From the ¶ Department of Biochemistry and Molecular
Biology, Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 17033 and the Department of Biochemistry and
Molecular Biology, Georgetown University Medical Center,
Washington, D. C. 20007
Received for publication, September 3, 2002, and in revised form, November 1, 2002
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ABSTRACT |
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Glycosylphosphatidylinositol (GPI)
anchors are crucial for the survival of the intraerythrocytic stage
Plasmodium falciparum because of their role in membrane
anchoring of merozoite surface proteins involved in parasite invasion
of erythrocytes. Recently, we showed that mannosamine can prevent the
growth of P. falciparum by inhibiting the GPI biosynthesis.
Here, we investigated the effect of isomeric amino sugars glucosamine,
galactosamine, and their N-acetyl derivatives on parasite
growth and GPI biosynthesis. Glucosamine, but not galactosamine,
N-acetylglucosamine, and N-acetylgalactosamine inhibited the growth of the parasite in a dose-dependent
manner. Glucosamine specifically arrested the maturation of
trophozoites, a stage at which the parasite synthesizes all of its GPI
anchor pool and had no effect during the parasite growth from rings to early trophozoites and from late trophozoites to schizonts and merozoites. An analysis of GPI intermediates formed when parasites incubated with glucosamine indicated that the sugar interferes with the
inositol acylation of glucosamine-phosphatidylinositol (GlcN-PI)
to form GlcN-(acyl)PI. Consistent with the non-inhibitory effect on
parasite growth, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine had no significant effect
on the parasite GPI biosynthesis. The results indicate that the enzyme
that transfers the fatty acyl moiety to inositol residue of GlcN-PI
discriminates the configuration at C-4 of hexosamines. An analysis of
GPIs formed in a cell-free system in the presence and absence of
glucosamine suggests that the effect of the sugar is because of direct
inhibition of the enzyme activity and not gene repression. Because the
fatty acid acylation of inositol is an obligatory step for the addition of the first mannosyl residue during the biosynthesis of GPIs, our
results offer a strategy for the development of novel anti-malarial drugs. Furthermore, this is the first study to report the specific inhibition of GPI inositol acylation by glucosamine in eukaryotes.
Malaria is a major health problem in many countries of the world.
Nearly 40% of the global population is vulnerable to this disease. In
Africa alone, malaria causes millions of deaths each year, mostly
children (1, 2). In recent years, the death toll due to malaria has
been rapidly increasing because of the widespread resistance of
parasites to chloroquine and other commonly used antimalarial drugs.
Therefore, there is an urgent need for the development of novel drugs.
Identification of new drugs targeting the metabolic pathways of
Plasmodium falciparum, the parasite that causes severe
malaria, has been the subject of study in several laboratories. The
recently studied parasite choline transporter, type II fatty acid
biosynthesis, dihydrofolate reductase-thymidylate synthase, and
glycosylphosphatidylinositol
(GPI)1 anchor biosynthesis
are some potential targets for drug design (3-7).
GPIs are a class of glycolipids involved in anchoring certain
functionally important proteins to the outer surfaces of cell membranes
(8). GPIs consist of a conserved trimannosylglucosaminyl core linked to
the inositol residue of a phosphatidylinositol (PI). GPIs from
different species or cell types vary widely in the type of fatty
acyl/alkyl residues and with respect to additional sugar residues
and/or ethanolamine phosphate attached to the glycan core (8). These
variations confer broad structural diversity to GPIs, resulting in a
wide range of biological activity (8, 9).
In the case of intraerythrocytic stage P. falciparum,
several proteins are anchored to the outer leaflet of the plasma
membrane through GPIs (10-13). These proteins include functionally
important molecules such as merozoite surface protein-1 (MSP-1), MSP-2, MSP-4, a 71-kDa protein of the heat shock family, the 102-kDa transferrin receptor, and a 75-kDa serine protease. Of these, MSP-1 is
crucial for recognition, binding, and invasion of erythrocytes (10-13). Furthermore, like many other parasites, P. falciparum synthesizes GPIs in severalfold excess than that
required for protein anchoring. Accumulated evidence suggests that
parasites use free GPIs for the regulation (stimulation/suppression) of the host immune system to assure their survival in the harsh
environments of the host (9). Therefore, GPIs are crucial for the
survival of P. falciparum in the host.
The GPIs of intraerythrocytic P. falciparum consist of
EtN-P-6(Man Materials--
RPMI 1640 culture medium and HEPES were purchased
from Invitrogen. TLCK, leupeptin, hypoxanthine,
p-aminobenzoic acid, saponin, galactosamine,
glucosamine, N-acetylglucosamine,
N-acetylgalactosamine, and jack bean Culturing of Parasite--
P. falciparum (FCR-3
strain) parasites were cultured in RPMI 1640 medium supplemented with
22 mM HEPES, 29 mM NaHCO3, 0.005% hypoxanthine, p-aminobenzoic acid (2 mg/liter), gentamycin
sulfate (50 mg/liter), and 10% human serum at 3-4% hematocrit (15). Cultures were maintained at 37 °C in an atmosphere of 90%
N2, 5% O2, and 5% CO2.
