From the School of Biological Sciences, University of
Liverpool, Life Sciences Building, Crown St., Liverpool L69 7ZB, United
Kingdom, the ¶ School of Tropical Medicine, University of
Liverpool, Pembroke Place, Liverpool L3 5QA, United Kingdom, and the
Department of Chemistry, University of Edinburgh, King's
Buildings, West Mains Rd., Edinburgh EH9 3JJ, United Kingdom
Received for publication, July 31, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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The amino acid leucine is efficiently used
by the trypanosomatid Leishmania mexicana for sterol
biosynthesis. The incubation of [2-13C]leucine with
L. mexicana promastigotes in the presence of ketoconazole gave 14 Parasitic trypanosomatid protozoa of the genus
Leishmania cause diseases in tropical and subtropical
regions of the world. The treatment of leishmaniasis still relies upon
the drugs introduced many years ago (1), which have toxic side effects,
and there is now a great need for more effective new chemotherapeutic
drugs (1, 2). This has prompted the search for new metabolic targets for drugs and has resulted in the recognition of sterol synthesis inhibitors as a potential candidate (2, 3). The importance of an active
sterol biosynthetic pathway in trypanosomatids for growth and viability
has been demonstrated using antifungal agents that are inhibitors of
sterol biosynthesis. Thus, the imadazole- and triazole-based drugs
(e.g. ketoconazole and itraconazole), which inhibit the
14 In our studies on sterol biosynthesis in Leishmania species,
we have recently demonstrated (12, 13) that leucine is the major source
of the carbon used for de novo sterol biosynthesis. By
contrast, acetate or substrates from which acetyl-CoA is generated by
metabolism (e.g. glucose, palmitic acid, alanine, serine,
and isoleucine) are very poorly incorporated into sterol, although they
are used efficiently for the synthesis of the fatty acid moieties of
triacylglycerol and phospholipid. The utilization of leucine for
sterol biosynthesis has been shown previously in animal tissues
(14-18), plants (19-21), and fungi (22, 23). In animals and plants, a
major route for leucine catabolism has been demonstrated to be located
in the mitochondrion (24, 25). The pathway proceeds through the
production of Chemicals--
[1-14C]Acetate, sodium salt (57 mCi/mmol), and [U-14C]leucine (299-314 mCi/mmol) were
obtained from Amersham Pharmacia Biotech. [2-13C]Leucine
(99% enrichment) was obtained from Promochem (Welwyn Garden
City, UK). [2-13C]Acetate,
[1-13C]glucose, ketoconazole, and mevalonic acid were
obtained from Sigma-Aldrich. Compound L-659,699 was a gift from Merck.
Cell Culture--
The strain of Leishmania used in
this study was L. mexicana (MNYC/62/BZ/M379). Promastigotes
were cultured at 26 °C in HO-minimum essential medium (26),
supplemented with 10% (v/v) heat-inactivated fetal calf serum.
14C- and 13C-labeled substrates were added to
the media to give the final concentrations indicated under
"Results." Cell density of cultures was determined by counting
using a Neubauer hemocytometer. Cultures were normally established with
an initial cell density of 106 cells/ml; at the termination
of the culture period, the cells were harvested by centrifugation as
described previously (12, 27). Lovastatin was administered to cultures
from a stock solution (2 mg/ml) in Me2SO; compound
L-659,699 was dissolved (2 mg/ml) in sterile H2O saturated
with NaHCO3 for administration to the cultures.
Amastigotes were obtained after the infection of macrophages isolated
from the peritoneal cavities of female CD1 mice with stationary phase
promastigotes (72 h of growth) (28, 29). Promastigotes were allowed to
infect macrophages for 24 h at 32 °C before the medium
overlying the macrophages was decanted. The cells were rinsed with
Locke's solution (containing the following per liter: 9 g of
NaCl, 0.42 g of KCl, 0.4 g of
CaCl2·H2O, 0.2 g of NaHCO3,
and 1 g of glucose) to remove any remaining extracellular promastigotes. RPMI supplemented 15% (v/v) with heat-inactivated fetal
calf serum and containing [2-13C]leucine in place of
unlabeled free leucine was then added to the infected macrophages, and
the cultures were left at 32 °C for 48 h. At this time,
amastigote forms were prepared by the method described by Haughan
et al. (29).
Lipid Extraction and Analyses--
Parasite lipids were isolated
after extraction with chloroform/methanol (2:1) as described previously
(12, 27). Radioactive lipid extracts were analyzed by analytical TLC
and radioscanning using silica gel TLC plates and chloroform/ethanol
(98:2) as the developing solvent. Sterols were isolated and analyzed by
GC or GC-MS as the TMS ether derivatives following previously described protocols (12, 27). Steryl acetates were prepared by treatment of the
free sterol with pyridine/acetic anhydride (1:1) followed by usual work
up of the steryl acetate. The steryl acetates were separated by
preparative TLC on silica gel impregnated with 10% AgNO3
and developed with freshly distilled chloroform. Sterols were
quantified by capillary GC analysis using 5 NMR Spectroscopy--
The NMR spectra were measured on
deuteriochloroform solutions using a Varian INOVA 600 spectrometer
operating at 599.9 MHz for protons and 150.9 MHz for 13C
nuclei. 1H NMR spectra were recorded using the following
parameters: 7000 Hz spectrum width, 3-s preacquisition delay, 90°
pulse, 5-s acquisition time, 10,000 transients, 64K data points No
weighting function was used before Fourier transformation. Broad band
proton-decoupled 13C NMR spectra were obtained using the
following parameters: 35,000-Hz spectrum width, 22° pulse, 0.936-s
acquisition time, 70,000 transients, 64K data points, line broadening
of 1 Hz before Fourier transformation.
