolamine metabolic pathway, is described and may provide the basis for further development of effective inhibitors for these pathways. Metabolic labeling and pulse-chase experiments provide evidence that trypanosomes utilize the Kennedy pathway for most of their PE biosynthesis. In addition to the phospholipid biosynthetic intermediates of the Kennedy pathway, phosphoethanolamine (P-Etn) and CDP-ethanolamine (CDP-Etn), two minor metabolites, dCDP-ethanolamine (dCDP-Etn) and glycerophosphoethanolamine (GPE), were found which may be important in other biosynthetic pathways.


EXPERIMENTAL PROCEDURES

Trypanosomes

Bloodstream forms of T. b. brucei variant 117 (MITat 1.4) or strain EATRO 110 were isolated from the blood of 3-day infected BALB/c mice or Sprague-Dawley rats by DEAE-cellulose chromatography as described previously(1) . These strains were used interchangeably. In experiments where ``slender'' and ``stumpy'' trypanosomes were compared, T. b. brucei strain TREU 667 (8) was used. In this case, C3H/HeJ mice received 200 mg/kg cyclophosphamide (Cytoxan, Mead Johnson) intraperitoneally 24 h prior to trypanosome injection(9) . Trypanosomes were isolated either 3-4 days after infection (log phase growth, 67-84% slender and dividing trypanosomes) or 7-8 days after infection (4-7% slender and dividing trypanosomes, 93-97% intermediate and stumpy trypanosomes).

Ethanolamine Transport and Incorporation into Metabolites

Transport of [H]ethanolamine was measured as described(1) . In experiments with ethanolamine analogs, the final concentrations in the incubation medium were 1 µC/ml of [H]ethanolamine (New England Nuclear, specific activity 8.8 Ci/mmol), 1.11 µM ethanolamine, and 500 µM analog.

Ethanolamine metabolites were extracted from trypanosomes incubated for 60 min at 37 °C in minimum Eagle's medium (MEM, Life Technologies, Inc.) containing the following final concentrations: 10 mg/ml of fatty acid-free albumin (Sigma), 1 µM ethanolamine, 5 µC/ml of [H]ethanolamine, and 2.5 10 trypanosomes/ml. Cells were washed twice by centrifugation and resuspension in large volumes of MEM, and finally resuspended in 0.5 ml of cold MEM containing 2% (w/v) bovine serum albumin (Sigma). Contamination by free [H]ethanolamine carried over from the initial incubation medium to the final washed, resuspended cells was negligible. In some cases trypanosomes were diluted with unlabeled trypanosomes after labeling but before washing.

For extraction of acid-soluble metabolites, 4 volumes of cold 0.6 N perchloric acid (PCA) was added to the washed trypanosome suspension(10) . After 30 min on ice, PCA-insoluble material was removed by centrifugation and the supernatant neutralized with 2.5 N KHCO, and kept on ice for another 30 min. The resulting KHClO precipitate was removed by centrifugation. It was important to use KHCO rather than KOH for neutralizing the PCA as neutralization with KOH sometimes led to significant losses of CDP-Etn.

The supernatant was lyophilized and subsequently reconstituted in a small volume of water, usually 0.1 ml, and stored at -20 °C. This PCA-soluble extract was analyzed on cellulose Chromagram sheets (Kodak) using two solvent systems: system A (butanol/glacial acetic acid/HO, 5:2:3) or system B (100% ethanol/concentrated ammonia/HO, 80:4:16). Authentic ethanolamine, P-Etn, and CDP-ethanolamine (all from Sigma) were used as standards. Metabolites were identified by ninhydrin spray (1% in acetone) or by fluorography at -70 °C following impregnation of the Chromagram sheet with EnHance (DuPont New England Nuclear).

Because dCDP-Etn is not commercially available but was needed as a standard in analysis of acid-soluble metabolites by thin layer chromatography (TLC), dCDP-Etn was synthesized by a modification of the procedure of Schneider et al.(11) . A 20% (w/v) rat liver homogenate in 0.145 M NaCl, 1 mM phenylmethylsulfonyl fluoride (Sigma), 0.1 mMp-tosyl-L-lysine chloromethylketone (Sigma) was centrifuged at 100,000 g for 90 min at 4 °C and the supernatant used as a source of phosphoethanolamine cytidylyltransferase. The reaction mix contained 10 mg protein/ml and 3 mM ATP, 3 mM MgCl, 20 mM 3-phosphoglycerate, 5 mM phosphoethanolamine, 20 mM Tris-succinate, pH 6.0, and either 2 mM CTP or 2 mM dCTP and was incubated at 30 °C for 2 h. The reaction was stopped by heating at 100 °C for 4 min and then centrifuged in a microfuge for 6 min. Aliquots (100 µl) of the supernatant were passed through activated charcoal columns, prepared in disposable pipette tips, to remove excess phosphoethanolamine and to facilitate subsequent TLC analysis of the products formed. After loading, charcoal columns were washed three times with distilled water, and then nucleotide-linked metabolites were eluted with 0.5 M ammonium hydroxide in 60% ethanol. The extracted material was taken to dryness in a SpeedVac and resuspended in 100 µl of water for chromatography. CDP-Etn and dCDP-Etn were identified by ninhydrin staining and UV absorption spectra and their R values on Chromagram sheets were determined in different solvent systems.