Treatment of Parasites with Hexosamines and
N-Acetylhexosamines--
The parasites were synchronized
4-6 h after erythrocyte invasion using 5% sorbitol (20).
The synchronous parasites were incubated in the culture medium
containing 1.25-10 mM of the test amino sugars. Parasites
incubated in culture medium without sugars served as control. At
various time intervals, thin smears of cultures were stained with
Geimsa and analyzed by light microscopy to assess the growth and
development of the parasites.
Radiolabeling of Parasite GPIs with Precursor Sugars--
To
investigate the effect of the sugars on the biosynthesis of GPIs, the
parasites were radiolabeled as follows: synchronous cultures of the
parasites with 15% parasitemia were treated with the inhibitor sugar
at 28 h after erythrocyte invasion. 2 h later, the medium was
replaced with one containing [3H]mannose (50 µCi/ml),
[3H]glucosamine (50 µCi/ml) or methionine (5 mg/liter),
and [35S]methionine (50 µCi/ml) and incubated for
6 h. For labeling with inositol, parasites with 15% parasitemia
were treated with inhibitors at 20 h after invasion (late rings)
and 2 h later were incubated with [3H]inositol (50 µCi/ml) for 12 h in regular medium (6). After radiolabeling, the
parasites were released from infected erythrocytes by suspending the
erythrocyte pellets in 0.025% saponin in 56 mM NaCl, 59 mM KCl, 1 mM NaH2PO4,
10 mM K2HPO4, 11 mM
NaHCO3, and 14 mM glucose, pH 7.4, and
incubated for 10 min in an ice bath. The suspension was passed through
a 26-G needle, centrifuged at 3800 rpm at 4 °C, and the pelleted
parasites washed with the buffer and stored at GPI Synthesis by Parasite Membranes and Inhibition by
Glucosamine--
Synchronous cultures of the parasites (1 ml) with
22% parasitemia were harvested at mid-trophozoite stage. Parasites
were released by lysis of erythrocyte membranes with 0.025% saponin as
described previously (6). The parasite pellet (~0.1 ml) was washed
with 50 mM sodium Bicine, 50 mM NaCl, 5 mM KCl, pH 8.0, containing 10 mg/ml glucose, and 1 mg/ml
bovine serum albumin. The parasites were lysed hypotonically by
suspending in 500 µl of water containing 0.1 mM TLCK, 1 µg/ml leupeptin, and homogenized with 20 strokes of a Dounce
homogenizer in an ice bath (21). To the lysate was added 500 µl of
200 mM sodium HEPES, 100 mM KCl, 20 mM MgCl2, pH 7.4, containing 0.1 mM
TLCK, and 1 µg/ml leupeptin, and the suspension was centrifuged at
15,000 × g for 10 min at 4 °C. The parasite
membrane pellet was suspended in 500 µl of 100 mM sodium
HEPES, 50 mM KCl, 10 mM MgCl2, pH
7.4, containing 0.1 mM TLCK, and 1 µg/ml leupeptin, and
the 100-µl suspension was used for each incubation. The membrane
suspensions were supplemented with 1 mM CoA, 1 mM ATP, 1 mM
UDP-N-acetylglucosamine, and 5 mM
MnCl2 and made up to 250 µl with the above buffer (21). Glucosamine (5 or 10 mM) was added, incubated at 37 °C
for 20 min, and then GDP-[3H]mannose (500,000 cpm) was
added and incubated at 37 °C for 2 h. The radiolabeled GPIs
formed were isolated and analyzed by HPTLC (see below).
Isolation of GPIs--
GPIs from parasites were extracted four
times with 5 volumes of CHCl3/CH3OH/water
(10:10:3, v/v/v). The extract was dried by SpeedVac and then
partitioned between water and water-saturated 1-butanol (22, 23). The
organic layer containing radiolabeled glycolipids was washed with water
and stored at Treatment of GPIs with Nitrous Acid--
The GPIs
(20,000-100,000 cpm) in 75 µl of 0.2 M NaOAc,
pH 3.75, 0.1% Nonidet P-40 were treated with 75 µl of 1 M NaNO2 (22, 23). After a 24-h incubation at
room temperature, the released lipid moieties were extracted with
water-saturated 1-butanol, dried, and analyzed by HPTLC. The glycan
moieties were recovered by chromatography on Bio-Gel P-4 (1 × 90 cm) in 100 mM pyridine, and 100 mM HOAc, pH
5.2.
