Glycosphingolipids in
Plasmodium falciparum
Presence of an active glucosylceramide synthase
Alicia S. Couto
1
, Carolina Caffaro
1
, M. Laura Uhrig
1
, Emilia Kimura
2
, Valnice J. Peres
2
, Emilio F. Merino
2
,
Alejandro M. Katzin
2
, Masae Nishioka
3
, Hiroshi Nonami
3
and Rosa Erra-Balsells
1
1
CIHIDECAR, Departamento de Quı
´
mica Orga
´
nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Argentina;

,
L
-threo-phenyl-2-palmitoylamino-
3-morpholino-1-propanol (PPMP). In addition, de novo
biosynthesis of glycosphingolipids was shown by metabolic
incorporation of [
14
C]palmitic acid and [
14
C]glucose in the
three intraerythrocytic stages of the parasite. The structure
of the ceramide, monohexosylceramide, trihexosylceramide
and tetrahexosylceramide fractions was analysed by UV-
MALDI-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.
Keywords: dihydroceramide; glucosylceramide synthase;
glycosphingolipids; malaria; Plasmodium falciparum.
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

thase (GCS)]. With regards to localization, as far as it is
known, GlcCer is special because it is the only glyco-
sphingolipid 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 Orga
´
nica, Pabello
´
n II, Facultad de Ciencias Exactas y Nat-
urales, 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, BODIPY-
dihydroceramide; BODIPY-Cer, BODIPY-ceramide;
D
,
L
-threo-PPMP,
D
,
L
-threo-phenyl-2-palmitoylamino-3-morpholino-
1-propanol, d18:0, 4-hydroxysphinganine; d20:0, 4-hydroxyicosa-
sphinganine; 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).

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 Tech-
nologies (New York, NY, USA). All solvents were of
analytical or HPLC grade. PercollÒ was purchased from
Pharmacia Chemicals (Uppsala, Sweden).
D
,
L
-threo-phenyl-
2-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) chloro-
form/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
NH
4

-[U-
14
C]glucose (Amersham, 291 mCiÆmmol
)1
,1,54
mCiÆmg
)1
) was incorporated at a concentration of
6.25 lCiÆmL
)1
in RPMI 1640 medium without addition
of 11 m
M
of glucose. Parasites (5.9% ring forms, 5.4%
trophozoites, 3.7% schizonts) were labeled for 18 h.
D
-[U-
14
C]galactose (Amersham, 306 mCiÆmmol
)1
,1,61
mCiÆmg
)1
) was incorporated at a concentration of
3.2 lCiÆmL
)1
in RPMI 1640 medium without addition of
11 m
M
of glucose. Parasites (9.8% ring forms, 3.0%

(0–48 h), precipitated with 12% (w/v) trichloroacetic acid,
and radioactivity was measured with a Beckman 5000
b-counter.
Treatment of parasites with
D
,
L
-
threo
-phenyl-2-
palmitoylamino-3-morpholino-1-propanol
Parasite cultures (6.4% ring forms, 2.4% trophozoites,
1.2% schizonts) were incubated with 5 l
MD
,
L
-threo-
phenyl-2-palmitoylamino-3-morpholino-1-propanol (
D
,
L
-
threo-PPMP). After 24 h of treatment, parasites were
labeled with [
14
C]palmitic acid or [
14
C]glucose for 18 h in
the presence of the drug. After the labeling period, each
stage was purified on a PercollÒ gradient as described

Monitoring, Horsham, PA, USA). Acidic lipids were also
concentrated through a Sep-Pack cartridge. Purification of
neutral glycosphingolipids was achieved by chromatogra-
phy 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-
14
C]palmitic acid
labeled parasites by TLC in solvent B. Spots corresponding
to the ceramide fraction (I), monohexosylceramide fraction
(II), trihexosylceramide fraction (IV) and tetrahexosylcera-
mide 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 °Cwith12
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 BF
3
/MeOH in dry toluene under nitrogen at 80 °Cfor
90 min [17].
Ceramide glycanase digestion

was performed with dipalmitoylphosphatidylcholine
and ceramide (palmitoyldihydrosphingosine or palmitoyl-
sphingosine) (10 : 1, v/v) containing 0.1 nmol of ceramide.
The constituent lipids were dissolved in chloroform/meth-
anol (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-[
14
C]glucose
(1 lCi, 319 mCiÆmmol
)1
, Amersham), 2 m
M
b-NAD and
the liposomal substrate (600 nmol lipid phosphorous) in
0.1
M
sodium phosphate buffer (pH 7.4). The cell homo-
genate (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 l
M
) was added to

visualized using a Fuji LAS1000 densitometer equipped with
IMAGE GAUGE
3.122, software, Fuji Film, Japan.
All protein determinations were performed using Brad-
ford’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 m
M
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-dihydroxy-
benzoic acid were obtained from Aldrich Chemical Co.
Calibrating chemicals for UV-MALDI-TOF analysis.
a-Cyclodextrin (cyclohexaamylose, M

¼ 337 nm; pulse width ¼ 3ns),
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 acceler-
ation voltage of 20 kV. Usually 50 spectra were accumu-
lated.
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; magnifica-
tion ·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 pre-
pared 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

harmane 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 [
14
C]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 (phosphatidylcho-
line, 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).