For identification of chloroform/methanol (CM)-soluble metabolites, [H]ethanolamine-labeled trypanosomes were extracted by the method of Folch et al.(12) . Lipid extracts were stored in chloroform/methanol (2:1) under N at -20 °C. One-dimensional TLC analysis was performed on Silica Gel G plates (Analtech) using the solvent system chloroform/methanol/glacial acetic acid/HO (25:15:4:2). Two-dimensional analysis was done on Redi-Coat 2D plates (Supelco), prewashed in acetone/petroleum ether (1:3), using the solvent systems of Turner and Rouser(13) . Metabolites were located by fluorography (see above for PCA-soluble material). Phosphate content of lipid extracts or of individual radioactive spots was quantitated by the Bartlett method(14) . Radioactivity of extracts was determined by counting small aliquots in Liquiscint (National Diagnostics) and of TLC spots by scraping the spot into a scintillation vial, adding 0.4 ml of HO, and incubating with shaking at 37 °C for 1 h before adding Liquiscint.

Ethanolamine Kinase Assays

Ethanolamine kinase was assayed by a modification of the method of Weinhold and Rethy(15) . This method separates the ethanolamine substrate from the P-Etn product by Dowex AG-50X8 [H] (Bio-Rad) chromatography. [C]Ethanolamine (New England Nuclear, specific activity 49 mCi/mmol) or [H]ethanolamine (New England Nuclear, specific activity 30 Ci/mmol) was repurified before use(15) . Trypanosome homogenates, made by lysing purified trypanosomes in 9 volumes of water or a high speed supernatant prepared from the homogenate (100,000 g, 60 min), were assayed for kinase activity. Incubation mixtures contained 3 mM MgCl, 200 mM KCl, 60 mM glycylglycine buffer, pH 8.5, 0.5 mM ethanolamine ([C]ethanolamine, 2 µCi/µmol), 3 mM ATP, and either homogenate (1.84 mg protein/ml) or 100,000 g supernatant (0.64 mg/ml). In some experiments, mixtures were supplemented with an ATP regenerating system consisting of 10 mM phosphoenolpyruvate and 6 units/ml pyruvate kinase. Reactions were incubated for 60 min at 30 °C. Protein was determined by the Bradford assay (16) using bovine serum albumin as a standard.


RESULTS

Ethanolamine Transport Inhibitors

Inhibition of ethanolamine transport might constitute a novel approach to the chemotherapy of trypanosomiasis. This would require a knowledge of the structural constraints on the ethanolamine substrate that might interfere with recognition by the ethanolamine transporter. Ethanolamine analogs, which differed from ethanolamine at either the amino or hydroxyl end or had different carbon backbones, were tested for their ability to inhibit ethanolamine transport by trypanosomes (). The results indicate that compounds with substitutions at the hydroxyl end do not inhibit ethanolamine transport as well as compounds with substitutions at the amino end. Thus, substitution of the hydroxyl group with a sulfhydryl group(2-aminoethanethiol), sulfonic acid moiety (taurine), hydrogen (ethylamine), or phosphate group (phosphoethanolamine) or substitution of the hydroxymethyl group by a carboxyl moiety (glycine) or by an aldehyde group (aminoacetaldehyde) had little or no effect on ethanolamine transport indicating that these compounds were not effectively recognized by the transport system. Increasing the length of the carbon backbone by a single methylene group (3-amino-1-propanol) dramatically decreased the ability of the analog to compete with ethanolamine. However, compounds in which one of the methylene hydrogens was replaced with a methyl group (2-amino-1-propanol) or an ethyl group (2-amino-1-butanol) were good inhibitors of ethanolamine transport. Analogs with either one or two methyl or ethyl substituted amino groups also effectively competed with ethanolamine in this assay. Other compounds which had little (less than 27% inhibition) or no inhibitory activity under the same experimental conditions are not listed in ; these were betaine, methylamine, dimethylamine, 2-(2-aminoethoxy)ethanol, N-(3-aminopropyl)diethanolamine, N,N-dimethylglycine,N,N-di-methylglycine ethyl ester, lysine, methylaminoacetaldehyde dimethylacetal, and dimethylaminoacetaldehyde diethylacetal. The acetals were converted to the free aldehydes by incubation in 0.1 N HCl for 30 min immediately before starting the assay. In summary, the trypanosome ethanolamine transporter seems to have a strong requirement for a hydroxymethyl group at one end of the substrate, a methylene group that can accommodate substitution of one of the hydrogens, but not both, by a small alkyl group (e.g. methyl, ethyl), and a preference for a primary amine at the other end of the substrate.