Treatment of GPIs with Aqueous Hydrofluoric Acid--
The GPIs
(100,000-200,000 cpm) were treated with 50% aqueous hydrofluoric acid
(50 µl) in an ice bath for 48 h (22, 23). The reaction mixture
was neutralized with frozen-saturated LiOH extracted with
water-saturated 1-butanol, dried, and analyzed by HPTLC.
Alkaline Hydrolysis of GPIs--
The GPIs in 100 µl of the
methanol-ammonia mixture (methanol, 30% ammonia, 1:1 (v/v), freshly
prepared) were incubated at 37 °C for 4 h. At the end of the
incubation, the reaction mixture was evaporated by SpeedVac and
analyzed by HPTLC.
Treatment of GPIs with Mannosidase--
The GPIs (25,000-50,000
cpm) were treated with jack bean High Performance Thin Layer Chromatography of GPIs--
The GPI
extracts were applied onto HPTLC plates developed with
chloroform/methanol/water (10:10:2.4, v/v/v). After air drying, the
plates were sprayed with En3Hance fluorographic spray and
exposed to x-ray films at SDS-PAGE and Fluorography--
The
[35S]methionine-labeled parasites were dissolved in 100 µl of SDS-PAGE sample, the lysates were heated in a boiling water bath for 5 min, and then they were electrophoresed under non-reducing conditions on 6-20% SDS-polyacrylamide gradient gels (25). The gels
were fixed, washed with water, soaked in 1 M sodium
salicylate solution for 30 min, dried, and exposed to x-ray films at
Effect of Hexosamines and N-Acetylhexosamines on the Survival of
Intraerythrocytic P. falciparum--
The synchronous cultures of the
parasites at the ring stage 8 h after the invasion of erythrocytes
were treated with various concentrations of the test sugars, and the
growth of the parasites was monitored through various stages of
development. Glucosamine inhibited the parasite growth in a
dose-dependent manner (Fig. 1
and Table I). The growth of parasites
treated with 1.25 mM glucosamine was similar to control
cultures during the first cell cycle. However, the efficiency of
erythrocyte invasion of merozoites that entered into the third cell
cycle was reduced by ~20% compared with that of the untreated
culture. The parasite treated with 2.5 mM glucosamine also
grew normally in the first cycle, but the growth was significantly
reduced during the second cell cycle. The efficiency of erythrocyte
invasion of merozoites formed during the second cell cycle and third
cell cycle was decreased by ~20 and ~60%, respectively (Table I).
In the case of 5 mM glucosamine, the rate of parasite
growth was significantly reduced during the first cell cycle itself.
Approximately 75% of the parasites developed into trophozoites and
schizonts, and 25% remained as early trophozoites. The invasion
efficiency of merozoites formed during the first cell cycle was ~53%
compared with that of the control culture. Continued treatment with 5 mM glucosamine completely arrested parasite growth in the
second life cycle, and all parasites died. Parasites treated with 10 mM glucosamine failed to develop into trophozoites, and
subsequently all parasites died.
To determine whether the observed effect of glucosamine on parasite
growth is the result of the inhibition of specific metabolic pathway or
nonspecific cell toxicity, parasites were treated at the ring stage (6 h post-erythrocyte invasion) with 5 and 10 mM glucosamine.
At various time intervals, glucosamine was withdrawn and the growth of
the parasites was monitored. In this case, treatment with 5 or 10 mM glucosamine during ring stage for 8-10 h had no effect
on the growth. At both concentrations, the parasites developed into
trophozoites and schizonts and the released merozoites invaded the
erythrocytes with the same efficiency as that of the untreated control
culture. However, treatment for 24 h (both the ring and trophozoite stages) with 5 mM glucosamine caused a 40-50%
reduction in parasitemia in the second cell cycle. The surviving
parasites developed normally during the second cell cycle.
In contrast to glucosamine, galactosamine had no effect on the growth
and development of the parasites. The growth of the parasites treated
with 2.5 or 5 mM galactosamine was similar to that of the
untreated culture (Fig. 1 and Table I). The parasite treated with 10 mM galactosamine also developed into late trophozoites and
schizonts, but the erythrocyte invasion of the merozoites formed was
73% compared with that of the control culture. Of the parasites that
entered into the second cycle in 10 mM
galactosamine-treated culture, ~73% developed into trophozoites
during the second cell cycle and the erythrocyte invasion efficiency of
the merozoites to enter into the third life cycle was markedly reduced.
The inhibition of parasite on prolonged treatment with 10 mM galactosamine is probably because of the metabolic
conversion of galactosamine into glucosamine 6-phosphate (26), which
could inhibit parasite growth in a manner similar to that by glucosamine.
