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J. Biol. Chem., Vol. 275, Issue 32, 24506-24511, August 11, 2000
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From the Department of Biochemistry and Molecular Biology,
Georgetown University Medical Center, Washington, D. C. 20007
Received for publication, March 13, 2000, and in revised form, May 17, 2000
Glycosylphosphatidylinositols (GPIs) are the
major glycoconjugates in intraerythrocytic stage Plasmodium
falciparum. Several functional proteins including merozoite
surface protein 1 are anchored to the cell surface by GPI modification,
and GPIs are vital to the parasite. Here, we studied the developmental
stage-specific biosynthesis of GPIs by intraerythrocytic P. falciparum. The parasite synthesizes GPIs exclusively during the
maturation of early trophozoites to late trophozoites but not during
the development of rings to early trophozoites or late trophozoites to
schizonts and merozoites. Mannosamine, an inhibitor of GPI
biosynthesis, inhibits the growth of the parasite specifically at the
trophozoite stage, preventing further development to schizonts and
causing death. Mannosamine has no effect on the development of either
rings to early trophozoites or late trophozoites to schizonts and
merozoites. The analysis of GPIs and proteins synthesized by the
parasite in the presence of mannosamine demonstrates that the effect is
because of the inhibition of GPI biosynthesis. The data also show that
mannosamine inhibits GPI biosynthesis by interfering with the addition
of mannose to an inositol-acylated GlcN-phosphatidylinositol (PI) intermediate, which is distinctively different from the pattern seen in
other organisms. In other systems, mannosamine inhibits GPI
biosynthesis by interfering with either the transfer of a mannose
residue to the Man Glycosylphosphatidylinositol
(GPI)1 anchors represent a
distinct class of glycolipids found covalently attached to proteins in
almost all eukaryotic cells (1, 2); they are particularly abundant in
parasites, in which they occur as free lipids or linked to proteins (3,
4). Although the primary function of GPIs is to anchor proteins to cell
surfaces, they have been implicated in a number of biological responses
including transmembrane signaling, apical targeting of proteins in
polarized cells, insulin mimetic activity, and endocytic mechanisms (1,
5, 6).
Plasmodium falciparum, the most virulent parasite among the
four species of plasmodial apicomplexan protozoa that infect man, causes millions of deaths each year in tropical and subtropical countries (7). In the past decades, chloroquine and other drugs have
been very useful in reducing mortality due to malaria. Although these
conventional drugs are still widely used for the treatment of parasite
infection, the parasite is rapidly becoming drug-resistant, raising the
death toll from malaria (7). Therefore, malaria is once again a global
concern, and novel antimalarial drugs are urgently needed.
In P. falciparum, GPIs are the major carbohydrate moieties,
whereas N- and O-linked carbohydrates or other
glycolipids are either absent or present only at very low levels (8,
9). More than 15 proteins of the intraerythrocytic stage P. falciparum including functionally important proteins such as
merozoite surface protein-1, -2, and -4, a 71-kDa heat shock family
protein, a 102-kDa transferrin receptor, and a 75-kDa serine protease
are modified with GPI anchors (8, 10, 11). These observations suggest that GPI biosynthesis is vital to the parasite.
The P. falciparum GPIs contain a conserved core
structure,
ethanolamine-phosphate-6Man During the 48-h intraerythrocytic life cycle of P. falciparum, the parasite undergoes several morphologically
distinct developmental stages, rings, trophozoites, schizonts, and
finally differentiates into merozoites, which upon release invade other
red blood cells. In this study, we show that intraerythrocytic P. falciparum synthesizes GPI anchors exclusively at the trophozoite
stage in a developmental stage-specific manner. We also investigated
the effect of the inhibition of GPI anchor biosynthesis on the growth
and development of the parasite using ManN, a known inhibitor of GPI
biosynthesis in eukaryotic cells (16-21). In eukaryotes, ManN can also
inhibit the assembly of N-linked oligosaccharides (17), and
it is a precursor for the synthesis of sialic acid (22). Because
P. falciparum contains little or no
N-glycosylation capacity, and the parasite does not
synthesize sialic acid, the effect of ManN on the parasite is because
of the inhibition of GPI biosynthesis. In the presence of ManN, the
growth of the parasite was arrested specifically at the trophozoite
stage, a developmental stage at which the parasite synthesizes all of
its GPIs; this resulted in the death of the parasite. Detailed study
demonstrated that the lethal effect of ManN on the parasite is because
of the inhibition of GPI biosynthesis, and that ManN interferes with
the addition of the first mannose residue to the inositol-acylated
GlcN-PI, a mechanism of inhibition that is different from that observed in other organisms.
