Báo cáo khoa học: Glycosphingolipids in Plasmodium falciparum Presence of an active glucosylceramide synthase pot

Eur. J. Biochem. 271, 2204–2214 (2004) Ó FEBS 2004

doi:10.1111/j.1432-1033.2004.04150.x

Glycosphingolipids in Plasmodium falciparum
Presence of an active glucosylceramide synthase
Alicia S. Couto1, Carolina Caffaro1, M. Laura Uhrig1, Emilia Kimura2, Valnice J. Peres2, Emilio F. Merino2,
Alejandro M. Katzin2, Masae Nishioka3, Hiroshi Nonami3 and Rosa Erra-Balsells1
1

CIHIDECAR, Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Argentina; 2Departamento de Parasitologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Brazil; 3College of
Agriculture, Ehime University, Matsuyama, Japan

Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore
developing new antimalarial drugs constitutes an urgent
challenge. Lipid metabolism has been attracting a lot of
attention as an application for malarial chemotherapeutic
purposes in recent years. However, little is known about
glycosphingolipid biosynthesis in Plasmodium falciparum.
In this report we describe for the first time the presence of
an active glucosylceramide synthase in the intraerythrocytic
stages of the parasite. Two different experiments, using
UDP-[14C]glucose as donor with ceramides as acceptors, or
UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the
parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with
low concentrations of D,L-threo-phenyl-2-palmitoylamino3-morpholino-1-propanol (PPMP). In addition, de novo

biosynthesis of glycosphingolipids was shown by metabolic
incorporation of [14C]palmitic acid and [14C]glucose in the
three intraerythrocytic stages of the parasite. The structure

of the ceramide, monohexosylceramide, trihexosylceramide
and tetrahexosylceramide fractions was analysed by UVMALDI-TOF mass spectrometry. When PPMP was added
to parasite cultures, a correlation between arrest of parasite
growth and inhibition of glycosphingolipid biosynthesis was
observed. The particular substrate specificity of the malarial
glucosylceramide synthase must be added to the already
known unique and amazing features of P. falciparum lipid
metabolism; therefore this enzyme might represent a new
attractive target for malarial chemotherapy.

Malaria is the most serious and widespread parasitic disease
in humans. Each year, approximately 300 million people
become infected and 2–3 million people die as a result. In
addition there is considerable morbidity associated with this
disease [1].
The glycobiology of Plasmodium falciparum has been
causing an increasing amount of interest in recent years. The
presence of N-linked glycoproteins in relation to schizogony
of the intraerythocytic stages [2] and glycosylphosphatidylinositols as the major carbohydrate protein modification

have been described in the human malaria parasite [3–6]. In
addition, lipid metabolism has also been attracting a lot
of attention with respect to basic biology and applications
for malarial chemoterapeutic purposes [7]. However, little
is known about glycosphingolipids (GSLs), a group of
ceramide-based lipids that in other systems regulate interactions of the cell with its environment and play a role in cell
signalling [8,9]. The first evidence of the presence of GSLs in
P. falciparum was obtained by metabolic incorporation
of [3H]serine and [3H]glucosamine. After labeling with the
carbohydrate precursor, hydrophilic glycosphingolipids

migrating slower than the penta-glycosylated ceramide
standard were detected [10]. More recently, the synthesis
of chloroplast galactolipids in apicomplexan parasites was
reported [11].
Biosynthesis of complex GSLs in mammalian cells
involves sequential glycosyltransferase reactions, starting
with the formation of glucosylceramide (GlcCer), and it has
been assumed that the various transferases used are
functionally organized within the Golgi [12,13]. It is known
that the key step involves the transfer of glucose to ceramide
from UDP-glucose, catalyzed by the action of a glucosylceramide transferase [EC 2.4.1.80: glucosylceramide synthase (GCS)]. With regards to localization, as far as it is
known, GlcCer is special because it is the only glycosphingolipid synthesized on the cytosolic leaflet in the early
Golgi but it is used for the synthesis of higher sphingolipids

Correspondence to A. S. Couto, CIHIDECAR, Departamento de
´
´
Quı´ mica Organica, Pabellon II, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, 1428, Argentina.
Fax/Tel.: + 54 11 4576 3346, E-mail:
Abbreviations: GSLs, glycosphingolipids; GlcCer, glucosylceramide;
GCS, glucosylceramide synthase; BODIPY-DHCer, BODIPYdihydroceramide; BODIPY-Cer, BODIPY-ceramide;
D,L-threo-PPMP, D,L-threo-phenyl-2-palmitoylamino-3-morpholino1-propanol, d18:0, 4-hydroxysphinganine; d20:0, 4-hydroxyicosasphinganine; C10:0, etc., decanoic acid, etc.; C10h:0, etc.,
hydroxydecanoic acid; C10-2h:0, dihydroxydecanoic acid;
C10-3h:0, trihydroxydecanoic acid, etc.
Enzyme: glucosylceramide synthase (EC 2.4.1.80).
(Received 4 December 2003, revised 26 February 2004,
accepted 6 April 2004)

Keywords: dihydroceramide; glucosylceramide synthase;
glycosphingolipids; malaria; Plasmodium falciparum.

Ó FEBS 2004

Glycosphingolipids in Plasmodium falciparum (Eur. J. Biochem. 271) 2205

in the lumenal leaflet [14]. Because glucosylceramide is a
pivotal precursor of numerous GSLs, this enzyme is
extremely important for understanding GSL function.
In this report, we describe for the first time the presence
of an active glucosylceramide synthase in the intraerythrocytic stages of P. falciparum. Two different experiments,
using UDP-[14C]glucose as donor or fluorescent ceramides
as acceptors were performed. In both cases, the enzyme
showed specificity for dihydroceramide as substrate.
The enzyme activity could be inhibited in vitro with
D,L-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, GSLs were shown by metabolic incorporation of [14C]palmitic acid and [14C]glucose
in the three intraerythrocytic stages of the parasite.
UV-MALDI-TOF mass spectrometry proved that four
fractions analysed corresponded to ceramides, monohexosylceramides, trihexosylceramides and tetrahexosylceramides, respectively. When PPMP was added to parasite
cultures, a correlation between arrest of parasite growth
and inhibition of GSL biosynthesis was shown. The
particular substrate specificity of the malarial GCS suggests
that this enzyme might represent a new attractive target for
malarial chemotherapy.