N-Acetylglucosamine and N-acetylgalactosamine
treatment was significantly less inhibitory on the growth and
development of the P. falciparum compared with the levels of
inhibition by glucosamine and galactosamine, respectively. At 2.5 mM concentrations, both N-acetylglucosamine and
N-acetylgalactosamine showed no noticeable inhibitory effect
on the parasite growth during the three cell cycles studied. Even at a
5 mM concentration, N-acetylglucosamine and
N-acetylgalactosamine caused no effect on the parasite
growth during the first cell cycle. In both cases, the merozoites
formed during the first cell cycle invaded the erythrocytes with same efficiency as that of untreated cultures, and the parasite developed to
the trophozoite stage normally in the second cell cycle (Table I).
However, merozoites formed from cultures treated with 5 mM N-acetylglucosamine invaded erythrocytes with only ~66%
efficiency compared with the control culture to enter the third cell
cycle; however, 5 mM N-acetylgalactosamine had
no significant effect. At 10 mM,
N-acetylglucosamine markedly inhibited parasite growth and
development; the erythrocyte invasion efficiency was ~80 and ~33%
for parasite entering the second and third cell cycles, respectively (Table I). In the case of N-acetylgalactosamine, 10 mM had no effect on the parasite in the first cell cycle.
The erythrocyte invasion efficiency of merozoites entering second life
cycle was comparable to the control culture. However, approximately
~87% of parasites developed into matured schizonts in the second
cell cycle, and the merozoites formed successfully invaded the
erythrocytes to enter into the third cell cycle.
Effect of Hexosamines and N-Acetylhexosamines on P. falciparum GPI
Biosynthesis--
We have previously shown that the intraerythrocytic
P. falciparum synthesizes all of its GPI pool in the
trophozoite stage (6). Therefore, to investigate the effect of amino
sugars on GPI biosynthesis, parasites were metabolically labeled with
[3H]mannose, [3H]glucosamine, and
[3H]inositol in the presence of glucosamine,
galactosamine, N-acetylglucosamine, and
N-acetylgalactosamine at the trophozoite stage. Parasites were treated with inhibitory sugars 2 h prior to the addition of
radioactive sugars to the culture medium and then labeled for 6 h
(12 h in the case of [3H]inositol) in the presence of
inhibitors. The GPIs synthesized by the treated and untreated parasites
were isolated and analyzed by HPTLC (Figs.
2 and 3).
The GPIs and biosynthetic intermediates formed were characterized by
specific degradation by HNO2 and hydrofluoric acid,
susceptibility to jack bean
When parasites were labeled with [3H]mannose in the
presence of glucosamine, GPI-biosynthetic intermediates containing 1-3 mannose were barely observed, indicating that the amino sugar inhibits
a biosynthetic step prior to the addition of the first mannose residue.
We have previously shown by labeling with [3H]glucosamine
that mannosamine, an epimer of glucosamine, inhibits GPI biosynthesis
in P. falciparum by inhibiting the transfer of the first
mannose residue to acylated GlcN-PI intermediate (6). However,
inhibition of GPIs by glucosamine cannot be studied using [3H] glucosamine, and labeling with
[3H]mannose will not furnish information regarding
inhibition prior to mannose addition. Therefore, the parasites were
labeled with [3H]inositol in the presence of 5 or 10 mM of either glucosamine or mannosamine and the products
were analyzed by HPTLC (Fig. 3). The identity of the GPI intermediates
was confirmed by specific degradation. HNO2 converted GPIs
and GPI intermediates into (acyl)PI, and Inhibition of GPI Synthesis by Glucosamine in the Cell-free
System--
To determine whether the effect of glucosamine on GPI
biosynthesis by in vitro cultured P. falciparum
is the result of the inhibition of the enzyme activity or interference
of the sugar with the gene expression, we studied GPI synthesis in a
cell-free system using parasite membranes (21). Membranes were prepared by harvesting the parasite at mid trophozoite stage, the developmental stage at which the GPIs are maximally synthesized by the
intraerythrocytic parasite. The membranes were incubated with
GDP-[3H]mannose in the presence of 5 and 10 mM glucosamine (21). Whereas GPIs and GPI intermediates
were efficiently synthesized by untreated membranes, glucosamine
effectively inhibited the synthesis of GPIs at the tested
concentrations (Fig. 4). A small but
significant amount of GPIs and intermediates was formed in
glucosamine-treated membranes. These were presumably synthesized from
the preexisting GlcN-(acyl)PI, which is expected to be present in
considerable amounts in the parasite because membranes were prepared
from parasites when GPI biosynthesis was maximal. These results
strongly suggest that glucosamine directly inhibits the enzyme activity
and that the effect of the sugar is not attributed to repression of the gene.