Isolation of
14 Incorporation of [2-13C]Leucine into the Sterols of
L. mexicana Promastigotes--
We have utilized
[2-13C]leucine to investigate if the leucine carbon
skeleton is incorporated by L. mexicana directly into the sterols by reduction of HMG-CoA to MVA. The major sterols of L. mexicana are cholesterol obtained from the medium, and the
biosynthesized ergosta-5,7,24(241)-trien-3
Preliminary experiments were first undertaken to determine the optimum
conditions for incubation of the L. mexicana promastigotes with ketoconazole to accumulate a 14
Ketoconazole at 0.1 µg/ml retarded growth by only about 5% compared
with the control, and the total sterol content of the treated cells was
~70% of the control value. At the higher ketoconazole concentration
(1.0 µg/ml), the growth was around 75% of the control, and the total
sterol was about 50% of the control. In a further experiment, the
L. mexicana promastigotes were incubated with ketoconazole
(0.1 µg/ml) and [U-14C]leucine (2.6 µCi) for 72 h to ensure that the accumulating 14
The above experiments showed that our approach to obtain a pure
sterol for the 13C NMR analysis was feasible. Therefore,
multiple cultures (50 × 50 ml) of L. mexicana
promastigotes were grown in HO-minimum essential medium (plus 10%
(v/v) heat-inactivated fetal calf serum) in which the free
(i.e. nonprotein) leucine was replaced with [2-13C]leucine, and 0.1 µg/ml ketoconazole was added.
The cells were cultured for 72 h and harvested, and the lipid was
extracted. The total sterol was isolated from the lipid, and the
14
The 13C labeling patterns predicted for sterol derived from
[2-13C]leucine by pathways involving either HMG-CoA
breakdown to the acetyl-CoA level or directly by reduction of HMG-CoA
to MVA are shown in Scheme 2. If the
incorporation proceeds indirectly through the intermediacy of
acetyl-CoA, the sterol will have 12 13C-enriched positions
(C-2, C-4, C-6, C-8, C-10, C-11, C-12, C-14, C-16, C-20, C-23, and
C-25). However, if incorporation results from direct conversion of
HMG-CoA to MVA, then only six positions will be enriched (C-2, C-6,
C-11, C-12, C-16, and C-23). Clearly, the mass spectral data revealing
labeled sterol species containing up to six 13C atoms
pointed to the latter labeling pattern. Accordingly, to determine the
exact number of 13C atoms and their locations in the
molecule, the labeled
14
The singlet signals at
Because the inhibitor ketoconazole was used to facilitate the
accumulation of the
14 Incorporation of [2-13C]Leucine into Sterol by
Amastigotes of L. mexicana--
The above experiments were performed
with L. mexicana promastigotes. We have previously reported
that the amastigote form of this parasite can also use
[U-14C]leucine for sterol biosynthesis (12). This has now
been confirmed by incubation of L. mexicana amastigotes
cultured in macrophages with [2-13C]leucine. The mass
spectrum of the isolated ergosta-5,7,24(241)-trien-3 Effects of an Inhibitor of HMG-CoA Synthase--
An inhibitor of
HMG-CoA synthase should block [1-14C]acetate
incorporation into isoprenoids but have no effect on the direct incorporation of [U-14C]leucine into sterol (Scheme 1).
Accordingly, [U-14C]leucine and
[1-14C]acetate were incubated separately with L. mexicana promastigotes in the presence and absence of the compound
L-659,699, a fungal metabolite, which is a competitive inhibitor of
HMG-CoA synthase (33, 34). An analysis of the labeled lipid by TLC and
radioscanning showed that after the incorporation of
[1-14C]acetate, about 2-3% of the radioactivity was in
sterol, with the remainder distributed between triacylglycerol
(~35%) and phospholipid (~60%) as observed previously (12).
However, incubation with [1-14C]acetate in the presence
of L-659,699 (20 µg/ml) resulted in the triacylglycerol and
phospholipid remaining labeled in about the same proportions as in the
control, but there was a complete abolition of label from the sterol
that was consistent with the inhibition of HMG-CoA synthase. The
results obtained with the [U-14C]leucine incubations are
presented in Table III and show that the
presence of L-659,699 had no apparent inhibitory effect on the
incorporation of radioactivity into total lipid and sterol or the
distribution of radioactivity between the labeled products even at the
highest concentration of L-659,699 (50 µg/ml). These results are
consistent with the view that in L. mexicana the
incorporation of leucine does not require breakdown to the acetyl-CoA
level as an essential step.
When cells were cultured with [2-13C]leucine and
increasing concentrations of the HMG-CoA synthase inhibitor L-659,699
followed by GC-MS analysis of the
ergosta-5,7,24(241)-trien-3 Effects of Lovastatin, an Inhibitor of HMG-CoA Reductase--
The
effects of an HMG-CoA reductase inhibitor on the incorporation of
leucine into sterol were tested using lovastatin, and the results are
presented in Table IV. Lovastatin
had little or no inhibitory effect on growth of the cultures at
concentrations of 5 and 10 µg/ml, but growth retardation became
apparent as the concentration was increased to 25 and 50 µg/ml.