To characterize further the nature of the inhibition of ethanolamine transport, the inhibitor constant (K) was determined for several of the most effective inhibitors in . Fig. 1A shows the competitive nature of N,N-dimethylethanolamine inhibition of ethanolamine transport (K = 90 µM). Similar results were obtained for N-methylethanolamine (K = 50 µM), L-2-aminopropanol (K = 58 µM), and both D- and L-2-amino-1-butanol (K = 11 µM). For comparison, the K for ethanolamine is 3.7 µM(1) . Fig. 1B shows that the transport system shows no stereospecificity at the methylene carbon of ethanolamine since both D- and L-2-amino-1-butanol give the same K.


Figure 1: Determination of inhibition constant (K) by Dixon plot analysis for N,N-dimethylethanolamine (A) or D- and L-2-amino-1-butanol (B). Cells were incubated as in Table I in either 3 µM ethanolamine (, , ,) or 6 µM ethanolamine () and increasing concentrations of inhibitor. In the experiment shown in A, the calculated K for ethanolamine was 1.8 µM. In B, 1/V was 6 (nmol/min/mg protein), as determined from a separate experiment.



Acid-soluble Metabolites

Chromatography of a [H]ethanolamine-labeled PCA extract on cellulose in an acid solvent system revealed one major and three minor spots (Fig. 2). These spots were identified as ethanolamine, P-Etn, dCDP-Etn, and CDP-Etn based on a comparison of their R values with authentic standards ( Fig. 2and Fig. 3, A and B). Radioactive material comigrating with P-Etn constituted 65-90% of the total radioactivity applied to the plate, while 3-30% corresponded to ethanolamine, and 4-10% to CDP-Etn and dCDP-Etn. In rat liver, the rate-limiting step in PE biosynthesis via the Kennedy pathway is the conversion of P-Etn to CDP-Etn, resulting in very low steady-state levels of CDP-Etn. The distribution of [H]ethanolamine radioactivity among the trypanosome acid-soluble metabolites (Fig. 2) is similar to that found in rat liver (17) and is consistent with the conclusion that also in trypanosomes the formation of CDP-Etn is rate-limiting.


Figure 2: Autoradiogram of [H]ethanolamine labeled PCA-soluble metabolites separated by chromatography on cellulose sheets and run in solvent system A, as described under ``Experimental Procedures.'' Lane 1, strain EATRO 110 labeled for 2 h at 37° C with 4 µC of [H]ethanolamine/ml; lane 2, strain TREU 667, 4-day infection, labeled for 1 h at 37° C with 3 µC of [H]ethanolamine/ml; lane 3, strain TREU 667, 7-day infection, labeled as trypanosomes in lane 2. Authentic standards were run in an adjacent lane, located by ninhydrin spray, and their R values determined. O = origin.




Figure 3: Identification of the major PCA-soluble metabolite as phosphoethanolamine. A, distribution of [H]ethanolamine counts in acid-soluble extract. Samples (5 µl) were analyzed on a cellulose Chromagram sheet using solvent system A. Standards were run as in Fig. 2. One-cm sections of the lane containing the PCA extract were placed in scintillation vials, extracted with 0.4 ml of HO for 30 min at 37° C, and 10 ml of Liquiscint added for scintillation counting. 92% of the spotted dpm were recovered. The region corresponding to phosphoethanolamine (hatched area) from an adjacent lane was scraped into a centrifuge tube and eluted by incubating in 1 ml of 1 mM phosphoethanolamine. The cellulose was sedimented by centrifugation, the supernatant lyophilized, and resuspended in a small volume of HO. Aliquots of the eluted material were taken for analysis by TLC and run in basic solvent system B (B) or in solvent system A after alkaline phosphatase digestion (C). B, distribution of radioactivity after chromatography of a 5-µl aliquot using the basic solvent system B, and analyzed as in A. C, another aliquot (50 µl) was incubated with 25 units of alkaline phosphatase (Sigma, Type VII-NL) in 10 mM Tris-HCl, pH 8.0, 1 mM MgCl, 0.1 mM ZnCl for 2 h at 37° C. A control aliquot was kept on ice. Portions of the digested and undigested samples were run in solvent system A. Chromagrams were analyzed as in A. Solid line, alkaline phosphatase digested; dashed line, control (no digestion). O = origin; F = front.