Materials and methods
Materials
Lipid standards and BSA were purchased from Sigma.
AlbuMax IÒ was obtained from Gibco BRL Life Technologies (New York, NY, USA). All solvents were of
analytical or HPLC grade. PercollÒ was purchased from

Pharmacia Chemicals (Uppsala, Sweden). D,L-threo-phenyl2-palmitoylamino-3-morpholino-1-propanol (PPMP) was
from Matreya (Pleasant Gap, PA, USA) and ceramide
glycanase from GlyKo, BODIPYÒ-sphingolipids used were
from Molecular Probes. Polyclonal antibodies against
human GCS were a kind gift of D. L. Marks and R. E.
Pagano, Mayo Clinic Foundation, Rochester, MN, USA.
TLC was performed on silica gel 60 precoated plates
(Merck) using the following solvent systems: (a) chloroform/methanol/water (65 : 25 : 3, v/v/v); (b) chloroform/
methanol/0.25% KCl (80 : 30 : 2, v/v/v); (b), chloroform/
methanol/1 M NH4OH (40 : 10 : 1, v/v/v); (d) chloroform/methanol/water (65 : 25 : 3, v/v/v); (e) chloroform/
methanol/water (80 : 20 : 2, v/v/v). In all cases, radioactive
samples were located by fluorography at )70 °C using
EN3HANCE (NEN) and Kodak X-OMAT AR films.

(Amersham, 291 mCiỈmmol)1, 1,54
mCiỈmg ) was incorporated at a concentration of
6.25 lCiỈmL)1 in RPMI 1640 medium without addition
of 11 mM of glucose. Parasites (5.9% ring forms, 5.4%
trophozoites, 3.7% schizonts) were labeled for 18 h.
14
)1
D-[U- C]galactose (Amersham, 306 mCiỈmmol , 1,61
)1
mCiỈmg ) was incorporated at a concentration of
3.2 lCiỈmL)1 in RPMI 1640 medium without addition of
11 mM of glucose. Parasites (9.8% ring forms, 3.0%
trophozoites, 1.6% schizonts) were labeled for 18 h.
The viability of the parasites was verified by microscopic
evaluation of Giemsa stained smears. Each stage was
purified on a 40/70/80% (w/v) discontinuous Percoll

gradient (15 000 g, 30 min, 25 °C). This procedure yielded
an upper band (40%) containing schizonts, another band
with trophozoites (70–80% interface) and a pellet of ring
forms [2].
A control containing a similar number of uninfected
erythrocytes was incubated with the different radioactive
precursors and further processed under the same conditions.
In order to evaluate biosynthesis of proteins, synchronous
P. falciparum ring-stage cultures, with a parasitemia of
around 5%, untreated or treated with 5 lM PPMP for 48 h,
were labeled with 25 lCiỈmL)1 of L-[35S]methionine
(> 1000 CiỈmmol)1) (Amersham) in 10 lM methioninedeficient RPMI medium, at the beginning or after 24 h
of treatment. Aliquots were collected at different times
(0–48 h), precipitated with 12% (w/v) trichloroacetic acid,
and radioactivity was measured with a Beckman 5000
b-counter.
14
D-[U- C]glucose
)1

Treatment of parasites with D,L-threo-phenyl-2palmitoylamino-3-morpholino-1-propanol
Parasite cultures (6.4% ring forms, 2.4% trophozoites,
1.2% schizonts) were incubated with 5 lM D,L-threophenyl-2-palmitoylamino-3-morpholino-1-propanol (D,Lthreo-PPMP). After 24 h of treatment, parasites were
labeled with [14C]palmitic acid or [14C]glucose for 18 h in
the presence of the drug. After the labeling period, each
stage was purified on a PercollÒ gradient as described
above and freeze-dried prior to lipid extraction. The effect
on parasite development was monitored by microscopy of
Giemsa-stained blood smears in two independent experiments.
In all cases, control cultures without the inhibitor and

a similar amount of uninfected erythrocytes were labeled
under the same conditions.

Cultures of P. falciparum and metabolic labeling

Isolation and purification of glycosphingolipids

An isolate (S20) of P. falciparum obtained from a patient
living in Porto Velho (Rondonia, Brazil) was used [15].
ˆ
Parasite cultures of P. falciparum were performed as
described [2].
[U-14C]Palmitic acid (Amersham 822 mCiỈmmol)1,
2.91 mCiỈmg)1) originally supplied in toluene was dried
under nitrogen, redissolved in ethanol, and coupled with
defatted BSA at a 1 : 1 (v/v) molar ratio. The final labeling
medium contained 6.25 lCiỈmL)1 of the radioactive precursor, 0.5% (w/v) Albumax and 0.05% (w/v) BSA.
Parasites were labeled for 18 h.

Each intraerythrocytic stage of P. falciparum was extracted
with chloroform/methanol 1 : 1 (3 · 1 mL). Each extract
was fractionated by anionic exchange chromatography on a
DEAE-Sephadex A-25 (acetate form) column, which was
eluted with chloroform/methanol/water (30 : 60 : 8, v/v/v)
to recover neutral GSLs and zwitterionic lipids. Anionic
lipids were bulk eluted with chloroform/methanol/0.8 M
NaAcO (30 : 60 : 8, v/v/v). The unbound fraction was
evaporated to dryness and treated with 0.1 M NaOH in
methanol (500 lL), at 37 °C for 3 h. The mixture was
neutralized with HCl 1 M in the presence of 1 M phosphate