Materials--
RPMI 1640 culture medium and HEPES were purchased
from Life Technologies, Inc. Hypoxanthine,
p-aminobenzoic acid, saponin, and ManN were purchased from
Sigma. Aspergillus saitoi Culturing of the Parasite--
P. falciparum (FCR-3
strain) 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 (8).
Cultures were maintained at 37 °C in an atmosphere of 90%
N2, 5% O2, and 5% CO2.
Effect of ManN on the Parasite--
The parasites were
synchronized 4-6 h after invasion using 5% sorbitol (23). The
cultures were treated with 1.25-10 mM ManN. At various
time periods, thin smears of cultures were prepared and stained with
Giemsa, and the growth and development of parasites was assessed under
light microscopy.
Labeling of the Parasites with Radioactive
Precursors--
Synchronous cultures of the parasites (30 h after
invasion, trophozoites) at 15% parasitemia were labeled with
[3H]GlcN (50 µCi/ml) for 6 h in a medium
containing 5 mM glucose. Labeling with
[35S]Met (50 µCi/ml) was performed in a medium
containing reduced Met (5 mg/liter). For inhibition experiments, the
radiolabeled precursors were added to the culture medium 2 h after
treatment with various concentrations of ManN. After labeling, the
parasites were released from infected erythrocytes by suspending the
erythrocyte pellets in 0.015% 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
incubation for 10 min in an ice bath. The cell suspension was passed
through a 26G needle and centrifuged at 3800 rpm at 4 °C (24), and
the parasites were washed with the buffer and stored at Isolation of the GPIs--
The parasites were lyophilized and
extracted four times with 5 volumes of chloroform/methanol/water
(10:10:3, v/v/v) (25, 26). The extract was dried by rotary evaporation
and then partitioned between water and water-saturated 1-butanol. The
organic layer, containing labeled GPIs, was washed with water and
stored at Nitrous Acid Treatment--
The [3H]GlcN-labeled
GPIs or their intermediates isolated by preparative TLC
(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 (25, 26). After a 24-h incubation at room
temperature, the released lipid moieties were extracted with
water-saturated 1-butanol, dried, and analyzed by HPTLC.
Treatment with Aqueous HF--
The
[3H]GlcN-labeled GPIs (30,000-50,000 cpm) were treated
with 50% aqueous HF (50 µl) in an ice bath for 48 h (25, 26). The reaction mixture was neutralized with frozen saturated LiOH, extracted with water-saturated 1-butanol, dried, and analyzed by
HPTLC.
Characterization of GPI Glycan Core--
The
[3H]GlcN-labeled GPIs or GPI intermediates
(50,000-100,000 cpm) were treated with HF and then with
HNO2 (25, 26). The samples were reduced with 100 µl of 1 M NaBH4 in 100 mM NaOH at room
temperature for 6 h. Excess NaBH4 was destroyed by
acidification with 2 M HOAc to pH 5 in an ice bath, and the
solutions were deionized with AG 50W-X16 (H+) and AG 4-X4
(base) resins, dried, and analyzed by HPTLC.
Treatment with Mannosidase--
The
[3H]GlcN-labeled GPIs (25,000-50,000 cpm) were treated
with jack bean Thin Layer Chromatography--
The
[3H]GlcN-labeled GPIs were applied onto HPTLC plates,
developed with chloroform/methanol/water (10:10:2.4, v/v/v), and dried.