Lovastatin at 5 µg/ml caused an inhibition of incorporation of
[U-14C]leucine into the total lipid and sterol. The
extent of inhibition became progressively greater as the lovastatin
concentration was increased to 50 µg/ml. This provided good evidence
for either the intermediacy of HMG-CoA and the action of HMG-CoA
reductase in the pathway or for the operation of a similar type of
reductive reaction to that catalyzed by HMG-CoA reductase, which is
sensitive to lovastatin inhibition. Analysis of the sterols recovered
from the incubations with lovastatin showed that there was a decline in
the amount of sterol in the cells, which paralleled the decline in
[U-14C]leucine incorporation. The decline in sterol
concentration was accompanied by a change in the composition of the
sterol mixture with a progressive increase in the proportion of
cholesterol (cholest-5-en-3
The addition of excess MVA to the L. mexicana culture was
tested to determine the effect on the incorporation of
[U-14C]leucine into sterol and on culture growth. The
incorporation of MVA into sterols by Leishmania species has
been demonstrated previously (4, 5, 11-13). Cultures (10 ml, 2 × 106 cells/ml) were incubated with
[U-14C]leucine (2 µCi) in the presence or absence of
MVA (1 mg/ml). The cultures were stopped after 72 h, the sterols
were extracted, and the radioactivity was determined. The addition of
MVA had a marked effect and significantly reduced (p = 0.005) the radioactivity incorporated from [U-14C]leucine
into the sterol by about 30% (control, 370 (S.D. = 33.4) dpm/106 cells (n = 4); plus MVA, 220 (S.D. = 37.2) dpm/106 cells (n = 3)). This
reduction in [U-14C]leucine incorporation would be
predicted if there is a considerable dilution of the cellular pool of
[14C]MVA derived from leucine metabolism. Moreover, the
growth inhibition of the cells by lovastatin was reversed by the
exogenous MVA (Fig. 3), showing that it
could enter the isoprenoid pathway to provide the requirements of the
cell for sterols and/or other essential isoprenoid-derived
compounds.
The incorporation of leucine into cholesterol by animal tissues
has been demonstrated (14-18), and it was reported that in rats this
proceeded with the prior breakdown of the leucine to acetate (14).
Likewise, the incorporation of leucine into a plant sterol was also
shown to require the catabolism of the leucine to acetyl-CoA before
being incorporated into the isoprenoid pathway (19). By contrast, we
have now established unequivocally by MS and 13C NMR
methods that the trypanosomatid L. mexicana can incorporate the leucine skeleton intact into the isoprenoid pathway for sterol production without breakdown first to acetyl-CoA. This could occur by a
pathway leading to HMG-CoA, and the HMG-CoA could then be directly
reduced to MVA by HMG-CoA reductase (Scheme 1). The fact that some
label from [U-14C]leucine appears in the fatty acids of
triacylglycerols and phospholipids (Tables III and IV) (12) is best
explained by the formation of intermediary HMG-CoA, which can yield
labeled acetyl-CoA by the action of HMG-CoA lyase. A mitochondrial
HMG-CoA reductase has recently been characterized in
Leishmania and Trypanosoma species (35, 36). The
existence of this enzyme could provide the opportunity for a portion of
any HMG-CoA produced from leucine metabolism to be reduced to MVA and
thus channeled directly into the isoprenoid pathway for sterol
biosynthesis. The operation of pathways from leucine and acetyl-CoA,
which merge at HMG-CoA, followed by reduction to MVA, is consistent
with the following observations. (i) Inhibition of HMG-CoA synthase
does not lower leucine incorporation into sterol (Table I). (ii)
Lovastatin inhibits the incorporation of leucine (Table IV) into
sterol. (iii) Labeled MVA is incorporated into Leishmania
sterol (4). (iv) Excess unlabeled MVA added to the culture lowers the
incorporation of [2-14C]leucine into sterol. (v) Added
MVA overcomes the inhibitory effect of lovastatin on
Leishmania growth (Fig. 3). However, there is an alternative
route for the direct incorporation of leucine into sterol that would
also be compatible with some of the above criteria. This requires the
reduction of dimethylcrotonyl-CoA to dimethylallyl alcohol, followed by
phosphorylation to yield dimethylallyl diphosphate, which is a
constituent of the isoprenoid pathway (Scheme 1) and is
interconvertible with isopentenyl diphosphate by an isomerase-catalyzed
reaction. This route would effectively be a reversal of the mevalonate
shunt that has been demonstrated in some animal tissues (24, 37, 38).
The reduction of dimethylcrotonyl-CoA to dimethylallyl alcohol would be
mechanistically similar to the conversion of HMG-CoA to MVA and could
perhaps be catalyzed by the HMG-CoA reductase or a very similar enzyme
that may also be susceptible to lovastatin inhibition. If leucine
carbon is being channeled along this route, the lowered incorporation
of [2-14C]leucine by added unlabeled MVA could be
explained in two ways. Either the conversion of MVA into an appreciable
unlabeled pool of dimethylallyl diphosphate/isopentenyl diphosphate
results in dilution of the leucine-derived radioactive dimethylallyl
diphosphate/isopentenyl diphosphate or the MVA is converted into excess
sterol that may inhibit leucine utilization by a feedback inhibition mechanism.