Ó FEBS 2004

2206 A. S. Couto et al. (Eur. J. Biochem. 271)

buffer pH 7 (50 lL) to avoid over-acidification. After
evaporation, salts were removed by reverse-phase chromatography, using a Sep-Pack C-18 cartridge (Worldwire
Monitoring, Horsham, PA, USA). Acidic lipids were also
concentrated through a Sep-Pack cartridge. Purification of
neutral glycosphingolipids was achieved by chromatography on silicic acid. The sample was dissolved in chloroform
and loaded into a column of Unisil (7 · 50 mm) which was
eluted with chloroform (20 mL), chloroform/methanol
(98 : 2, v/v, 20 mL) and chloroform/methanol (1 : 3, v/v,
25 mL) [16].
In another experiment, total lipids from schizont forms
were extracted and purified as described above. The purified
neutral GSL fraction was analysed in parallel with an
analogous fraction obtained from [U-14C]palmitic acid
labeled parasites by TLC in solvent B. Spots corresponding
to the ceramide fraction (I), monohexosylceramide fraction
(II), trihexosylceramide fraction (IV) and tetrahexosylceramide fraction (V) were extracted from the plate and
analysed by UV-MALDI-TOF MS.
Acid methanolysis and methylation
The sample was hydrolysed for 18 h at 80 °C with 12 M
HCl/MeOH/water (3 : 29 : 4, v/v/v). The hydrolysate was
dried and the acid eliminated by several evaporations with
addition of water. Methylation of fatty acids was carried out
with BF3/MeOH in dry toluene under nitrogen at 80 °C for
90 min [17].

Ceramide glycanase digestion
Samples were dissolved in 250 mM phosphate buffer pH 5.0
(100 lL) containing 1% (w/v) sodium cholate. Ceramide
glycanase (from Macrobdella decora) (0.3 mU) was added
and digestion was performed at 37 °C for 18 h. Lipids
were extracted with chloroform/methanol (1 : 1, v/v) and
analysed by TLC.
Glucosylceramide synthase assay
Parasite homogenates were prepared in 0.1 M sodium
phosphate buffer (pH 7.4) containing 5 mM MgCl2,
25 mM KCl, 1 mM phenylmethanesulfonyl fluoride, 1 mM
N-a-tosyl-L-lisine chloromethyl ketone hydrochloride
(TLCK) and 10 lgỈmL)1 leupeptine by probe sonication
three times with 10 pulses while on ice. Liposomal substrate
was performed with dipalmitoylphosphatidylcholine
and ceramide (palmitoyldihydrosphingosine or palmitoylsphingosine) (10 : 1, v/v) containing 0.1 nmol of ceramide.
The constituent lipids were dissolved in chloroform/methanol (1 : 1, v/v), vortexed and dried under nitrogen. Lipids
were dispersed in 0.1 M sodium phosphate buffer pH 7.4
by sonication at 0 °C.
The reaction mixture consisted of UDP-[14C]glucose
(1 lCi, 319 mCiỈmmol)1, Amersham), 2 mM b-NAD and
the liposomal substrate (600 nmol lipid phosphorous) in
0.1 M sodium phosphate buffer (pH 7.4). The cell homogenate (50–100 lg protein per tube) was added making a
total volume of 15 lL. The mixture was incubated at 37 °C
for 5 h with shaking. Incubations were stopped by freezing
and the mixtures were cleaned by passage through C18

cartridges. Lipids were eluted with chloroform/methanol
(1 : 1, v/v) and further analysed by TLC. When the
inhibition test was performed, PPMP (5 lM) was added to

the reaction mixture. Spots were quantified using a phosphoimager (Molecular Analyst, Bio-Rad) with MOLECULAR
ANALYST software.
In another experiment, BODIPY analogues of ceramides
were used. BODIPY-dihydroceramide (BODIPY-DHCer)
was synthesized from dihydrosphingosine and BODIPY
acid according to Kok & Hoekstra [18]. The enzyme assay
was performed in tubes precoated with dipalmitoylphosphatidylcholine (15 nmoles added in chloroform and dried
down under nitrogen) by adding the fluorescent ceramide
(300 ng per tube) precoupled to BSA, the parasite lysates
(50–100 lg protein) and the assay buffer (0.1 M sodium
phosphate buffer pH 7.4 containing 25 mM KCl, 5 mM
MgCl2, 2.5 mM UDP-glucose and 2 mM b-NAD) in a final
volume of 150 lL. The reaction was incubated at 37 °C for
5 h with shaking. The mixture was extracted with chloroform/methanol (1 : 1, v/v) and analysed by TLC. Spots were
visualized using a Fuji LAS1000 densitometer equipped with
IMAGE GAUGE 3.122, software, Fuji Film, Japan.
All protein determinations were performed using Bradford’s method [19].
Immunoprecipitation
Parasite lysates (1–2 mg protein) were incubated with GCS
1.2 antibody (which recognizes a region near the GCS
C-terminus) [20] in buffer Tris/HCl pH 8.0 containing
150 mM NaCl, 0.5% (w/v) sodium deoxycholate and 0.1%
(w/v) SDS, for 2 h at 5 °C. Protein A-Sepharose (10% in the
same buffer, 100 lL) was added and it was incubated for a
further 60 min. The mixture was centrifuged at 10 000 g
and the immunoprecipitate was washed (3 · 100 lL). The
immunoprecipitates were dissolved in sample buffer and
subjected to SDS/PAGE in 10% gels. Western blot to
poly(vinylidene difluoride) membrane was performed and
blots were probed with anti-peptide polyclonal antibodies

GS-5.1 (1/1500) which recognizes a region near the GCS
N-terminus [20] followed by an anti-rabbit horseradish
peroxidase secondary antibody and visualized using ECLÒ
(Amersham) enhanced chemiluminescence reagent.
UV-MALDI-TOF MS analysis
Matrices for UV-MALDI-TOF MS. The b-carboline
(9H-pyrido[3,4-b]indole), nor-harmane and 2,5-dihydroxybenzoic acid were obtained from Aldrich Chemical Co.
Calibrating chemicals for UV-MALDI-TOF analysis.
a-Cyclodextrin (cyclohexaamylose, Mr 972.9), b-cyclodextrin (cycloheptaamylose, Mr 1135.0), c-cyclodextrin (cyclooctaamylose, Mr 1297.1), angiotensin I (Mr 1296.49),
neurotensin (Mr 1672.96) and bovine insulin (Mr 5733.5)
were purchased from Sigma-Aldrich.
Solvents. Methanol, ethanol, acetonitrile (Sigma-Aldrich
HPLC grade) and trifluoroacetic acid (Merck) were used as
purchased without further purification. Water of very low
conductivity (Milli Q grade; 56–59 nSỈcm)1 with PURIC-S
(ORUGANO Co., Ltd, Tokyo, Japan) was used.