The GPI bands were viewed by fluorography using
En3HanceTM (8). The glycan cores obtained by
treatment of GPIs with HF and nitrous acid/NaBH4 and their
mannosidase digestion products were analyzed by HPTLC using
1-propanol/acetone/water (10:6:4, v/v/v). The GPIs were identified by
comparison of the RF values of the glycan cores
with those of a standard 2,5-anhydromannitol to
Man4-2,5-anhydromannitol ladder (8).
SDS-PAGE and Fluorography--
The [3H]GlcN- or
[35S]Met-labeled parasites were dissolved in nonreducing
SDS-PAGE sample buffer, and the lysates were heated in a boiling water
bath for 5 min and then electrophoresed under nonreducing conditions on
6-20% SDS-polyacrylamide gradient gels (28). The gels were fixed,
washed with water, soaked in 1 M sodium salicylate in water
for 30 min, dried, and exposed to x-ray films at Effect of ManN on the Survival of P. falciparum at Different Stages
of the Intraerythrocytic Development--
We investigated the
developmental stage-specific biosynthesis of GPIs in intraerythrocytic
P. falciparum and the effect of inhibition of the GPI
biosynthetic pathway on the survival of the parasite. Synchronous
cultures of the parasites were treated with 1.25-10 mM
ManN at the early ring stage (8 h after invasion of erythrocytes), and
the growth of parasites was monitored through various stages of
intraerythrocytic development. ManN inhibited parasite growth in a
dose-dependent manner (Fig. 1
and Table I). Parasites treated with 10 mM ManN developed normally during the ring stage, but their
growth was markedly impaired at the trophozoite stage; although the
treated parasites differentiated into trophozoites, they looked
unhealthy with little or no pigment formation, were unable to become
schizonts, and died. Parasites treated with 5 mM ManN
developed into trophozoites and schizonts, but the rate of growth was
significantly reduced during the trophozoite stage. The merozoites
formed during 5 mM ManN treatment invaded red blood cells
with significantly lower efficiency compared with the untreated parasites. Continued treatment with 5 mM ManN through the
second cell cycle completely arrested parasite growth at the
trophozoite stage and all parasites died. The parasites treated with
1.25 mM or 2.5 mM ManN grew similar to control
cultures during the first cell cycle, but the efficiency of erythrocyte
invasion of merozoites formed during the second cell cycle was
significantly reduced (~90 and ~75%, respectively) compared with
that of the control culture. The survival of parasites treated with <5
mM ManN is because of the formation of significant amounts
of GPIs, which were used for the GPI anchoring of proteins (see
below).
To examine whether the effect of ManN on the parasite is because of the
inhibition of a specific metabolic pathway at the trophozoite stage or
to nonspecific toxicity, the parasites were treated (6 h after
invasion) with 5 or 10 mM ManN. At various time intervals,
ManN was withdrawn. Treatment with either 5 or 10 mM ManN
during the ring stage only had no significant effect on parasite
growth; the parasites developed into trophozoites and schizonts, and
the invasion efficiency of the merozoites was similar to that of the
untreated parasites. However, treatment with 5 mM ManN for
24 h, extending the treatment period to the trophozoite stage,
caused about a 60% reduction in parasitemia in the second cell cycle;
the surviving parasites then grew at the same efficiency as that of the
control culture. When treated with 10 mM ManN for 24 h, the growth was arrested at the mid-trophozoite stage, and
subsequently all parasites died.
Treatment with 10 mM ManN starting at the early trophozoite
stage (28 h after invasion or earlier) caused death of the parasites in
the first cell cycle. However, when ManN treatment was started 36 h after invasion (late trophozoite stage) or later, the growth of the
parasites was not affected. The parasites differentiated normally to
schizonts, which formed merozoites that efficiently invaded the
erythrocytes; these developed into late rings even in the continued
presence of 10 mM ManN. However, in the continued presence
of 10 mM ManN, parasite growth was completely stopped at
the trophozoite stage in the second cell cycle. These results suggest
that ManN affects the growth of P. falciparum only at the
trophozoite stage, and this is because of the inhibition of GPI
biosynthesis (see below).