The utilization of the intact leucine skeleton for sterol production
may make an important contribution to the metabolic economy of the
Leishmania cell. The use of leucine could spare the need for
acetyl-CoA produced from glucose or fatty acid catabolism, which
would therefore remain available for energy production or other
biosynthetic reactions. However, amino acids derived from the breakdown
of exogenous proteins are recognized as important energy and carbon
sources in trypanosomatids (39-43). Leucine and other amino acids
(glutamate, proline) are reported to be taken up readily from the
growth medium by Leishmania sp. and T. cruzi and
catabolized to provide acetyl CoA or other metabolites that can be
oxidized to provide energy (39-43). Our work has shown that several
Leishmania species, Trypanosoma cruzi, and
Endotrypanum monterogeii can all utilize leucine as a carbon
source not only for sterol production but also for fatty acid
biosynthesis (12, 13). Part of the HMG-CoA produced from leucine could
be channeled into breakdown by HMG-CoA lyase (Scheme 1) to produce the
acetyl-CoA needed for the synthesis of fatty acids. Additionally, this
route of leucine catabolism could provide some acetyl-CoA for oxidation in the tricarboxylic acid cycle (41, 43, 44) or to support the
mitochondrial acetate-succinate CoA transferase cycle for the
generation of ATP and acetate (45). We have demonstrated that the
metabolism of [U-14C]leucine by L. mexicana
promastigotes produces
14CO2.2
Part of this 14CO2 will arise from the
decarboxylation of the labeled leucine (Scheme 1) and by C-4
demethylation of a labeled sterol intermediate (4, 32), but some could
arise from the oxidation of acetyl-CoA generated from the leucine (43,
44). Clearly, there must be coordinated regulation of the metabolism of
amino acids, glucose, and fatty acids to maintain the balance of
acetyl-CoA needed for cell metabolism under conditions of varying
availability of these substrates to the promastigote or amastigote
forms of the Leishmania parasite.
The key position of HMG-CoA in the catabolism of leucine and in the
production of isoprenoids poses questions regarding the cellular
compartmentation and regulation of the pathways and enzymes involved in
HMG-CoA metabolism. In animals and plants, leucine breakdown is a
mitochondrial event (24, 25). It has been reported that leucine
aminotransferase and -methylergosta-8,24(241)-3
-ol as the
major sterol, which was shown by mass spectrometry to contain up to six
atoms of 13C per molecule. 13C NMR analysis of
the 14
-methylergosta-8,24(241)-3
-ol revealed that it
was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and
C-23. This established that the leucine skeleton is incorporated intact
into the isoprenoid pathway leading to sterol; it is not converted
first to acetyl-CoA, as in animals and plants, with utilization of the
acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An
inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of
[1-14C]acetate into sterol but had no inhibitory effect
on [U-14C]leucine incorporation. The HMG-CoA reductase
inhibitor lovastatin inhibited promastigote growth and
[U-14C]leucine incorporation into sterol. The addition of
unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of
growth and also diluted the incorporation of
[1-14C]leucine into sterol. These results are compatible
with two routes by which the leucine skeleton may enter intact into the
isoprenoid pathway. The catabolism of leucine could generate HMG-CoA
that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be
directly reduced to produce an isoprene alcohol followed by
phosphorylation to enter the isoprenoid pathway post-MVA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-methylsterol 14-demethylase, and the allylamines (e.g.
terbinafine), which inhibit squalene epoxidase (2-11), have been shown
to block sterol synthesis in a number of Leishmania and
Trypanosoma species with retardation of growth and death of the parasite.
-ketoisocaproate, isovaleryl-CoA,
3-methylcrotonyl-CoA, and 3-methylglutaconyl-CoA (Scheme 1) to give
3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA),1 which is then
cleaved by a lyase to produce acetyl CoA and acetoacetate. The
acetyl-CoA generated in this way can be either fed into the citric acid
cycle or alternatively transported out of the mitochondrion into the
cytosol, where the acetyl-CoA may be utilized for the biosynthesis of a
range of compounds including fatty acids and isoprenoids such as
sterols. The entry of the acetyl-CoA into the isoprenoid pathway
requires the regeneration of HMG-CoA, which is then reduced to
mevalonic acid (Scheme 1). Conclusive
evidence that leucine enters isoprenoids in plants by this indirect
route and involving production of acetyl-CoA has been provided by
incubation of 13C-labeled leucine with a callus culture of
Andrographis paniculata (19, 20). 13C NMR
analysis of the 13C-enriched sesquiterpenoid and
phytosterols (19, 20) produced by the callus showed unequivocally that
the leucine was metabolized to acetyl-CoA and acetoacetate prior to
incorporation into the isoprenoid pathway. We have demonstrated
previously (12, 13) that [U-14C]leucine incubated with
Leishmania mexicana and other trypanosomatid species was
very efficiently incorporated into sterol and to some limited extent
into fatty acids. However, by contrast, [1-14C]acetate
was readily incorporated into fatty acids but poorly utilized for
sterol production. These observations are incompatible with a route in
Leishmania that requires leucine degradation to proceed to
acetyl CoA before reutilization for isoprenoid production (19, 20). We
have therefore undertaken the studies described here to investigate the
metabolic route whereby L. mexicana uses leucine for sterol
biosynthesis, since this may produce evidence for a target for new
antileishmanial drug development.