Ó FEBS 2004

Glycosphingolipids in Plasmodium falciparum (Eur. J. Biochem. 271) 2207

UV-MALDI-TOF-MS experiments. Measurements were
performed using a Shimadzu Kratos, Kompact MALDI 4
(pulsed extraction) laser-desorption time-of-flight mass
spectrometer (Shimadzu, Kyoto, Japan) equipped with a
pulsed nitrogen laser (kem ¼ 337 nm; pulse width ¼ 3 ns),
tunable pulse delay extraction (PDE), post source decay
(PSD) (MS/MS device) and a secondary electron multiplier.
Experiments were first performed using the full range

setting for laser firing position in order to select the optimal
position for data collection, and secondly fixing the laser
firing position in the sample sweet spots. The samples were
irradiated just above the threshold laser power for obtaining
molecular ions and with higher laser power for studying
cluster formation. Thus, the irradiation used for producing
a mass spectrum was analyte-dependent with an acceleration voltage of 20 kV. Usually 50 spectra were accumulated.
All samples were measured in the linear and the reflectron
modes, in both the positive- and the negative-ion mode.
The stainless steel polished surface 2 sample-slides were
purchased from Shimadzu Co., Japan (P/N 670-19109-01).
Polished surface slides were used in order to get better
images for morphological analysis with a stereoscopic
microscope (NIKON Optiphot, Tokyo, Japan; magnification ·400) and with a high-resolution digital microscope
(Keyence VH-6300, Osaka, Japan; magnification ·800).
Sample preparation. Matrix stock solutions were made by
dissolving 1 mg of the selected compound in 0.5 mL of 1 : 1
(v/v) methanol/water. Analyte solutions were freshly prepared by dissolving the samples (0.05 mg) in chloroform/
methanol, 1 : 1 (v/v) (0.025 mL).
To prepare the analyte-matrix deposits two methods were
used. Method A; thin-film layer method (sandwich
method). Typically 0.5 lL of the matrix solution was placed
on the sample probe tip, and the solvent removed by
blowing air at room temperature. Subsequently, 0.5 lL of
the analyte solution was placed on the same probe tip
covering the matrix and partially dissolving it, and the
solvent was removed by blowing air. Then, two additional
portions (0.5 · 2 lL) of the matrix solution were deposited
on the same sample probe tip, producing a partial dissolution of the previously deposited thin-film matrix and
analyte layers. The matrix to analyte ratio was 3 : 1 (v/v)

and the matrix and analyte solution loading sequence was:
(a) matrix, (b) analyte, (c) matrix and (d) matrix. Method B;
mixture method. The analyte stock solution was mixed with
the matrix solution in 1 : 1–1 : 12 (v/v) ratio. A 0.5 lL
aliquot of this analyte-matrix solution was deposited onto
the stainless steel probe tip and dried with a stream of forced

room temperature air. Then, an additional portion of
0.5 lL was applied to the dried solid layer on the probe,
causing it to redissolve partially, and the solvent was
removed by blowing air.
The resulting solid partially crystalline layers were
found to be relatively homogeneous in both cases. norHarmane and 2,5-dihydroxybenzoic acid as matrices
showed signals of higher quality by using Method A.
Thus, the results shown and discussed in the present
article are those obtained using this sample preparation
method for each analyte, in the optimum experimental
conditions.
Spectra were calibrated using external calibration reagents: (a) commercial proteins (neurotensin; angiotensin I;
bovine insulin) and (b) a-, b- and c-cyclodextrins with norharmane as matrix, in positive- and in negative-ion mode.
The KRATOS KOMPACT calibration program was used.

Results
Metabolic labeling of GSLs
Cultures of P. falciparum with parasitemia around 10%
(4.5% ring forms, 2.7% trophozoites and 1.6% schizonts)
were metabolically labeled with [14C]palmitic acid for 18 h.
The different stages were purified on a Percoll gradient and
extracted with chloroform/methanol (1 : 1, v/v). A control
of uninfected erythrocytes was analysed in parallel

(Table 1). The different extracts were further fractionated
by DEAE-Sephadex A-25 (ACO–) column chromatography
into neutral and acidic lipids. TLC analysis of the unbound
fraction showed that the radioactive precursor was mainly
incorporated into diacyl-phospholipids (phosphatidylcholine, phosphatidylethanolamine and their lyso-derivatives)
as reported previously [21] (not shown). The acidic fraction
corresponding to the schizont stage showed a significantly
high incorporation in comparison with ring and trophozoite
stages (Table 1); thus similar amounts of radioactivity of
each fraction was applied to the TLC plate. Acidic lipids
analysed in solvent A showed main spots corresponding
to phosphatidylinositol, phosphatidic acid and fatty acids
(Fig. 1A).
The unbound fraction of each stage was treated with
0.1 M NaOH in methanol to hydrolyse non ceramide-based
lipids and after purification, the samples were analysed
by TLC in solvent B (Fig. 1B). These lipidic components
migrated close to standards of GSLs. A spot with the
mobility similar to a standard of sphingomyelin was also
shown. Even though the sample of control erythrocytes
used was enhanced, only faint bands were observed. In

Table 1. Incorporation of radioactive precursors (CPM per 108 parasites) in the different fractions of lipids obtained from the three intraerytrocytic
stages of P. falciparum. C, control uninfected erythrocytes; R, rings; T, trophozoites; S, schizonts.
[14C]palmitic acid