To ascertain that the major soluble radioactive metabolite was indeed P-Etn, material having an R = 0.43 in solvent system A (Fig. 3A) was eluted from the chromatogram and rerun in solvent system B (Fig. 3B). 83% of the counts had the same R as authentic P-Etn run in an adjacent lane. Similarly, material comigrating with P-Etn in solvent system B, eluted and rerun in solvent system A, again comigrated with P-Etn (data not shown). Identity of the major radioactive metabolite in Fig. 3A as P-Etn was confirmed by alkaline phosphatase digestion. After alkaline phosphatase digestion, 72% of the radioactivity in the digest migrated with an R = 0.68, equivalent to that of ethanolamine (Fig. 3C).

Fig. 3B reveals another minor metabolite (labeled with an asterisk) which comigrates with P-Etn under acid conditions but which can be separated from it under basic conditions. The R of this material in these two solvent systems suggest that it is GPE. GPE would be insensitive to alkaline phosphatase digestion and thus may also account for the apparent ``undigested'' P-Etn in Fig. 3C. GPE could be an intermediate in a lipid biosynthetic pathway, similar to the postulated involvement of glycerophosphorylcholine in the synthesis of lung phosphatidylcholine(18) . Alternatively, GPE could be a degradation product resulting from the deacylation of lysoPE by phospholipase A (19). In either case, it does not contribute significantly to the radioactivity in the P-Etn fraction and should not affect the interpretation of the data of pulse-chase experiments to be described below.

While P-Etn was always the major ethanolamine metabolite, variability was found in the relative amounts of the other ethanolamine metabolites. To test whether this variability in Etn content may be due to the stage of the growth cycle at which the parasites are isolated, the acid-soluble metabolites of slender and stumpy trypanosomes were compared. Using the pleomorphic T. brucei strain 667, parasites isolated from the logarithmic, rapidly dividing phase had relatively less ethanolamine and dCDP-Etn (Fig. 2, lane 2), while these same metabolites were more abundant in cell populations containing large numbers of stumpy (non-dividing) trypanosomes (Fig. 2, lane 3). Thus, the intracellular pools of ethanolamine metabolites seems to vary according to the morphological subtypes of trypanosomes in the population and may reflect different biosynthetic needs of these different trypanosome populations.

Ethanolamine Kinase

The formation of P-Etn is likely due to an ethanolamine kinase, which serves to trap the newly transported ethanolamine in the cell. Consequently, the properties of this enzyme in trypanosomes were characterized, using the assay developed by Weinhold and Rethy(15) . Trypanosome ethanolamine kinase is likely to be a cytosolic enzyme since enhanced kinase specific activity was found in the high speed supernatant of a trypanosome homogenate. The mean specific activity in four experiments was 228 ± 27 pmol/min/mg protein for freshly prepared homogenates and 526 ± 48 pmol/min/mg protein for a high speed supernatant. In the absence of ATP, kinase activity was only 3-4% of that found in reaction mixtures containing ATP. Kinase activity was further enhanced over 2-fold when an ATP-generating system was present in addition to ATP, suggesting that ATP serves as a phosphate donor. The activity was somewhat unstable since freezing led to an approximately 50% loss of activity.

The trypanosome ethanolamine kinase had a broad pH optimum of 8-8.5 (data not shown). The kinetic properties of this enzyme were determined and the K for ethanolamine and the V were found to be 2.75 µM and 32.4 pmol/min/mg protein, respectively (Fig. 4). Choline, which does not inhibit ethanolamine transport in trypanosomes, had very little effect on trypanosome ethanolamine kinase (K > 600 µM for choline) (Fig. 5). This is in contrast to the rat liver ethanolamine kinase which is effectively inhibited by choline(20) . However, N,N-dimethylethanolamine, another rat liver ethanolamine kinase inhibitor (20) and an inhibitor of ethanolamine transport in trypanosomes, inhibited the trypanosome kinase with a K of 22.5 µM (Fig. 5). Thus, trypanosomes seem to have evolved an ethanolamine uptake system which is uniquely designed to both transport and trap ethanolamine, even in the presence of the much more abundant metabolite choline.