Analysis of GPI Biosynthesis at Different Developmental Stages of
Intraerythrocytic P. falciparum--
The level of GPI biosynthesis at
various stages of intraerythrocytic P. falciparum was
determined by measuring the incorporation of [3H]GlcN
into GPIs. Synchronous cultures of the parasites were treated with
[3H]GlcN 10 h after invasion, and then at 6 h
intervals, the parasites were harvested, and the GPIs formed were
analyzed. The parasites did not synthesize GPIs during the ring stage
(until 16 h after invasion); low levels of GPIs were synthesized
by early trophozoites (16-28 h after invasion) (Fig.
2). However, the parasites synthesized almost all of their GPIs during development from early trophozoites to
late trophozoites (28-40 h of the parasite erythrocytic life cycle).
Little or no GPI synthesis occurred during the differentiation of
trophozoites to schizonts (40-48 h of the cell cycle). These results,
taken together with mannosamine inhibition data demonstrate that GPIs
are critically required for the development of trophozoites to
schizonts.
The [3H]GlcN-labeled GPIs and their intermediates were
identified by their susceptibility to nitrous acid and HF and by
analysis of their glycan cores before and after digestion with
In P. falciparum, EtN-P-Man4-GlcN-PI is the
major GPI, and it is exclusively found in GPI-anchored parasite
proteins (8, 14). Among various GPI intermediates formed during
biosynthesis, the formation of Man4-GlcN-PI that lacks the
ethanolamine phosphate substituent (see also Ref. 13), in addition to
EtN-P-Man3-GlcN-PI, the conserved GPI structure in all
eukaryotes, is noteworthy. The presence of both these GPI species in
significant amounts suggests that the formation of the matured GPI,
EtN-P-Man4-GlcN-PI, occurs through two different pathways:
(i) by transfer of a mannose residue to EtN-P-Man3-GlcN-PI,
and (ii) by the addition of mannose to Man3-GlcN-PI and
then substitution with EtN-P. As far as we know, the latter reaction
sequence is novel. The operation of this pathway is evident by nearly
complete conversion of the [3H]GlcN-labeled intermediate
to the matured GPI when [3H]GlcN-containing medium was
replaced with regular medium at the midtrophozoite stage and the
parasites were grown to
schizonts.3
To determine the time required for the incorporation of exogenous GlcN
into the matured GPI molecules, the parasites were metabolically
labeled at their trophozoite stage with [3H]GlcN, and the
radiolabeled GPIs formed were analyzed at various time intervals. A
significant amount of radioactivity was incorporated into matured GPIs
within 30 min after the addition of [3H]GlcN to the
culture medium, with maximum incorporation reached at 4 h (Fig.
3). Therefore, the observed absence of
GPI synthesis during ring stage development is not because of any delay
in the uptake or utilization of [3H]GlcN by the
parasite.
Effect of ManN on P. falciparum GPI Biosynthesis--
Because, in
intraerythrocytic P. falciparum, the GPIs are synthesized
only at the trophozoite stage, the parasites were treated with varying
concentrations of ManN at the early trophozoite stage (28 h after
invasion), and 2 h later, they were labeled with
[3H]GlcN for 6 h in the continued presence of ManN.