[14C]glucose

Total lipids
C

R
T
S

Neutral lipids

Acidic lipids

Sphingolipids

Total lipids

Neutral lipids

Acidic lipids

Sphingolipids

21800
719300
1011400
6269500

21500
457300
808300
5192900

–
5400

7400
73800

300
22800
30200
115900

3600
6047
7834
87854

2700
7300
5100
71200

–
300
400
2500

–
700
200
3800


2208 A. S. Couto et al. (Eur. J. Biochem. 271)

Ó FEBS 2004

Fig. 1. Incorporation of [14C]palmitic acid into
lipids of Plasmodium falciparum. (A) TLC
analysis in chloroform/methanol/water
(65 : 25 : 3, v/v/v) of the acidic lipids. Samples
obtained from 4.8 · 107 ring forms (lane 1),
2.06 · 107 trophozoites (lane 2) and
4.38 · 106 schizonts (lane 3) were spotted in
order to apply similar amounts of radioactivity. PtdGr, phosphatidyl glycerol; PtdH,
phosphatidic acid; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; lysoPtdIns,
lysophosphatidylinositol. (B) The unbound
fractions of the DEAE-Sephadex column were
saponified and analysed by TLC. Samples
corresponding to: 2.4 · 108 ring forms
(lane 1); 1.0 · 108 trophozoites (lane 2);
0.4 · 108 schizonts (lane 3); 7.0 · 108 noninfected erythrocytes (lane 4) were analysed in
chloroform/methanol/0.25% KCl (80 : 30 : 2,
v/v/v); I, ceramide; II, glucosylceramide; III,
lactosylceramide; IV, globotriaosylceramide;
V, globotetraosylceramide; VI, sphingomyelin.

order to ensure that the labeled components corresponded
to ceramide-based lipids, the spot comigrating with the
standard of GbOse3Cer (IV) from the schizont fraction
(Fig. 1B, lane 4), was eluted from the plate and digested
with ceramide glycanase. As expected, hydrolysis was not
complete, however, a new spot comigrating with a standard
of ceramide was obtained (Fig. 2A).

Fig. 2. Hydrolysis of the GSLs. The spot comigrating with GbOse3Cer
(IV) from Fig. 1B (lane 4) was incubated with ceramide glycanase for
18 h, extracted with choroform/methanol and further subjected to
TLC in chloroform/metanol/0.25% KCl (80 : 30 : 2, v/v/v). Cer, ceramide; GbOse3Cer, globotriosylceramide. The glycosphingolipid
fraction metabolically labeled with [14C]palmitic acid was subjected to
methanolysis, further treated with BF3/methanol and analysed by TLC
in chloroform/metanol/1 M NH4OH (40 : 10 : 1, v/v/v).C18-Sph, C18sphingosine; C18-sSph, C18- dihydrosphingosine.

A sample of the saponified neutral lipids obtained from
schizont stages, was further purified by Unisil column
chromatography. Three fractions (CHCl3, CHCl3/MeOH
(98 : 2, v/v), and CHCl3/MeOH (1 : 3, v/v)) were eluted.
The latter, containing the glycosphingolipids, was hydrolysed with HCl/MeOH/water (3 : 29 : 4, v/v/v), treated
with BF3/MeOH to methylate the rest of the fatty acids
that could interfere, and analysed by TLC in solvent C
(Fig. 2B). Two spots, migrating in the region where long
chain bases are resolved, were detected. One of them (RF
0.33) with the mobility of an authentic standard of C18sphinganine, the other one (RF 0.38) migrating slightly
above, would correspond to C20-sphinganine. A similar
result was obtained when the spot comigrating with
GbOse3Cer was analysed under the same conditions (not
shown).
In another experiment a [14C]glucose incorporation was
tried. The three sugar labeled stages were extracted as above
and the extracts were fractionated by DEAE-Sephadex
column chromatography and saponified. Although recoveries in ring and trophozoite forms extracts were low, the
purified glycosphingolipid fraction obtained from the
schizont stage showed a significant higher incorporation
of the radiolabeled sugar than uninfected erythrocytes

(Table 1). This extract was analysed by TLC in solvent B
in comparison with an analogous [14C]palmitic acid labeled
fraction (Fig. 3A). Four spots with RF similar to those
obtained by [14C]palmitic acid labeling were detected.
Cerebroside (RF 0.85) was clearly resolved in the sugar
labeled sample (Fig. 3A, lane 2). When a similar experiment
was performed using [14C]galactose as precursor, a faint
band corresponding to galactosylceramide was also detected
(Fig. 3B).
In order to further analyse each GSL fraction, extracts
obtained from schizont stages were fractionated as above
and the neutral GSL fraction was subjected to TLC in
parallel with an analogous [14C]palmitic acid labeled


Ó FEBS 2004

Glycosphingolipids in Plasmodium falciparum (Eur. J. Biochem. 271) 2209

Fig. 3. Incorporation of [14C]glucose and [14C]galactose into glycosphingolipids of Plasmodium falciparum. (A) TLC analysis in chloroform/methanol/0.25% KCl (80 : 30 : 2, v/v/v) of the unbound
fractions after mild alkaline treatment. Lane 1, [14C]glucose labeled
control erythrocytes (7 · 108 cells); lane 2, [14C]glucose labeled glycosphingolipids from schizonts (4 · 108 cells); lane 3, [14C]palmitic
acid labeled glycosphingolipids from schizonts (0.4 · 108 cells).
I, ceramide; II, glucosylceramide; III, lactosylceramide; IV, globotriaosylceramide; V, globotetraosylceramide; VI, sphingomyelin. (B)
TLC analysis in chloroform/methanol/0.25% KCl (80 : 30 : 2, v/v/v)
of the glycosphingolipid fraction purified from schizonts (1.4 · 108
parasites) after metabolic incorporation of [14C]galactose. GalCer,
galactosylceramide.

fraction. Spots corresponding to the ceramide fraction (I),

monohexosylceramide fraction (II), trihexosylceramide
fraction (IV) and tetrahexosylceramide fraction (V) were
extracted from the plate and analysed by UV-MALDI-TOF
MS (Fig. 4). Table 2 shows the m/z values (mass numbers)
and possible sphingoid-fatty acid-sugar combinations of
ceramides for the signals obtained from each fraction,
taking into account the results obtained by TLC analysis of
the long chain bases.
Presence of an active glucosylceramide synthase
In a first approach, the activity of the enzyme in a parasite
lysate was examined using UDP-[14C]glucose as donor and
two different ceramides, palmitoyldihydrosphingosine and
palmitoylsphingosine as acceptors (Fig. 5A). A spot comigrating with glucosylceramide was obtained but, interestingly, although dihydroceramides are poor substrates for
the mammalian enzymes [22], P. falciparum enzyme seemed
to be active only with the saturated compound (Fig. 5A,
lane 2). In order to confirm the substrate specificity of the
plasmodial enzyme, the enzymatic assay was performed
using fluorescent ceramides and UDP-glucose as donor.