Figure 4: Kinetics of P-Etn formation by ethanolamine kinase in a high speed supernatant fraction of a trypanosome lysate. A, initial rates of phosphoethanolamine formation measured as described under ``Experimental Procedures.'' The assay mix contained 12.6 µCi/ml [H]ethanolamine. B, reciprocal plot of S/VversusS where S is the ethanolamine concentration and V is the initial rate of uptake (pmol/min/mg protein). The V and K values derived from this plot by linear regression analysis (correlation = 0.995) are 32.4 pmol/min/mg protein and 2.75 µM, respectively.




Figure 5: Dixon plot analysis of inhibition of ethanolamine kinase activity by N,N-dimethylethanolamine and choline. The incubation tubes contained 2.3 µM [H]ethanolamine (5.55 µCi/nmol) and increasing concentrations of inhibitor. A high speed supernatant fraction of a trypanosome lysate was used as a source of the enzyme. V (32.4 pmol/min/mg protein) was derived from Fig. 4B. Data was analyzed by linear regression analysis (correlation = 0.988). The K for N,N-dimethylethanolamine derived from this plot is 22.5 µM.



Chloroform/Methanol-soluble Metabolites

Thin layer chromatography of a Folch extract of [H]ethanolamine-labeled trypanosomes is shown in Fig. 6. By one-dimensional analysis (Fig. 6A) 64% of the total counts migrated as PE, 6% as dimethyl-phosphatidylethanolamine (DMPE), and 30% in the region where phosphatidylcholine (PC) and lysophosphatidylethanolamine (LPE) overlap. Two-dimensional TLC confirmed the identity of these radioactive lipids as PE, DMPE, and LPE (Fig. 6B). No radioactivity was seen in PC or any other lipids with labeling periods up to 2 h. If, instead, the Bligh and Dyer (21) method was used to extract labeled trypanosomes, some additional radioactive spots of unknown identity, constituting less than 2.5% of the total radioactivity, appeared which migrated ahead of PE in a one-dimensional analysis (data not shown). A comparison of the relative lipid labeling pattern of slender and stumpy trypanosomes (T. brucei strain 667) showed no difference, although the specific activity (dpm/µg phosphate) of the stumpy extract as well as of individual lipids (PE and LPE) was 64-69% of that of slender trypanosomes.


Figure 6: Autoradiograms of 1-D (A) and two-dimensional (B) thin layer chromatography of a Folch extract of [H]ethanolamine-labeled trypanosomes. Trypanosomes (strain 110) were labeled for 90 min at 37° C in MEM containing 2.6 µC of [H]ethanolamine/ml and 2.5 10 trypanosomes/ml. After the labeling period, cells were diluted with MEM containing 1 mM ethanolamine, washed two times by centrifugation and resuspension, and finally resuspended in 2% albumin. PCA was added as described under ``Experimental Procedures,'' and the PCA-insoluble material was extracted by the Folch method. In A, standard lipids (all from Sigma) were run in adjacent lanes, while in B a standard serum lipid mixture (Supelco) was run simultaneously on a separate plate. In A the solvent system was chloroform/methanol/acetic acid/H0 (25:15:4/2); in B, the solvent systems were chloroform/methanol/28% aqueous ammonia (65:30:5) in the first direction and chloroform/acetone/methanol/acetic acid/H0 (30:40:10:10:5) in the second direction. Standards were located by iodine vapor. LPE = lysophosphatidylethanolamine, SP = sphingomyelin, LPC = lysophosphatidylcholine, PI = phosphatidylinositol, PS = phosphatidylserine, PA = phosphatidic acid, O = origin.



Time Course of [H]Ethanolamine Incorporation

Incorporation of [H]ethanolamine incorporation into total PCA-soluble and PCA-insoluble, chloroform/methanol-soluble material occurs with no appreciable lag (Fig. 7A). A more detailed analysis of the incorporation at shorter times is seen in Fig. 7B for PCA-soluble metabolites and Fig. 7C for lipid metabolites. A very short lag may be seen for PE (1 min) and LPE (2 min), while the lag for DMPE is difficult to ascertain due to the low amounts of radioactivity incorporated into this metabolite. Essentially no lag was seen for P-Etn, CDP-Etn, or Etn.