ManN inhibited GPI synthesis in a dose-dependent manner
(Fig. 4). The parasites treated with
1.25, 2.5, 5, and 10 mM ManN synthesized ~30, 18, 12, and
6% of GPIs, respectively, compared with the amount of GPIs synthesized
by the untreated parasites. This dose-dependent inhibition
of parasite GPI biosynthesis by ManN parallels the concentration-dependent inhibition of parasite growth by
ManN. Whereas significant proportions of both the GlcN-PI and
inositol-acylated GlcN-PI intermediates were observed in untreated
parasites, inositol-acylated GlcN-PI was the major intermediate in
ManN-treated parasites (Fig. 4). However, whereas untreated parasites
contain a small amount of Man1-GlcN-PI, this GPI
intermediate is below the detectable level in the ManN-treated
parasites (compare Fig. 2C with Fig. 4). Furthermore, both
untreated and ManN-treated parasites contain small but similar
proportions of Man2-GlcN-PI. Together these results suggest
that although inositol-acylated GlcN-PI formed in a significant amount,
this intermediate is not utilized rapidly for mannosylation. Thus, ManN
inhibits the addition of the first mannose residue to the
inositol-acylated GlcN-PI intermediate. This is in contrast to the
mechanism of inhibition of GPI biosynthesis by ManN in other organisms,
where either the accumulation of Man2-GlcN-PI or the
formation of ManN-Man-GlcN-PI was observed (5, 17-21).
The matured GPIs synthesized by parasites treated with ManN appear to
be utilized efficiently for the anchoring of proteins. SDS-PAGE
fluorography of the parasite lysates showed significant amounts of
GPI-modified proteins in parasites treated with 1.25 or 2.5 mM ManN (Fig. 5); this agrees
with the growth of the treated parasites through several cycles. The
parasites treated with 5 mM ManN contained low levels of
GPI-anchored proteins, and those treated with 10 mM ManN
almost completely lacked GPI-anchored proteins. These results suggest
the essential role of GPI-anchored proteins in parasite survival.
Effect of ManN on P. falciparum Protein Synthesis--
To examine
whether ManN treatment also affected protein synthesis, the parasites
were labeled with [35S]Met, and the cell lysates were
analyzed by SDS-PAGE fluorography. The results show that the parasites
treated with 1.25-10 mM ManN for 5-6 h all synthesized
similar levels of proteins compared with the untreated parasites (not
shown). Protein synthesis, however, was significantly reduced when
parasites were treated with 10 mM ManN during the entire
trophozoite stage because of slow growth or growth arrest (not shown).
Four important findings emerge from this study: 1)
intraerythrocytic P. falciparum synthesizes GPI anchors in a
developmental stage-specific manner exclusively at the trophozoite
stage; 2) GPI biosynthesis is critical to the differentiation of
trophozoites into schizonts, and thus the inhibition of GPI
biosynthesis causes parasite growth arrest at the trophozoite stage; 3)
in P. falciparum, ManN inhibits GPI biosynthesis by
interfering with the attachment of first Man to the inositol-acylated
GlcN-PI intermediate, a mechanism that is distinctively different from
those in other organisms, including man; and 4) The matured GPI,
EtN-P-Man4-GlcN-PI, is formed by two different reaction
sequences: transfer of a mannose residue to
EtN-P-Man3-GlcN-PI and to Man3-GlcN-PI;
Man4-GlcN-PI is then substituted with ethanolamine phosphate.
This study shows that ManN inhibits the growth of P. falciparum specifically at the trophozoite stage by inhibiting GPI
biosynthesis. 1) The parasite synthesizes almost all of its GPI pool at
the trophozoite stage. 2) ManN treatment does not affect the growth of
the parasite at either the ring or the schizont stage. Rings treated
with even 10 mM ManN developed into early trophozoites, and
late trophozoites became schizonts and functional merozoites even in
the continued presence of 10 mM ManN. 3) When ManN
treatment was withdrawn at the late ring stage, the parasites developed normally to schizonts despite having been treated during most of the
ring stage. However, continued treatment with 5 or 10 mM ManN either significantly reduced the growth rate or caused complete growth arrest and death of parasites at the midtrophozoite stage. 4)
ManN inhibits GPI biosynthesis and the GPI-anchor modification of
proteins in a dose-dependent manner. 5) ManN does not
significantly affect protein synthesis in the parasite. In several
eukaryotic systems, ManN has been shown to be a specific inhibitor of
GPI and N-linked oligosaccharide biosynthesis (14). Because
intraerythrocytic P. falciparum has little or no
N-glycosylation capacity and the parasite treated with
tunicamycin (an inhibitor of N-glycosylation) during the
trophozoite stage, synthesized normal levels of GPIs, and developed
into schizonts,2 the effect of ManN is not because of the
inhibition of N-glycosylation of parasite proteins. These
observations conclusively establish that the effect of ManN on the
growth of trophozoite stage P. falciparum is specifically
because of the inhibition of GPI biosynthesis.