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Scheme 1.
The metabolic pathway for the conversion of
leucine into 3-hydroxy-3-methylglutaryl-CoA and the further production
of acetyl CoA or mevalonic acid and sterol. The sterol shown,
ergosta-5,5,24(241)-trien-3 -ol, is the major sterol of
L. mexicana promastigotes.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cholestane as a standard.
-Methylergosta-8,24(241)-dien-3
-ol--
Promastigotes
were cultured for 72 h in the presence of 0.1 mg/ml ketoconazole
(administered from a stock solution of 1 mg/ml in Me2SO) in
HO-minimum essential medium. The sterols were isolated as previously
described (12, 26), and the
14
-methylergosta-8,24(241)-dien-3
-ol was
characterized by GC-MS and 1H NMR analyses. For the
experiments studying [2-13C]leucine incorporation into
the sterol, the medium contained [2-13C]leucine (78 mg/liter) in place of the unlabeled free leucine normally present. In
total, 50 cultures (50 ml in each culture) were grown, and the cells
were harvested in batches and extracted with 3 × 100 ml of
chloroform/methanol (2:1) using procedures similar to the those used
for the smaller scale extraction (12, 27). The 13C-labeled
N14
-methylergosta-8,24(241)-dien-3
-ol was then
purified from the lipid by reversed-phase HPLC using an Econosphere
C18 column (250 × 4.6 mm; inner diameter, 5 mm;
supplied by Alltech) to separate it from cholesterol and other minor
sterols. Compounds were eluted isocratically using acetonitrile/water
(9:1), and sterols were detected by UV absorbance at 215 nm. The
14
-methylergosta-8,24(241)-dien-3
-ol was eluted at
26-30 min, and cholesterol was eluted at 32-36 min. The solvent
volume of the eluate was carefully reduced by rotary evaporation, and
the sterol was extracted into petroleum ether before taking to dryness
and storage at
20 °C. The purity of the isolated
13C-labeled
14
-methylergosta-8,24(241)-dien-3
-ol (~3 mg) was
checked by GC-MS and then analyzed by 13C NMR
spectroscopy
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-ol (4, 12,
13) with smaller variable amounts of ergosta-5,7,22-trien-3
-ol,
stigmasta-5,7,24(241)-trien-3
-ol, and precursors such as
ergosta-7,24(241)-dien-3
-ol. A sterol mixture of this
nature presents certain problems for the type of 13C NMR
study envisaged. First, the complexity of the sterol mixture demands
careful purification of one of the biosynthesized ergosta types
of sterol so that signal assignments to 13C-enriched
carbons can be made with accuracy and without ambiguity. Second, the
major sterols of L. mexicana are
5,7-compounds that are notoriously unstable in small
amounts due to oxidation, and this may cause difficulties in the
purification, storage, and NMR analysis of these sterols. This problem
has been considered in detail by Schroepfer et al. (30),
specifically in relation to the NMR analysis of
5,7-sterols. Finally, the success of the study depends
upon the extent of enrichment of the biosynthesized sterol with
13C. A large pool of preexisting sterol from the inoculum
will dilute the 13C-enriched sterol species and could make
13C-enriched carbons difficult to detect. Accordingly, we
looked for a new approach to the problem and decided to use an
inhibitor of sterol biosynthesis. An inhibitor was required that would
cause the accumulation of a large amount of a relatively stable sterol intermediate that would normally occur in only trace amounts in the
parasite, so dilution of the newly synthesized
[13C]sterol by preexisting material would not be
significant. Sterol biosynthesis inhibitors suited to this purpose are
the imadazole and triazole types of antifungal drugs. These compounds
block the action of the cytochrome P450-dependent
14
-methylsterol 14-demethylase with the result that the normal
sterols are depleted and one or more 14
-methylsterols accumulate,
often in large amounts (31). It has been demonstrated previously that
antifungal imidazoles and triazoles also inhibit the
14
-demethylation step in sterol biosynthesis in several
Leishmania species with the resulting appearance of
4
,14
-dimethylergosta-8,24(241)-dien-3
-ol and
14
-methylergosta-8,24(241)-dien-3
-ol in appreciable
amounts (4, 6). These 14
-methylsterols are considerably more stable
during isolation, storage, and NMR analysis than are
5,7-sterols. Consequently, we decided that using the
14-demethylase inhibitor ketoconazole offered the best opportunity for
the isolation of a pure 13C-enriched sterol undiluted by
preexisting endogenous sterol, which was required for the
13C NMR analysis to determine the route of incorporation of
leucine into the isoprenoid pathway.