Figure 7: Time course of incorporation of [H]ethanolamine into individual metabolites. Trypanosomes (EATRO 110) were incubated at 37° C in 2.6 µCi of [H]ethanolamine/ml. At each time point, duplicate aliquots equivalent to 10 cells were removed and added to 1.5 volumes ice-cold MEM containing 1 mM ethanolamine. After several washes, the cells were resuspended in 0.5 ml 2% (w/v) albumin. 0.4 ml was used for PCA extraction as described under ``Experimental Procedures.'' PCA-insoluble material was extracted by the Folch method (=CM soluble fraction). Each time point is the average disintegrations/min of duplicate extractions. A, PCA-soluble and -insoluble material. The data represent the total radioactivity associated with the acid-soluble or -insoluble (CM soluble) fraction of 8 10 trypanosomes. B = acid soluble metabolites and C = chloroform/methanol-soluble metabolites. Detailed analysis of the first 15 min of incubation. 10-µl aliquots of the PCA extracts and chloroform/methanol extracts were separated and quantitated by thin layer chromatography as in Figs. 2 and 6, respectively.



The P-Etn pool appeared to saturate by 10 min and so in pulse-chase experiments to be described below, the chase period was started at 10 min. The leveling off of incorporation into P-Etn was not due to exhaustion of radioactive ethanolamine in the incubation medium as the amount of ethanolamine (1.1 µM) is sufficient for several hours of uptake at a rate of 132 pmol/min/mg protein(1) .

Using the concentration (1.1 µM) and the specific radioactivity (4.2 µC/nmol) of [H]ethanolamine in the incubation medium, we can calculate from the data in Fig. 7, B and C, that during the initial 10 min of incubation the rate of uptake of ethanolamine into the acid-soluble pool is 49 pmol/min/mg protein while the rate of uptake into lipid is 64.5 pmol/min/mg protein. The rate-limiting step of ethanolamine incorporation into phospholipid is therefore unlikely to be due to the enzymes involved in phospholipid biosynthesis but rather due to the rate of transport of ethanolamine into the cell.

Pulse-chase experiments revealed a very rapid turnover of the PCA-soluble metabolites (Fig. 8A). Immediately upon adding cold ethanolamine, the radioactivity in the PCA-soluble pool decreased with a half-time of 5 min (Fig. 8B), while the radioactivity in the chloroform/methanol extract continued to increase for 2 min after the start of the chase period. The immediate decrease in the PCA-soluble pool is the result of an immediate decrease in the radioactive P-Etn and other PCA-soluble metabolites (Fig. 8C).


Figure 8: Pulse-chase analysis of [H]ethanolamine incorporation into PCA-soluble and chloroform/methanol-soluble fractions. Cells were labeled at 37° C in 5 µCi of [H]ethanolamine/ml, 1.1 µM final ethanolamine, as in Fig. 7. At 10 min, the chase was initiated by adding to the incubation medium 1/20 volume of prewarmed 0.1 M ethanolamine in MEM to give a final ethanolamine concentration of 5 mM. Duplicate aliquots were removed at the indicated times and processed as in Fig. 7. A, disintegrations/min derived from 2 10 trypanosomes are plotted. B, PCA-soluble radioactivity from the first 10 min of the chase period in A replotted to estimate the half-life of radioactivity in this fraction. Aliquots of the PCA-soluble and chloroform/methanol-soluble fractions at each time point were analyzed by TLC as in Fig. 7, B and C. C, PCA-soluble metabolites: D, CM-soluble metabolites.



Closer analysis of the radioactive chloroform/methanol-soluble metabolites (Fig. 8D) suggests that the continued incorporation of radioactivity into the CM-soluble metabolites for 2 min after the start of the chase period as well as the subsequent decrease was reflected in the radioactive PE fraction. In contrast, no decrease in radioactivity during the chase period was found in LPE or DMPE, suggesting that these metabolites continue to be synthesized at the expense of PE.

It is interesting to note that while trypanosomes are able to methylate PE to form DMPE ( Fig. 6and Fig. 8), further methylation to PC does not occur to any appreciable extent. This is surprising since it is believed that a single enzyme is involved in the sequential methylation of PE to form PC(22) . Maybe the formation of the methyl donor, S-adenosylmethionine, is compromised under our incubation conditions. Alternatively, since PE methyl transferases are influenced by binding of boundary lipids to the enzyme(23) , under our in vitro incubation conditions the catalytic activity might be compromised due to changes in the relative abundance of various types of phospholipid species.