Effect of Glucosamine on P. falciparum Protein Synthesis--
To
further ascertain that the observed effect of glucosamine on parasite
growth and GPI inhibition in vitro was not attributed to
nonspecific inhibition of protein synthesis, the parasites were
metabolically labeled with [35S]methionine at the
trophozoite stage for 5-6 h in the presence of 1.25, 2.5, 5, and 10 mM glucosamine. After radiolabeling, the parasites were released from the infected erythrocytes by saponin treatment and the parasite proteins were analyzed by SDS-PAGE fluorography. The parasites treated with 1.25-10 mM
glucosamine synthesized similar levels of proteins compared with those
by untreated parasites (data not shown). However, in the case of 10 mM glucosamine, treatment for extended periods
significantly reduced protein synthesis because of the growth arrest.
A previous study from our laboratory showed that mannosamine
prevents the growth and development of P. falciparum by
inhibiting the GPI biosynthesis (6). It has been previously shown that in mammalian cells and other microorganisms that mannosamine
inhibits GPI biosynthesis by interfering with the addition of the
third mannose residue of the conserved glycan core (17-19). In
contrast, we found that in P. falciparum, mannosamine
blocked the addition of the first mannose residue (6). In this study,
we show that glucosamine, an epimer of mannosamine, can also inhibit
the growth of intraerythrocytic P. falciparum by
specifically blocking the biosynthesis of GPI anchors. However, unlike
mannosamine, glucosamine inhibits the parasite GPI biosynthesis by
interfering with the fatty acylation of the inositol residue of GlcN-PI
intermediate. This is a novel mechanism of inhibition of GPI
biosynthesis (see below). Our data also show that galactosamine, a C-4
isomer of glucosamine, and N-acetylglucosamine and
N-acetylgalactosamine do not inhibit either the parasite
growth or the GPI biosynthesis.
Several lines of evidence demonstrate that glucosamine can arrest the
growth of P. falciparum by specifically inhibiting GPI biosynthesis. Glucosamine inhibits the parasite GPI biosynthesis in a
dose-dependent manner; however, the sugar had no effect on the protein synthesis. The inhibition of parasite growth by glucosamine is developmental stage-specific, i.e. the sugar specifically
inhibits the maturation of trophozoites, the developmental stage at
which the intraerythrocytic parasite synthesizes its entire GPI pool. Glucosamine had no effect on the growth of the ring or schizont stage
parasites. The parasites treated with 10 mM glucosamine during most of the ring stage developed normally into early
trophozoites, which became schizonts and functional merozoites. The
parasites also developed into normal schizonts and functional
merozoites when treated with 10 mM glucosamine after the
formation of late trophozoites. However, treatment with 10 mM glucosamine during the trophozoite stage caused complete
growth arrest and death at the mid-trophozoite stage. Treatment of
parasite with 5 mM glucosamine during the entire first cell
cycle caused a significant reduction in the formation of mature
schizonts, and the erythrocyte invasion of merozoite was ~50%
compared with that of untreated culture. Continued presence of 5 mM glucosamine caused complete cell death during the second
cell cycle. Thus, the disruption of P. falciparum
trophozoite maturation with concomitant inhibition of GPI biosynthesis
by glucosamine clearly indicates that the GPIs are essential for the
growth of parasites.
Although glucosamine specifically arrested the growth of
intraerythrocytic P. falciparum at the trophozoite stage by
inhibiting the GPI biosynthesis, galactosamine did not inhibit either
parasite growth or GPI biosynthesis. Unlike glucosamine, which at 5 and 10 mM concentrations inhibited parasite GPI biosynthesis by
36 and 13%, respectively, 5 and 10 mM galactosamine had no
noticeable effect on GPI biosynthesis. Galactosamine-treated parasites
synthesized similar levels of GPIs compared with untreated culture (see
Fig. 2 and Table II). Consistent with these results and in agreement with our finding that GPIs are essential for parasite growth and development, galactosamine had no effect on the parasite growth. Thus,
in contrast to parasite culture treated with 5 mM
glucosamine, which caused complete cell death during the second
cell cycle, the parasites treated with 5 mM
galactosamine grew normally through all of the three cell cycles
measured. Whereas 10 mM glucosamine completely prevented
the trophozoite development in the first cell cycle, parasites treated
with 10 mM galactosamine developed into trophozoites and
schizonts and entered the second life cycle; however, the merozoite
invasion efficiency was somewhat decreased. This moderate inhibitory
effect on parasite growth by 10 mM galactosamine could be
the result of the metabolic conversion of significant amount of
galactosamine to glucosamine-6-phosphate (25), which is likely to cause
the inhibition of GPI synthesis similar to that by glucosamine.
Our finding on the inhibitory effect of hexosamines on parasite GPI
biosynthesis is in agreement with a previous observation by Pan
et al. (17) that glucosamine but not galactosamine inhibits the biosynthesis of GPIs in MDCK cells. Treatment of the MDCK cells
with 5 mM glucosamine caused a 67% reduction in the GPI biosynthesis, whereas similar amounts of galactosamine had no effect on
the levels of GPI biosynthesis (17). However, the mechanism of
inhibition of GPI synthesis by glucosamine in MDCK cells has not been
investigated. Based on the results of this study (discussed below), it
is likely that in MDCK and other mammalian cells, glucosamine also
inhibits the inositol acylation of GlcN-PI.