Lysates from each stage were assayed and the products were
analysed by TLC. As expected, in the three stages, only
parasite lysates incubated with BODIPY-DHCer as acceptor synthesized fluorescent glucosylceramide (Fig. 5B, lanes
1–3). No fluorescent product was obtained when the
unsaturated ceramide was used (Fig. 5B, lanes 4–6). The
special substrate specificity assures the parasite origin of
the detected enzyme activity.
In order to show the presence of the GCS, immunoprecipitation of parasite lysates from each stage was performed
using polyclonal GCS 1.2 antibody [20]. The immunoprecipitates were subjected to SDS/PAGE and electrotransferred to poly(vinylidene difluoride) membranes. When the
membranes were developed with the GCS 5.1 antibody, a
band at a molecular mass of 48 kDa was detected in the

three intraerythrocytic stages (Fig. 5C, lanes 2–4). Nevertheless, the possibility that the 48 kDa band can be due to a
cross-reacting parasite protein not related with the GCS
cannot be ruled out.
In mammalian cells, low concentrations of D,L-threoPPMP have no effect on sphingomyelin synthase but can
inhibit the synthesis of glucosylceramides [23–26]. In order
to establish if the plasmodial enzyme activity was affected,
the experiment was performed in the presence of 5 lM
PPMP (Fig. 5D). TLC analysis revealed that the presence of
threo-PPMP efficiently inhibited the synthesis of glucosylceramides (52%). Additionally, the primary effect of PPMP
seemed to be specifically on GSLs, because no difference in
bulk protein synthesis was seen when comparing whole
[35S]methionine labeled precipitate of identical number of
parasites that were left untreated or treated with 5 lM
PPMP (Fig. 5D).
Previous reports showed that treatment of parasite
cultures with PPMP resulted in a potent inhibition of the
intraerythrocytic development of P. falciparum [27–30].
In order to determine the effect of the inhibitor in GSLs
synthesis, treatment of parasite cultures with threo-PPMP
for 24 h was performed followed by incorporation of
[14C]glucose or [14C]palmitic acid in the presence of the
drug. Parasite development was monitored by microscopy
of Giemsa-stained blood smears (Table 3). As expected,
treatment with threo-PPMP showed inhibition of the
intraerythrocytic development at the ring stage as described
by Haldar et al. [26]. Each labeled stage from treated and
nontreated parasites was purified by Percoll gradient and
further fractionated as above to achieve purified GSLs.
Comparison of the incorporation of [14C]glucose in the
same number of treated and nontreated parasites, showed a

clear reduction in the ring stage (Fig. 6A). As a result of the
arrest on development, a low amount of treated trophozoite
and schizont stages were obtained, this fact joined to a low
incorporation of the sugar precursor precluded further
analysis of these stages. Fractions corresponding to the
same number of [14C]glucose-labeled ring forms were
analysed by TLC in solvent A (Fig. 6B). While the fraction
obtained from nontreated ring forms showed spots corresponding to the labeled GSLs, no spots were detected in the
fraction obtained from PPMP-treated ring forms.
As regards the palmitic acid labeled parasites, the same
analysis was carried out. In accordance, when the incorporation of palmitic acid was compared in treated and
nontreated parasites, inhibition of the precursor incorpor-


Ó FEBS 2004

2210 A. S. Couto et al. (Eur. J. Biochem. 271)

Fig. 4. UV-MALDI-TOF mass spectra in
positive ion mode of the different GSLs fractions. Values indicate m/z of sodium adducted
molecular ions, [M + Na]+, in nominal
mass. Posible ceramide species are listed in
Table 2. (A) UV-MALDI-TOF MS of ceramides (fraction I) in reflectron mode; matrix:
nor-harmane; (B) UV-MALDI-TOF MS of
monohexosylceramides (fraction II) in linear
mode; matrix: nor-harmane; (C) UV-MALDITOF MS of globotriaosylceramides (fraction IV) in reflectron mode; matrix: 2,5-dihydroxybenzoic acid; (D) UV-MALDI-TOF
MS of globotetraosylceramides (fraction V) in
reflectron mode; matrix: 2,5-dihydroxybenzoic
acid.

Table 2. Mass numbers and possible sphingoid-fatty acid-sugar combinations of ceramides in the different fractions of GSLs obtained from
schizont forms after TLC analysis. Molecular related ions, [M+Na]+
are expressed as nominal mass. Listed ceramide species were deduced
from UV-MALDI-TOF MS spectra (Fig. 4). Spectra are shown in
Fig. 6. m/z, Data from UV-MALDI-TOF MS.
Spectra m/z

Proposed structures

A

494.9
522.9
537.2
550.8
553.2
569.0

d18:0-C10h:0
d20:0-C10h:0
d20:0-C10-2h:0
d18:0-C14h:0
d20:0-C10-3h:0
d18:0-C14-2h:0

522.5
550.7
568.0
656.6
686.3

d20:0-C10h:0
d18:0-C14h:0
d18:0-C14-2h:0
d18:0-C10h:0(+hex)
d20:0-C10h:0 (+hex)

B

d18:0-C12h:0
d18:0-C12-2h:0
d20:0-C12h:0
d18:0-C12-3h:0
d20:0-C12-2h:0
d18:0-C12h:0
d20:0-C12h:0
d20:0-C12-2h:0
d18:0-C12h:0 (+hex)

C

1036.5 d18:0-C14h:0(+3hex)
d20:0-C12h:0(+3hex)
1052.3 d18:0-C14-2h:0(+3hex) d20:0-C12-2h:0(+3hex)
1068.3 d18:0-C14-3h:0(+3hex) d20:0-C12-3h:0(+3hex)

D

1038.2 d18:0-C14h:0(+3hex)
d20:0-C12h:0(+3hex)

1052.8 d18:0-C14-2h:0(+3hex) d20:0-C12-2h:0(+3hex)
1185.0 d18:0-C10h:0
(3hex+hexNAc)

ation in the three stages was also observed (Fig. 6C). The
lipidic precursor is incorporated more efficiently probably
as a result of the development of the membrane network.
Consequently, even when a low amount of schizont stages
was obtained, the comparison on TLC could be carried out
(Fig. 6D). Interestingly, treated schizonts showed no glycosphingolipid components in contrast with the nontreated
samples.