DISCUSSION

For trypanosomes the major source of ethanolamine is the host's plasma, where the free ethanolamine concentration is around 12 µM(24, 25) . The properties of the ethanolamine transport system of trypanosomes distinguishes it from the mammalian and yeast ethanolamine transport systems. In some mammalian cells ethanolamine and choline, a structurally related phospholipid headgroup, are transported by separate transporters(26, 27) , whereas in other cells they share the same transporter(27, 28, 29) . In yeast, genetic evidence favors a single protein for uptake of both ethanolamine and choline. In mutants defective for choline transport, ethanolamine uptake was also decreased(30) . Reintroduction of the yeast choline transporter gene into choline transport-deficient yeast restored both choline and ethanolamine transport activity(31) . In trypanosomes, however, ethanolamine transport is barely affected by a 500-fold molar excess of choline (). Moreover, choline transport studies with bloodstream trypanosomes over a wide range of external choline concentrations (0.02 µM to 2 mM) showed no saturable transport process for the uptake of choline.()The difference between trypanosomes and other cells may be related to the cells' need to have uptake of these phospholipid precursors tightly regulated with certain biosynthetic processes. Thus, in neural cells, where the biosynthesis of acetylcholine is crucial to the correct functioning of the neuron, a choline transport system is well-documented(27) . In trypanosomes, PE serves as the P-Etn donor in the biosynthesis of the VSG glycolipid anchor(3) . Because trypanosomes depend on protective layer of 10 VSG molecules on their surface for survival in the mammalian host, trypanosomes may have modified a choline-type transporter to exclusively transport ethanolamine to suit their particular biosynthetic need for ethanolamine.

The substrate structure requirements of the trypanosome ethanolamine transport system share some properties with choline transport systems in other cells(27, 32, 33) . Analog studies have shown that both ethanolamine and choline transporters have an essential requirement for the substrate hydroxyl group ( and Refs. 32, 33). Any alteration, such as substitution with a thiol group, carboxyl group, hydrogen or halogen atom, resulted in very weak inhibition of transport, indicating that such molecules are not recognized by the transporter. A possible explanation would invoke a role for the hydroxyl group as a hydrogen bridge donor to some amino acid side chain of the transport protein. At the amine end of the substrate, quaternary amines are the best inhibitors of choline transport(32, 33) . Other amines exhibited decreasing efficacy of inhibition in the order tertiary (N,N-dimethylethanolamine) > secondary (N-methylethanolamine) > primary (ethanolamine). However, the degree of inhibition of trypanosome ethanolamine transport by such analogs, as described in this paper, is in the reverse order, i.e.N-methylethanolamine (K= 50 µM) is a more effective transport inhibitor than N,N-dimethylethanolamine (K = 90 µM). Choline was an extremely poor inhibitor and even in 500-fold molar excess only decreased ethanolamine transport by 23% ().

Phosphorylation represents a general mechanism whereby cells take up and trap substances which are not taken up by active transport(34) . Trypanosomes rapidly convert ethanolamine to P-Etn (Fig. 8). The trypanosome ethanolamine kinase, like other ethanolamine kinases(15, 20, 35) , is an ATP-requiring, soluble enzyme with a broad pH optimum around pH 8. Rat liver and yeast each have two ethanolamine kinase activities(15, 35) . Ethanolamine kinase II phosphorylates both choline and ethanolamine but with a higher affinity for choline, while a minor activity, ethanolamine kinase I, exclusively uses ethanolamine as a substrate. The trypanosome activity most resembles the rat liver ethanolamine kinase I activity in that it is not inhibited by choline. Our data also suggest that trypanosomes do not appear to have an ethanolamine kinase II-like activity.

Thus, whereas other cells have a transporter for choline, which also transports ethanolamine, and a choline kinase activity, which also phosphorylates ethanolamine, trypanosomes seem to have developed a system exclusively for the transport of ethanolamine and its phospohorylation in order to efficiently deliver ethanolamine from the extracellular milieu to phospholipid biosynthetic pathways.

Deoxycytidine metabolites, such as dCDP-choline or dCDP-Etn, have been described in a variety of mammalian tissues(36, 37) . The function of the deoxyribonucleotide metabolites is not known, although their relative levels varies in different tissues. Because enzymes in the phospholipid biosynthetic pathway do not discriminate between the deoxyribonucleotide and ribonucleotide moiety(38) , it has been proposed that the ratio of dCDP-Etn to CDP-Etn is simply a reflection of the ratio of dCTP to CTP in the cell(38, 39) . Alternatively, dCDP-Etn and CDP-Etn may be used in different compartments of the cell (for example, nucleus versus cytoplasm) for PE biosynthesis (40). In rat liver, ethanolamine phosphotransferase is found in both the endoplasmic reticulum and in the Golgi fraction(41) . It has been postulated that the Golgi synthesizes phospholipid subspecies that are used for addition to secretory lipoproteins(42) . In trypanosomes, the presence of dCDP-Etn and CDP-Etn may reflect a compartmentalization of PE biosynthesis such that ethanolamine metabolites necessary for VSG biosynthesis are efficiently routed to the site of VSG biosynthesis (e.g. endoplasmic reticulum) while bulk phospholipid synthesis takes place in a separate cellular compartment. Alternatively, different intermediates may be used for diacylglycerophospholipid and for plasmalogen biosynthesis, which in trypanosomes are synthesized in peroxisomes(43) . The variability in the relative amounts of these cytidine-linked metabolites in trypanosomes might also reflect the different biosynthetic needs of different morphological forms, i.e. slenders and stumpies (Fig. 2).