-methylsterol in sufficient amount for isolation and 13C NMR analysis. The incubation
of promastigotes of L. mexicana with ketoconazole (0.1 and
1.0 µg/ml) for 72 h followed by isolation and GC-MS examination
of the sterols showed that, as anticipated, the
ergosta-5,7,24(241)-trien-3
-ol found in the control was
replaced by 14
-methylergosta-8,24(241)-dien-3
-ol in
the ketoconazole-treated cultures (Table
I). Cholesterol taken up from the
medium was present in both the control and treated cells. The
14
-methylergosta-8,24(241)-dien-3
-ol was identified
by the mass spectrum of the TMS ether and by the 1H NMR
spectrum (6, 32). MS m/z (rel. intensity): 484 [M]+ (43), 469 [M-methyl]+ (100), 385 [M-methyl-part side chain]+ (13), 379 [M-methyl-TMSOH]+ (79), 303 (36), 295 [M-TMSOH-methyl-part side chain]+ (29), 281 (17), 227 [M-TMSOH-side chain and ring D]+ (28), 213 [M-methyl-TMSOH-side chain and ring D]+ (41).
1H NMR (chloroform-d):
0.70 s
(H3-18), 0.94 s (H3-19), 0.92 d (H3-21), 0.88 s
(H3-32), 1.01 d and 1.02 d
(H3-26 and H3-27), 4.65 br s and 4.70 br
s (H2-241).
The effects of ketoconazole on the growth and sterol composition of
promastigotes of L. mexicana
-methylsterol was being
biosynthesized from leucine derived from the medium rather than from
some internal source of unlabeled precursor(s). The lipids were
extracted and found to contain 4.2% of the radioactivity added to the
culture medium, while analytical TLC with radioscanning showed that the
14
-methylsterol was the major labeled material. Recovery of the
labeled sterol from the TLC plate, acetylation, and rechromatography by
TLC on silver nitrate-impregnated silica gel showed that the
radioactivity accompanied a material with the same
Rf as
14
-methylergosta-8,24(241)-dien-3
-yl acetate.
-methylergosta-8,24(241)-dien-3
-ol was then
separated from the cholesterol and other minor sterols by HPLC (see
"Materials and Methods"). The cholesterol was shown by GC-MS
analysis to contain only the natural abundance of 13C, and
there was no detectable labeling from the [2-13C]leucine.
This observation established unequivocally that the cholesterol in
L. mexicana must be taken up from the medium and that it is
not the product of de novo synthesis in the parasite. The
purity of the isolated
14
-methylergosta-8,24(241)-dien-3
-ol (~3 mg) was
95% as judged by GC analysis. The mass spectrum of the TMS ether
showed clusters of ions for the molecular and fragment ions arising
from several labeled species of the sterol containing from one to six
13C atoms (Fig. 1). Ions due
to unlabeled sterol were very minor, showing that there was excellent
incorporation of [2-13C]leucine into the sterol in accord
with our previous studies with [U-14C]leucine
incorporation, which had revealed that at least 80% of the sterol
carbon originated from leucine (12, 13). The molecular ion region
comprised a cluster of ions at m/z 484 (unlabeled sterol),
485, 486, 487, 488, 489, and 490, with the last two predominating (Table II). The fragment ion clusters at
m/z 469-475 and 379-385, which arise by loss of a methyl
and methyl and TMSOH from the [M]+ ion,
respectively, showed a similar distribution of molecular species
containing 1-6 13C-enriched positions (Fig. 1). The ions
at m/z 304-307 showed fragments containing up to five
13C-enriched positions. The ions arising by the McLafferty
loss of the terminal part of the side chain were at m/z
295-300 with the strong ion at m/z 300 showing the presence
of five 13C-labeled positions in the fragment. The ion
cluster at m/z 213-217 [M+-side ring D-TMSOH]
revealed up to four 13C-enriched carbons in the remaining
fragment. The mass spectral results for the 13C-labeled
sterol were compatible with the incorporation of up to six molecules of
[2-13C]leucine into the sterol. Moreover, the
fragmentation ions had labeling patterns revealing that one labeled
position was in the side chain, one in the carbons of ring D, and the
remaining four in the rings A, B, and C.
View larger version (20K):
[in a new window]
Fig. 1.
The mass spectrum of the TMS ether of
14 -methylergosta-8,24(241)-dien-3
-ol
isolated from promastigotes of L. mexicana cultured
for 72 h in the presence of ketoconazole (0.1 µg/ml) and
[2-13C]leucine. The identities of the ions are
described under "Results."
Mass spectra of the major sterols isolated after incorporation of
[2-14C]leucine into the major sterol of (a) L. mexicana
promastigotes incubated with ketoconazole (0.1 µg/ml), (b) L. mexicana control promastigotes, (c) L. mexicana amastigotes
-methylergosta-8,24(241)-dien-3
-ol was purified by
preparative HPLC and examined by 13C NMR spectroscopy (Fig.
2). The spectrum showed a very high
enrichment of the compound with 13C and displayed six
strong signals indicating the positions specifically labeled from the
[13C]leucine. The assignments of these carbon signals
were made by comparison with the reported 13C NMR spectra
of other sterols (32).
View larger version (15K):
[in a new window]
Scheme 2.
The 13C labeling patterns
predicted in
14 -methylergosta-8,24(241)-dien-3
-ol
produced by L. mexicana promastigotes from
[2-13C]leucine if the biosynthetic pathway proceeds
either indirectly from HMG-CoA to acetyl-CoA with subsequent
regeneration of HMG-CoA from the acetyl-CoA and conversion to mevalonic
acid (MVA) (a) or directly by
reduction of HMG-CoA to MVA without the intermediacy of acetyl-CoA
(b).
View larger version (14K):
[in a new window]
Fig. 2.