Compared with glucosamine, N-acetylglucosamine is
significantly less inhibitory on parasite growth and GPI biosynthesis.
Treatment with 5 and 10 mM N-acetylglucosamine
caused 21 and 40% decrease, respectively, in GPI synthesis compared
with untreated parasites. Consistent with only modest inhibition of GPI
synthesis, 5 mM N-acetylglucosamine had no
significant inhibitory effect on parasite growth in the first cell
cycle, although there was a low to moderate levels of inhibition in the
second and third cell cycles. Treatment with 10 mM
N-acetylglucosamine throughout the first cell cycle caused a
25% reduction in parasite growth. The inhibitory effect of
N-acetylglucosamine on treatment with 10 mM
sugar or prolonged treatment at 5 mM sugar could be
attributed to either the formation of a significant amount of
glucosamine 6-phosphate (25) or another intermediate from
N-acetylglucosamine that may be inhibitory or cause a
nonspecific effect.
The data presented here show that glucosamine inhibits a specific step
in the biosynthetic pathway of GPIs and that the enzyme involved in
this step discriminates the configurational structures of hexosamines.
As shown in Fig. 3A, inositol-acylated GlcN-PI is barely
formed in parasite treated with glucosamine, suggesting that the
glucosamine specifically inhibits the parasite GPI-biosynthetic pathway
by interfering with the inositol acylation of GlcN-PI intermediate to
form GlcN-(acyl)PI. The parasites treated with glucosamine synthesized
significant levels of GlcN-PI, suggesting that the effect of
glucosamine on P. falciparum biosynthesis is not
at or before the N-deacetylation of
N-acetylglucosamine-PI. In contrast, galactosamine or
mannosamine has no effect on inositol acylation. Thus, the inositol
fatty acid-transferring enzyme exhibits strict specificity to the
equatorial hydroxyl group at C-4 of glucosamine in GlcN-PI, and the
results of this study offer a strategy for the development of specific
inhibitors for GPI synthesis.
The results of this study also demonstrate that the effect of
glucosamine on GPI biosynthesis is because of the inhibition of
inositol-acylating enzyme activity but not because of the gene repression. This is evident by the synthesis of significant levels of
GPIs in isolated parasite membranes and the marked decrease in the
levels of newly synthesized GPIs when glucosamine was added to the
membrane incubation mixture (Fig. 4).
In summary, the data presented here show that glucosamine but not
galactosamine can specifically inhibit the biosynthesis of GPIs in
intraerythrocytic P. falciparum. Unlike mannosamine, an
epimer that has been previously shown to inhibit GPI biosynthesis in
the parasite by interfering with the addition of mannose to GlcN-(acyl)PI intermediate, glucosamine inhibits GPI biosynthesis by
preventing the transfer of fatty acyl moiety to GlcN-PI, a novel mode
of inhibition. Our data also show that glucosamine directly inhibits
the inositol-acylating enzyme activity but does not repress the gene
expression. Thus, glucosamine and mannosamine inhibitory steps of
parasite GPI biosynthesis can be differential targets for the
development of anti-malarial drugs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-2)Man
1-2Man
1-6Man
1-4GlcN linked
to the inositol residue of PI. The PI moiety is heterogeneous with
regard to fatty acyl substituents at the sn-1 and
sn-2 positions and at C-2 of the inositol residue
(14, 15). P. falciparum synthesizes GPIs exclusively during
the trophozoite stage in a developmental stage-specific manner. We have
previously shown that mannosamine inhibits the growth of P. falciparum specifically at the trophozoite stage and thereby
causes the death of the parasite (6). In P. falciparum, mannosamine prevents the addition of the first mannose to the inositol-acylated GlcN-PI intermediate. The mechanism by which mannosamine inhibits GPI biosynthesis in P. falciparum is
different from that observed in other organisms (16-19). In this
study, we investigated the effect of two isomeric amino sugars,
glucosamine and galactosamine, and their N-acetyl
derivatives on the parasite growth and GPI biosynthesis. The data
presented here show that of these amino sugars, only glucosamine
inhibits the growth of P. falciparum and parasite GPI
biosynthesis. The sugar inhibits fatty acid acylation of the inositol
residue of GlcN-PI, a novel inhibition mechanism.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase (30 units/mg)
were purchased from Sigma. Gentamycin sulfate was from Biofluids
(Rockville, MD). O-type human blood was obtained from the Georgetown
University Hospital. O-type human serum was from Interstate Blood Bank
(Memphis, TN). [2-3H]Mannose (18 Ci/mmol),
[6-3H]glucosamine (23 Ci/mmol),
[35S]methionine (1000 Ci/mmol), and
14C-labeled protein molecular weight markers were from
Amersham Biosciences. [1,2-3H]Inositol (30-80
Ci/mmol) and GDP-[2-3H]mannose (40 Ci/mmol) were from
American Radiolabeled Chemicals (St. Louis, MO). Silica Gel 60 HPTLC
plates were from Whatman (Clifton, NJ).