The rate of the extremely rapid appearance of radioactive ethanolamine into a lipid fraction (64.5 pmol/min/mg protein) (Fig. 7C) exceeds that which one would expect from the rate of formation of P-Etn (49 pmol/min/mg protein) (Fig. 7B). Therefore, in addition to PE biosynthesis by the Kennedy pathway, it is likely that some direct incorporation of ethanolamine into PE occurs without formation of P-Etn, possibly involving headgroup exchange between PE and an existing phospholipid headgroup.()A third known pathway for synthesis of PE is the decarboxylation of phosphatidylserine. Experiments by Menon et al.(3) have shown that in trypanosomes radioactive serine can be incorporated into PE. Thus, trypanosomes appear to have the ability to synthesize PE by all the known biosynthetic pathways. As in other eukaryotic cells, the Kennedy pathway accounts for the majority of PE biosynthesis in trypanosomes.

Because membrane phospholipids determine membrane fluidity and the surface charge of cell surfaces, both important parameters for the proper functioning of cell membranes, ethanolamine utilization and phosphatidylethanolamine biosynthesis controls a variety of cellular processes. It has been reported that in the absence of ethanolamine, growth of epithelial cells in vitro stops(44) . In ethanolamine-deficient medium, the ratio of PC to PE increases, and protein kinase C does not function normally presumably due to abnormal association of the enzyme with PE-deficient/PC-excess membranes(45) . The binding of ligands, such as growth factors, to cell surface receptors is also affected by the relative levels of PE, which can either mask or unmask hidden receptors(46) . Gross changes in membrane fluidity can affect glucose transport(47, 48) . Thus, it is likely that inhibition of ethanolamine uptake and/or utilization by trypanosomes could severely compromise their growth and thus constitute a future area of attack for novel chemotherapic agents.

  
Table: Inhibition of ethanolamine transport by analogs

Ethanolamine transport by T. brucei was determined at 37 °C by the method of Rifkin and Fairlamb (1). The incubation medium contained 1 µC of [H]-ethanolamine/ml, 1.11 µM ethanolamine, and 500 µM analog. All analogs were added from a stock 10 mM solution in KRB, adjusted to pH 7. Four time points were taken over a 90-s period. The slope of ethanolamine uptake was determined by linear regression analysis. Correlation coefficients in all cases were >0.93, and usually >0.97. Percent inhibition refers to the decrease in ethanolamine uptake with added analog (or ethanolamine) relative to no addition. In the table below, ethanolamine has been broken down into three parts: A = amino end (HN&cjs0810;), B = methylene group (&cjs0810;CH&cjs0810;), C = hydroxymethyl end (&cjs0810;CHOH), in order to show substitutions of each moiety separately. Analogs which have substitutions in two or more parts of the molecule are mentioned in the text. All chemicals were obtained from Sigma, Aldrich, ICN Pharmaceuticals, or Chemical Dynamics Corp.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants AI-20324 and AI-21429. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-4941; Fax: 212-860-9279.

Present address: Dept. of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel St., London, WC1E 7HT, United Kingdom.

The abbreviations used are: VSG, variant surface glycoprotein; Etn, ethanolamine; P-Etn, phosphoethanolamine; CDP-Etn, CDP-ethanolamine; MEM, minimum Eagle's medium; dCDP-Etn, dCDP-ethanolamine; PCA, perchloric acid; TLC, thin layer chromatography; PE, phosphatidylethanolamine; CM, chloroform-methanol; GPE, glycerophos-phoethanolamine; PC, phosphatidylcholine; DMPE, dimethylphosphatidylethanolamine; LPE, lysophosphatidylethanolamine.

M. R. Rifkin and C. Strobos, manuscript in preparation.

M. R. Rifkin, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Jules Feledy and Helen Shio for technical assistance.


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