Discussion
Glycosphingolipids seem to be a general feature of eukaryotic cells. However, the physiological functions of these
glycolipids have only been documented in mammalian cells,
whereas very little information is available of their roles in
other systems [31]. In this report we show for the first time
the presence of an active glucosylceramide synthase in the
intraerythrocytic stages of P. falciparum. Incorporation of
[14C]palmitic acid and [14C]glucose allowed the analysis of
purified glycosphingolipids. When the long chain base
component of these GSLs was investigated, using [14C]palmitic acid as a precursor, labeled sphinganine was obtained
(Fig. 3) in contrast with the major long chain base present in
erythrocytes, indicating clearly the parasite origin of the
detected compound. Degradation of host sphingomyelin to
produce ceramide for parasite growth has been suggested,
supported by the existence of sphingomyelinase in P. falciparum [30,32,33]. However, although the amount of cera-


Ó FEBS 2004

Glycosphingolipids in Plasmodium falciparum (Eur. J. Biochem. 271) 2211

Fig. 5. Glucosylceramide synthase analysis. (A) The enzymatic assay was performed using UDP-[14C]glucose as marker in 0.1 M sodium phosphate
buffer (pH 7.4), 2 mM b-NAD and a liposomal substrate consisting in dipalmitoylphosphatidylcholine and ceramide (10 : 1) (containing 0.1 nmol
of ceramide). The mixture was purified and analysed by TLC in chloroform/methanol/water (65 : 25 : 2, v/v/v). Lane 1, palmitoylceramide; lane 2,
palmitoyldihydroceramide. (B) The enzyme assay was performed using the fluorescent ceramide precoupled to BSA, UDP-glucose (2.5 mM) and
2 mM b-NAD. The parasite lysates (50–100 lg protein) in 0.1 M sodium phosphate buffer (pH 7.4). The mixture was extracted with chloroform/
methanol (1 : 1) and analysed by TLC in chloroform/methanol/water (65 : 25 : 2, v/v/v). Lanes 1, 2 and 3 are rings, trophozoites and schizonts,
respectively, using BODIPY-dihydroceramide; lanes 4, 5 and 6, the same using BODIPY-ceramide. (C) Immunoprecipitation of parasite lysates
performed using polyclonal GCS 1.2 antibody. The immunoprecipitates were subjected to SDS/PAGE and electrotransferred to poly(vinylidene
difluoride) membranes. The membranes were developed with the GCS 5.1 antibody followed by ECL. 1, control erythrocytes; 2, ring forms; 3,
trophozoites; 4, schizonts. Molecular mass of markers is indicated (in kDa) at the right side of the figure. The arrow at the left side shows the band at
48 kDa. (D) The enzymatic assay was performed using schizonts as enzymatic source as in (A), without (lane 1) or with (lane 2) 5 lM PPMP as
inhibitor. GlcCer, glucosylceramide; R, ring forms; T, trophozoites; S, schizonts. (E) Incorporation of L-[35S]methionine in proteins obtained from
parasites treated or nontreated with 5 lM PPMP. Aliquots were collected at different times (0–48 h), precipitated with 12% (w/v) trichloroacetic
acid and radioactivity was measured.

mide produced is low, our results confirm that the de novo
biosynthetic pathway of ceramides is active in this parasite.
Nevertheless, the last step of the ceramide biosynthesis,
involving the dehydrogenation of N-acylsphinganine to
N-acylsphingenine would be absent in P. falciparum. Additionally, [14C]galactose incorporation showed the presence
of galactosylceramide as reported recently [11].

The sphingolipid structure of the different products
obtained in the GSL fraction was proven by UVMALDI-TOF mass spectrometry. The spectra showed
that the predominant components of Fraction I were
ceramides involving long chain bases d18:0 or d20:0 and
hydroxy fatty acids C10:0, C12:0 and C14:0 bearing one,

two or three hydroxy residues (Fig. 4A, Table 2). This


Ó FEBS 2004

2212 A. S. Couto et al. (Eur. J. Biochem. 271)

Table 3. Effect of 5 lM threo-PPMP on parasite development. Parasite
cultures were incubated with 5 lM DL-threo-PPMP. After 24 h of
treatment, parasites were labeled with [14C]palmitic acid or [14C]glucose for 18 h in the presence of the drug. Control cultures without the
inhibitor were labeled under the same conditions. The effect on parasite development was monitored by microscopy of Giemsa-stained
blood smears in two independent experiments. R, ring forms;
T, trophozoites; S, schizonts.
[14C]glucose

[14C]palmitic acid

Control
%R
%T
%S

Fig. 6. Inhibition of GSL synthesis by threo-PPMP treatment. GSLs
were purified from threo-PPMP treated and nontreated parasites and
were further analysed by TLC in chloroform/methanol/water
(65 : 25 : 3, v/v/v). (A) Comparison of the radioactivity recovered in
the GSL fractions obtained from treated (unfilled bars) and nontreated
(filled bars) [14C]glucose incorporated parasites. R, ring forms;
T, trophozoites; S, schizonts; C, control uninfected erythrocytes. (B)
Lane 1, control [14C]glucose labeled ring stage (4.2 · 108 parasites);

lane 2, threo-PPMP-treated [14C]glucose labeled ring stage (5.5 · 108
parasites). (C) Comparison of the radioactivity recovered in the GSL
fractions obtained from treated (unfilled bars) and nontreated (filled
bars) [14C]palmitic acid incorporated parasites. R, ring forms;
T, trophozoites; S, schizonts; C, control uninfected erythrocytes. (D)
Lane 1, control [14C]palmitic acid labeled schizonts (0.13 · 108 parasites); lane 2, threo-PPMP-treated [14C]palmitic acid labeled schizonts
(0.16 · 108 parasites). In all cases, at each stage, a similar number of
parasites was compared.