The 13C NMR spectrum of the
14 -methylergosta-8,24(241)-dien-3
-ol
isolated from promastigotes of L. mexicana cultured
for 72 h in the presence of ketoconazole (0.1 µg/ml) and
[2-13C]leucine. Insets A,
B, and C are expansions of the signals for C-11,
C-12, and C-16, respectively.
25.2, 31.2, and 31.5 ppm were readily
assigned to C-6, C-2, and C-23, respectively. The three signals centered at
21.7 (Fig. 2, inset A) comprised
a singlet due to C-11 in molecules with no 13C enrichment
at the adjacent positions (C-9 and C-12) and a doublet arising from
coupling with C-12 in molecular species that were 13C-enriched at this position. Similarly, the signals
centered at
30.9 were assigned to C-12 with a singlet in those
molecular species lacking 13C-enrichment at C-11 or C-13
and a doublet due to coupling in molecules with 13C at
position C-11. However, as shown (Fig. 2, inset
B), each of the three signals arising from C-12 was further
split by another 13C-13C long range coupling.
The labeled position responsible for this coupling was assigned to
C-16, the signal for which was at
28.1 (Fig. 2, inset
C). Expansion of the signal at
28.1, which at first
sight appeared to be a singlet, showed it was composed of a singlet
plus a doublet. The splitting to give the doublet must have resulted
from long range coupling in molecules labeled with 13C at
C-12 as well as at C-16. The 13C labeling pattern
determined in the
14
-methylergosta-8,24(241)-dien-3
-ol was therefore
entirely consistent with the leucine skeleton remaining intact during
metabolism and incorporation into the isoprenoid pathway (Schemes 1 and
2). This is in striking contrast to leucine utilization in plants,
where it is first degraded to acetyl-CoA before utilization in
isoprenoid production (19, 20).
-methylergosta-8,24(241)-dien-3
-ol used for the NMR
study, there was perhaps a possibility that this drug could have
perturbed the metabolic pathways through HMG-CoA. For example, it may
have caused an overexpression of HMG-CoA reductase in response to the
decline in the normal sterol (i.e.
ergosta-5,7,24(241)-trien-3
-ol). This could have
resulted in HMG-CoA being rapidly reduced before it could be cleaved by
HMG-CoA lyase to acetyl-CoA and acetoacetate. To check this point,
promastigotes were cultured with [2-13C]leucine in the
absence of ketoconazole. GC-MS analysis of the major sterols as their
TMS ether derivatives showed that
ergosta-5,7,24(241)-trien-3
-ol had a molecular ion
cluster (m/z 469-475), indicating species of the sterol
molecule containing from one up to a maximum of six 13C
atoms (Table II) with a distribution of molecular species fairly similar to that found previously for
14
-methylergosta-8,24(241)-dien-3
-ol. The various
fragment ion clusters were also consistent with the presence of labeled
species containing 13C in the positions predicted from the
labeling pattern determined for
14
-methylergosta-8,24(241)-dien-3
-ol. The mass
spectra of the ergosta-5,7,24(241)-trien-3
-ol TMS ether
labeled from either [2-13C]acetate or
[1-13C]glucose contained the molecular ion at
m/z 468 for unlabeled compound, presumably produced from
leucine, and a series of further molecular ions of diminishing
abundance containing 1-10 13C atoms (above this, the ions
were too weak to determine whether molecular species with the
theoretical maximum of 12 13C atoms were present). The
incorporation of acetate and glucose into the sterol was consistent
with our previous investigations (12, 13), which indicated that acetate
can provide up to 20% of the carbon needed for sterol production in
L. mexicana but with the major portion (~80%) arising
from leucine.
-ol,
analyzed as the TMS ether, had a strong molecular ion at m/z
468 for unlabeled sterol, but this was accompanied by an ion of similar
abundance at m/z 473 for sterol with five atoms of
13C, together with less abundant ions for molecules
containing one, two, three, four, and six atoms of 13C
(Table II) in proportions similar to that seen in promastigotes. The
unlabeled ergosta-5,7,24(241)-trien-3
-ol must be from
the preexisting sterol pool in the amastigotes produced prior to
exposure to [2-13C]leucine. In the 48-h incubation used
for this experiment, the amastigotes will have undergone about two cell
divisions; therefore, the amount of newly synthesized sterol labeled
with 13C must be insufficient to dilute the unlabeled
sterol to the extent seen with the promastigotes (Table II). Thus, it
can be concluded that the promastigote and amastigote forms of the
parasite both utilize leucine as a main carbon source for sterol
biosynthesis that proceeds by the direct route.
The effect of L-659,699 on the incorporation of
[U-14C]leucine into the lipids of L. mexicana promastigotes
-ol, the results provided
evidence for the dual sources of carbon from either leucine metabolism
or acetyl-CoA to fuel the isoprenoid pathway. In the absence of the
inhibitor, the predominant molecular species of the sterol TMS ether
(M+ at m/z 473) had five 13C atoms
rather than six, indicating a contribution from unlabeled precursors
probably via the acetyl-CoA route. At an L-659,699 concentration of 1 µg/ml, the [M+] ion at m/z 474 (containing
six atoms of 13C) showed a small enhancement, while at 10 and 20 µg/ml concentration of inhibitor, the m/z 474 ion
was the major one. This increase by 1 mass unit in response to
inhibitor treatment was also seen in the main fragmentation ions,
e.g. [M+-TMSOH] at m/z 383 (no
inhibitor) increased to 384 (plus inhibitor), [M+-TMSOH-Me] at m/z 368 up to 369, m/z 341 up to 342, and [M+-side chain-ring
D-TMSOH] at m/z 214 up to 215. These results can be
explained by the inhibition of the HMG-CoA synthase, resulting in no
contribution from the acetyl-CoA pool in the cell and all of the sterol
then being derived from 13C-labeled leucine. Moreover, they
are also consistent with unlabeled carbon introduced in the absence of
HMG-CoA synthase inhibitor being largely derived from acetyl-CoA
obtained from a carbohydrate, fatty acid, or ketogenic amino acid
source rather than directly from unlabeled leucine of protein origin.