En3HanceTM fluorographic spray for TLC plates
was from PerkinElmer Life Sciences.
80 °C.
20 °C.
-mannosidase (30 units/ml) in 40 µl of 100 mM NaOAc, 2 mM Zn2+, pH
5.0, containing 0.1% sodium taurodeoxycholate at ~20 °C for 2 h and then at 37 °C for 22 h (24). The solutions were
heated in a boiling water bath for 5 min, extracted with
water-saturated 1-butanol, and analyzed by HPTLC.
80 °C (10).
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of hexosamines and
N-acetylhexosamines on the growth and development of
P. falciparum. Equal aliquots of synchronous cultures of the
parasites (4% parasitemia) 8 h after erythrocyte invasion were
separately treated with 1.25-10 mM of the indicated sugars
in complete medium. An aliquot of untreated parasites was cultured in
parallel as controls. Parasitemia was measured by counting the cells in
Geimsa-stained thin smears using light microscope. The percent
inhibition in parasitemia in cultures treated with various amino sugars
with respect to the level of parasitemia in the control culture was
plotted against the concentrations of amino sugars in the culture
medium. A, percent decrease in parasitemia in cultures after
48-h (8 h after parasites enter into the second cell cycle) treatment
with the indicated amounts of sugars. White,
gray, striped, and black bars indicate
the cultures with 1.25, 2.5, 5, and 10 mM
sugars, respectively. B, percent decrease in parasitemia in
cultures treated with the 5 mM sugars for various time
periods. White, gray, striped, and
black bars indicate the cultures treated for 24, 48, 72, and
96 h, respectively.
The effect of hexosamines and N-acetylhexosamines on parasitemia and
growth of intraerythrocytic P. falciparum
-mannosidase, and identification of
products by HPTLC (Fig. 3) (data not shown) (see also Refs. 6 and 10).
In both labeling procedures tested, glucosamine inhibited the GPI
biosynthesis in a dose-dependent manner. The parasites
treated with 1.25, 2.5, 5, and 10 mM glucosamine had
synthesized 95, 69, 36, and 13% GPIs, respectively, compared with
those synthesized by the untreated parasites (Fig. 2 and Table
II). At high concentrations,
N-acetylglucosamine also inhibited GPI synthesis. Thus, the
parasites treated with 5 and 10 mM
N-acetylglucosamine synthesized 79 and 60% GPIs,
respectively (Table II). This finding suggests that the inhibition of
the growth and development of P. falciparum by
N-acetylglucosamine noted above (Table I) is because of the
interference of the sugar in the biosynthesis of GPIs. In contrast, the
levels of the mature GPIs and intermediates synthesized by parasites
treated with 5 or 10 mM galactosamine were not
significantly different from the amounts of GPIs synthesized by the
untreated parasites (Fig. 2 and Table II).
N-Acetylgalactosamine caused little or no inhibition.
Parasites treated with 10 mM sugar synthesized ~92% GPIs
compared with untreated parasites (Table II).
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Fig. 2.
Analysis of [3H]mannose-labeled
GPIs synthesized by P. falciparum treated with
hexosamines and N-acetylhexosamines. Equal
aliquots of parasite cultures (1 × 107 cells) with
15% parasitemia were treated with 1.25, 2.5, 5, or 10 mM
amino sugars at 28 h after erythrocyte invasion (trophozoite
stage) in complete medium. After 2 h, [3H]mannose
(50 µCi/ml) was added to the culture in the presence of the
respective amounts of amino sugars and incubated for 6 h. An
aliquot of untreated parasites was cultured in parallel as controls.
The parasites were harvested, and free GPIs and GPI intermediates were
isolated, analyzed by HPTLC, and visualized by fluorography.
A, lane 1, GPIs synthesized by untreated
parasites. Lanes 2-5, GPIs synthesized by
parasites treated with 1.25, 2.5, 5, and 10 glucosamine, respectively.
B, lane 1, GPIs synthesized by untreated
parasites. Lanes 2 and 3, GPIs synthesized by
parasites treated with 5 and 10 mM galactosamine;
lanes 4 and 5, GPIs synthesized by parasites
treated with 5 and 10 mM N-acetylglucosamine;
lanes 6 and 7, GPIs synthesized by parasites
treated with 5 and 10 mM N-acetylgalactosamine.
The identity of the GPIs and their intermediates are indicated.