finding is in accordance with a previous report showing
that the de novo biosynthetic pathway of fatty acids in
P. falciparum involved C10:0 to C14:0, some of them

Treated

Control

Treated

5.0
3.1
1.4

5.2
2.7
0.1

4.5
2.7
1.5

4.4
2.1
0.1

hydroxylated [34]. Fraction II migrated very near Fraction I and was shown to be a mixture of ceramides and
monohexosyl ceramides, not very well resolved. The latter
were mainly monohexosylceramides of d18:0 and d20:0
acylated with C10h:0 and C12h:0 (Fig. 4B, Table 2).
Spectrum C (Fig. 4) showed that the predominant component of Fraction IV is a trihexosylceramide (m/z
1036.5) with a possible sphingoid-fatty acid combination
d18:0-C14h:0 (or d20:0-C12h:0). On the other hand,
Fraction V (Fig. 4, spectrum D) showed less intense
signals than the others. However, it was very interesting
to detect a component of m/z 1185.0 corresponding to a
tetrahexosylceramide bearing an N-acetylhexosamine residue. This result agrees with the fact that incorporation of
tritiated glucosamine led to the preferential detection of
GSLs migrating as highly glycosylated species [10].
Biosynthesis of GSLs in P. falciparum pointed to the
presence of an active glucosylceramide transferase. When
the enzyme activity was searched in parasite lysates using
UDP-[14C]glucose as marker as well as using fluorescent
ceramides, activity was found only when the dihydroceramide was used as substrate. This is in good agreement with
the result described above and would explain earlier reports
showing that the parasites were not competent to the
formation of glucosylceramide when using unsaturated
ceramides [28].
GCS from different eukaryotic kingdoms have been
cloned; remarkably their sequences present only a few
conserved amino acids and the overall similarity between

the enzymes from species with remote evolutionary relationship is rather low [31]. In particular for P. falciparum,
we were unable to find any sequences related to GCS. This is
not rare as it has been suggested that enzymes are more
difficult to identify in P. falciparum by sequence similarity
methods. The difficulty has been attributed either to the
great evolutionary distance between P. falciparum and
other well studied organisms or to the high A + T content
of the genome [35]. Nevertheless, we detected a potential
gene for GCS (GenBankTM, accession number NP_701286)
with conserved domains for glycosyltransferases [36].
However, in an attempt to detect the presence of the
plasmodial enzyme in a parasite lysate, immunoprecipitation with polyclonal antibodies against the human GCS was
tried. A band of molecular mass near 48 kDa was
recognized in the three stages of the parasite. This band


Ó FEBS 2004

Glycosphingolipids in Plasmodium falciparum (Eur. J. Biochem. 271) 2213

was absent in the control erythrocytes. The apparent Mr
resembles the predicted molecular mass of the human and
rat GCS polypeptides although the empirical molecular
mass described is  38 kDa [20].
In mammalian cells, low concentrations (1–5 lM) of D,Lthreo-PPMP have no effect on sphingomyelin synthase but
can inhibit the synthesis of glucosylceramides. In P. falciparum, PPMP has been described as a potent inhibitor of the
intraerythrocytic maturation leading to an arrest of the
parasites at ring stage. Rings formed in the presence of
the drug contain no tubular structures. On the contrary,
mature trophozoites and schizonts that contain a fully

extended tubular network were not affected by the drug
[26,27,29,30]. When we tried the action of PPMP in vitro on
the GCS, using UDP-[14C]glucose as marker, the enzyme
activity which resulted was clearly reduced (Fig. 5D). In
another experiment, when the inhibitor was added in parasite
cultures, we observed an arrest on parasite development.
Parasites collected at the ring stage had been treated with
PPMP at the trophozoite stage ( 40 h before), and resulted
unaffected (Table 3). On the contrary, parasites collected at
the schizont stage that had received the inhibitor at the ring
stage, were not able to evolve and died. When the [14C]glucose labeled GSL fraction purified from PPMP treated and
from control parasites collected at the ring stage were
compared by TLC, disappearance of GSLs was shown
(Fig. 6B). This fact indicates that although parasites are able
to evolve to the ring stage, no new GSLs are biosynthesized.
Using [14C]palmitic acid as precursor, the analysis could
also be performed with the schizont stage. Likewise, parasites treated with PPMP showed disappearance of GSLs
(Fig. 6D). In this case two hypotheses may be postulated:
PPMP is also acting on the glucosyltransferase and although
there is de novo synthesis of ceramides, the glycosylating step
is blocked; or, parasites that overcome treatment are so
stressed that the tubovesicular membrane network is not able
to import the lipidic precursor. Anyway, the possibility of
both events taking place simultaneously must be considered.
In conclusion, we have isolated and characterized the
major GSL structures present in the intraerythrocytic forms
of P. falciparum by UV-MALDI-TOF mass spectrometry.
A glucosylceramide synthase activity with specificity for
saturated ceramides which can be inhibited by low concentrations of PPMP was identified for the first time. The
inhibitor, used in cultures, arrests parasite development with

a concomitant depletion of GSLs. The special feature
presented by the plasmodial GCS, joined to the expanding
number of cellular functions that may be glycosphingolipid
dependent, makes this enzyme a promising target for
antimalarial drug development. Studies are underway for
the characterization of the enzyme and its intracellular
location in P. falciparum.

Acknowledgements
This work was supported by grants from: CONICET, Universidad de
´
Buenos Aires and Agencia Nacional de Promocion Cientı´ fica y
´
Tecnologica (Pict 06-06545), Argentina. FAPESP, CNPq, PRONEX,
Brazil, UNDP/World Bank/WHO (TDR). A. S. C. and R. E.-B. are
members of Research Council CONICET (Argentina) and C. C.,
ANPCyT fellow. Mass spectrometry was performed as part of the
Academic Agreement between R. E.-B. and H. N. with the facilities of

the High Resolution Liquid Chromatography-integrated Mass Spectrometer System Laboratory of the United Graduate School of
Agricultural Sciences (Ehime University, Japan) and partially supported by Heiwa Nakajima Foundation.

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