(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).
A sample of the saponified neutral lipids obtained from
schizont stages, was further purified by Unisil column
chromatography. Three fractions (CHCl
3
,CHCl
3
/MeOH
(98 : 2, v/v), and CHCl
3
/MeOH (1 : 3, v/v)) were eluted.
The latter, containing the glycosphingolipids, was hydro-
lysed with HCl/MeOH/water (3 : 29 : 4, v/v/v), treated
with BF
3
/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 (R
F
0.33) with the mobility of an authentic standard of C
18
-
sphinganine, the other one (R
F
0.38) migrating slightly
above, would correspond to C

labeled sample (Fig. 3A, lane 2). When a similar experiment
was performed using [
14
C]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 [
14
C]palmitic acid labeled
Fig. 1. Incorporation of [
14
C]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 · 10
7
ring forms (lane 1),
2.06 · 10
7
trophozoites (lane 2) and
4.38 · 10
6
schizonts (lane 3) were spotted in
order to apply similar amounts of radioacti-
vity. PtdGr, phosphatidyl glycerol; PtdH,
phosphatidic acid; PtdIns, phosphatidylinosi-

fraction metabolically labeled with [
14
C]palmitic acid was subjected to
methanolysis, further treated with BF
3
/methanol and analysed by TLC
in chloroform/metanol/1
M
NH
4
OH (40 : 10 : 1, v/v/v).C
18
-Sph, C
18
-
sphingosine; C
18
-sSph, C
18
- dihydrosphingosine.
2208 A. S. Couto et al.(Eur. J. Biochem. 271) Ó FEBS 2004
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.

theless, 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
-threo-
PPMP 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 l
M
PPMP (Fig. 5D). TLC analysis revealed that the presence of
threo-PPMP efficiently inhibited the synthesis of glucosyl-
ceramides (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
[
35
S]methionine labeled precipitate of identical number of
parasites that were left untreated or treated with 5 l
M
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

poration of palmitic acid was compared in treated and
nontreated parasites, inhibition of the precursor incorpor-
Fig. 3. Incorporation of [
14
C]glucose and [
14
C]galactose into glyco-
sphingolipids of Plasmodium falciparum. (A) TLC analysis in chloro-
form/methanol/0.25% KCl (80 : 30 : 2, v/v/v) of the unbound
fractions after mild alkaline treatment. Lane 1, [
14
C]glucose labeled
control erythrocytes (7 · 10
8
cells); lane 2, [
14
C]glucose labeled gly-
cosphingolipids from schizonts (4 · 10
8
cells); lane 3, [
14
C]palmitic
acid labeled glycosphingolipids from schizonts (0.4 · 10
8
cells).
I, ceramide; II, glucosylceramide; III, lactosylceramide; IV, globo-
triaosylceramide; 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 · 10
8

mitic 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. falci-
parum [30,32,33]. However, although the amount of cera-
Fig. 4. UV-MALDI-TOF mass spectra in
positive ion mode of the different GSLs frac-
tions. Values indicate m/z of sodium adducted
molecular ions, [M + Na]
+
,innominal
mass. Posible ceramide species are listed in
Table 2. (A) UV-MALDI-TOF MS of cera-
mides (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-MALDI-
TOF MS of globotriaosylceramides (frac-
tion IV) in reflectron mode; matrix: 2,5-di-
hydroxybenzoic 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 combi-
nations 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

of galactosylceramide as reported recently [11].
The sphingolipid structure of the different products
obtained in the GSL fraction was proven by UV-
MALDI-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
Fig. 5. Glucosylceramide synthase analysis. (A) The enzymatic assay was performed using UDP-[
14
C]glucose as marker in 0.1
M
sodium phosphate
buffer (pH 7.4), 2 m
M
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 m
M
)and
2m
M
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

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-[
14
C]glucose as marker as well as using fluorescent
ceramides, activity was found only when the dihydrocera-
mide 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 rela-
tionship 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 (GenBank
TM
, accession number NP_701286)
with conserved domains for glycosyltransferases [36].

14
C]palmitic acid labeled schizonts (0.13 · 10
8
para-
sites); lane 2, threo-PPMP-treated [
14
C]palmitic acid labeled schizonts
(0.16 · 10
8
parasites). In all cases, at each stage, a similar number of
parasites was compared.
Table 3. Effect of 5 l
M
threo-PPMP on parasite development. Parasite
cultures were incubated with 5 l
MDL
-threo-PPMP. After 24 h of
treatment, parasites were labeled with [
14
C]palmitic acid or [
14
C]glu-
cose for 18 h in the presence of the drug. Control cultures without the
inhibitor were labeled under the same conditions. The effect on para-
site development was monitored by microscopy of Giemsa-stained
blood smears in two independent experiments. R, ring forms;
T, trophozoites; S, schizonts.
[
14
C]glucose [

C]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 [
14
C]glu-
cose 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 [
14
C]palmitic acid as precursor, the analysis could
also be performed with the schizont stage. Likewise, para-
sites 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

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