M4Gn-(A)PI,
Man4-GlcN-(acyl)PI;
EM3Gn-(A)PI,
EtN-P-Man3-GlcN-(acyl)PI;
EM4Gn-(A)PI,
EtN-P-Man4-GlcN-(acyl)PI.
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Fig. 3.
Analysis of
[3H]inositol-labeled GPIs synthesized by P. falciparum treated with glucosamine and
mannosamine. Equal aliquots of parasite cultures with 15%
parasitemia were treated with 5 and 10 mM sugars 20 h
after erythrocyte invasion (late rings and early trophozoites) in
complete medium. After 2 h, [3H]inositol (50 µCi/ml) was added to the culture in the presence of glucosamine or
mannosamine and incubated for 12 h. An aliquot of untreated
parasites was cultured in parallel as controls. The parasites were
harvested, and free GPIs and GPI intermediates were isolated, analyzed
by HPTLC, and visualized by fluorography. A, lane
1, GPIs synthesized by control parasite culture. Lanes
2 and 3, GPIs synthesized by parasites treated with 5 and 10 mM glucosamine, respectively; lanes 4 and
5, GPIs synthesized by parasites treated with 5 and 10 mM mannosamine, respectively. B, GPIs
synthesized by control parasite culture. Lane 1, untreated
GPIs; lanes 2 and 3, GPIs treated with nitrous
acid and jack bean -mannosidase, respectively. The identity of the
GPIs and their intermediates are indicated.
(A)PI, (acyl)PI;
Gn-(A)PI, GlcN-(acyl)PI;
GnAc-PI, N-acetylglucosamine-PI;
M2Gn-(A)PI,
Man2-GlcN-(acyl)PI;
M4Gn-(A)PI,
Man4-GlcN-(acyl)PI;
EM3Gn-(A)PI,
EtN-P-Man3-GlcN-(acyl)PI;
EM4Gn-(A)PI,
EtN-P-Man4-GlcN-(acyl)PI. PI*, PI
without acyl substituent on the inositol residue.
The effect of hexosamines and N-acetylhexosamines on GPI biosynthesis
by intraerythrocytic P. falciparum
-mannosidase converted
EtN-P-Man4-GPI into
EtN-P-Man3-GPI and converted the intermediates
lacking ethanolamine into GlcN-(acyl)PI (Fig. 3B). These
results revealed that although inositol-acylated GlcN-PI intermediate
is present in significant amounts in parasites labeled with mannosamine
(Fig. 3A, lanes 4 and 5), the amount of inositol-acylated GlcN-PI is dramatically decreased in
glucosamine-treated parasites (Fig. 3A, lanes 2 and 3). In contrast, significant levels of nonacylated
GlcN-PI were present in both glucosamine- and mannosamine-treated parasites. These results demonstrate that glucosamine inhibits the
inositol acylation of GlcN-PI intermediate.
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Fig. 4.
Inhibition of GPI synthesis by glucosamine in
P. falciparum cell-free system. The freshly
prepared parasite membranes were incubated with
GDP-[3H]mannose in the presence of glucosamine as
described under "Experimental Procedures." The GPIs formed were
isolated by extraction with 1-butanol, washed with water, dried,
analyzed by HPTLC, and visualized by fluorography. Lane 1,
GPIs synthesized by control parasite membranes. Lane 2 and
3, GPIs synthesized by parasite membranes treated with 5 and
10 mM glucosamine, respectively. The identity of the GPIs
and GPI intermediates are indicated in the left margin.
DPM, dolichol phosphate mannose;
M2Gn-(A)PI,
Man2-GlcN-(acyl)PI;
M3Gn-(A)PI,
Man3-GlcN-(acyl)PI;
M4Gn-(A)PI,
Man4-GlcN-(acyl)PI;
EM3Gn-(A)PI,
EtN-P-Man3-GlcN-(acyl)PI;
EM4Gn-(A)PI,
EtN-P-Man4-GlcN-(acyl)PI.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. V. P. Bhavanandan (Pennsylvania State College of Medicine) for critical reading of the paper.
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FOOTNOTES |
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* The study was supported by NIAID, National Institutes of Health Grant AI41139.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.
§ Present Address: Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, MD.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Pennsylvania State University
College of Medicine, 500 University Dr., Hershey, PA 17033. Tel.:
717-531-0992; Fax: 717-531-7072; E-mail: gowda@psu.edu.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M208976200
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ABBREVIATIONS |
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The abbreviations used are:
GPI, glycosylphosphatidylinositol;
MSP, merozoite surface protein;
TLCK, N-p-tosyl-L-lysine
chloromethyl ketone;
GlcN, glucosamine;
N-acetylgalactosamine, PI, phosphatidylinositol;
EtN, ethanolamine;
HPTLC, high performance thin-layer chromatography;
MDCK, Madin-Darby canine kidney;
Bicine, N,N-bis(2-hydroxyethyl)glycine.
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