From the
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
[
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)
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
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.
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 [
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
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/H
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
For identification of chloroform/methanol
(CM)-soluble metabolites, [
To characterize
further the nature of the inhibition of ethanolamine transport, the
inhibitor constant (K
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
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.
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
Using the concentration (1.1 µM)
and the specific radioactivity (4.2 µC/nmol) of
[
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).
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.
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 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
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.
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.
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 [
We thank Jules Feledy and Helen Shio for technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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.
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.
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.
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.
, 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.
O, 5:2:3) or system
B (100% ethanol/concentrated ammonia/H
O, 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 En
Hance (DuPont
New England Nuclear).
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.
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/H
O
(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 H
O, 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.
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.
) 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 H
O 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 H
O. 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).
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.
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.
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/H
0 (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/H
0
(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 [
Incorporation of
[H]Ethanolamine
Incorporation
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) .
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.
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.
(
)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.
= 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% ().
(
)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.
Table: Inhibition of ethanolamine transport by analogs
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 (H
N&cjs0810;), B =
methylene group (&cjs0810;CH
&cjs0810;), C =
hydroxymethyl end (&cjs0810;CH
OH), 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.