-ol) and corresponding drop in the amount
of ergosta-5,7,24(241)-trien-3
-ol. Cholesterol is not
synthesized by the parasite but is derived from the fetal calf serum of
the culture medium (12, 13), whereas the
5,7-sterols
with a C-24 substituent in the side chain are produced by de
novo biosynthesis in the protozoa (4). It was noticeable that in
these experiments the proportion of
stigmasta-5,7,24(241)-trien-3
-ol was observed to
increase up to a concentration of 25 µg/ml lovastatin. This could be
accounted for by the utilization of the 24-methylenesterols as
substrate by the second C-241-transmethylase; the
24-methylenesterols were then not being replaced because of the block
in sterol production imposed by the lovastatin. Leishmania
sp. may have a growth requirement for a sterol with the structural
features of the endogenous sterols (i.e. a
5,7-ring system and a C-24 methylene, methyl, or
ethylidine side chain) or alternatively perhaps only newly synthesized
sterol can play some important role in sustaining cell growth (27). However, the inhibition of HMG-CoA reductase by lovastatin could also
inhibit the production of other isoprenoid-derived compounds (e.g. dolichols, prenylated proteins, ubiquinone side
chain). This could lead to growth inhibition by starvation of the cell of other vital compounds (e.g. dolichols, prenylated
proteins) in addition to the sterols.
The effect of lovastatin on the incorporation of
[U-14C]leucine into the sterols and other lipids of L. mexicana promastigotes and the sterol composition of the protozoa
View larger version (19K):
[in a new window]
Fig. 3.
The effects of lovastatin and lovastatin plus
mevalonic acid (MVA) on the growth of L. mexicana promastigotes. Cells were cultured as
described under "Materials and Methods." Either lovastatin (30 µg/ml) or lovastatin (30 µg/ml) plus MVA (1 mg/ml) was added to
cultures at the start of the growth period, and samples were withdrawn
at intervals to determine the growth by cell counting using a Neuberger
hemocytometer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-ketoisocaproate dehydrogenase are present in
cytosolic and mitochondrial preparations from T. cruzi (46).
Similarly, we have found that leucine aminotransferase is located in
the mitochondrion of L. adleri.3 These facts
suggest that leucine catabolism to produce HMG-CoA could be located in
the mitochondrion of trypanosomatids. HMG-CoA lyase is a mitochondrial
enzyme in the mammalian liver cell (47), and there is an HMG-CoA
synthase also located in the mitochondrion of mammalian cells (47), but
these enzymes have not yet been studied in trypanosomatids. The
remaining enzyme of HMG-CoA metabolism, HMG-CoA reductase, is
associated mainly with the endoplasmic reticulum in mammalian cells.
The HMG-CoA reductase of trypanosomatids has been studied in
Trypanosoma brucei (48), T. cruzi (49, 50), and
Leishmania major (35, 51) and variously described as either a microsomal, a glycosomal, or a soluble enzyme. However, recent investigations have now revealed that the HMG-CoA reductase of T. cruzi and L. major (51) and T. brucei (36)
are predominantly located in the mitochondrion. Thus, the mitochondrion
may be perhaps a major cellular site for the first stages in the
production of isoprenoids in trypanosomatids. Certainly, a
mitochondrial location of HMG-CoA reductase could provide for an
efficient integration of the isoprenoid pathway with a mitochondrial
leucine degradation sequence of reactions and thus facilitate the
efficient utilization of leucine carbon for sterol synthesis.
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ACKNOWLEDGEMENT |
---|
The 150-MHz 13C NMR spectra were obtained at the Engineering and Physical Sciences Research Council-supported National Ultrahigh Field NMR Center at Edinburgh University.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Biotechnology and Biological Sciences Research Council research studentship. Present address: Wellcome Unit of Molecular Parasitology, The Anderson College, University of Glasgow, Church St., Glasgow, G11 5SR, United Kingdom.
** To whom correspondence should be addressed: School of Biological Sciences, University of Liverpool, Life Sciences Bldg., Crown St., Liverpool L69 7ZB, United Kingdom. Tel.: 44 151 794 4343; Fax: 44 151 794 4349; E-mail: ljgo@liv.ac.uk.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M006850200
2 M. L. Ginger, M. L. Chance, and L. J. Goad, unpublished observations.
3 B. Smythe, A. Jones, M. L. Chance, and L. J. Goad, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: HMG, 3-hydroxy-3-methylglutaryl; GC, gas chromatography; MS, mass spectrometry; HPLC, high pressure liquid chromatography; TMS, tetramethylsilyl; TMSOH, tetramethylsilol.
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