The data presented here demonstrate that the biosynthesis of GPIs in
P. falciparum occurs exclusively at the trophozoite stage of
the parasite. Metabolic labeling of the parasites with
[3H]GlcN at various stages of intraerythrocytic
development and analysis of the labeled GPIs indicated that the GPIs
are not synthesized during the ring stage. As shown in Fig. 2, all of
the GPIs were synthesized during the trophozoite stage (between 28 and
38 h after erythrocytic invasion), and little or no GPIs were
formed during the schizont stage. The absence of GPI biosynthesis
during the ring stage, however, is not because of a delay in the
passage of [3H]GlcN through erythrocyte membranes and its
entry into the parasites, because trophozoites synthesize significant
amounts of GPIs within 30 min after the addition of
[3H]GlcN to the culture medium.
Although GPI structures of all eukaryotes contain
EtN-P-Man3-GlcN-PI, a conserved core structure, the sugar
backbone is variously modified with additional sugars and
oligosaccharide moieties, which are usually attached to the third
and/or first mannose residues (12, 15). Among known substituents, the
presence of a fourth mannose residue appears to be the common feature
of many eukaryotic GPIs (15). The biosynthetic sequence by which the
fourth mannose is added is not clear. It is believed that the formation
of EtN-P-Man4-GlcN-PI occurs through the transfer of a
mannose residue to EtN-P-Man3-GlcN-PI. However, the results
of this study suggest that, in P. falciparum, EtN-P-Man4-GlcN-PI is formed via two distinct reaction
sequences: 1) by transfer of a mannose residue to
EtN-P-Man3-GlcN-PI, and 2) by the addition of mannose to
Man3-GlcN-PI followed by the transfer of a ethanolamine
phosphate moiety. If the latter sequence is unique to P. falciparum GPIs, then understanding of the relative regulation of
the two reaction sequences may provide a parasite-specific target for
the development of antimalarial agents.
The inhibition of parasite growth at the trophozoite stage is not
because of nonspecific ManN toxicity. Parasites treated with 10 mM ManN after the midtrophozoite stage develop normally to
schizonts, and the formed merozoites successfully invaded erythrocytes. Withdrawal of ManN from the parasite cultures treated up to late ring
stage also allowed normal parasite development and protein synthesis.
The data establish that the deleterious effect of ManN on the
trophozoite stage of P. falciparum is specifically because of the inhibition of GPI biosynthesis and agrees with the synthesis of
GPIs occurring exclusively during trophozoite development.
The effect of ManN specifically at the trophozoite stage of P. falciparum, i.e. the failure of the parasite to develop
into schizonts with concomitant inhibition of GPI biosynthesis,
strongly suggests that the GPIs are essential for the differentiation
of trophozoites into schizonts. Complex carbohydrates in the forms of
glycoproteins and/or glycolipids are present in almost all eukaryotic
organisms where they have critical roles in development and
differentiation. In animals, GPIs have been shown to be vital for
normal development (11), although they are present at markedly lower
levels compared with those in parasites.
Analysis of the GPI intermediates formed in P. falciparum
treated with ManN suggested that ManN affects the parasite biosynthetic pathway by a mechanism distinctly different from those in other organisms. In several eukaryotic systems including trypanosomes, Leishmania, Madin-Darby canine kidney cells, and HeLa cells,
ManN has also been shown to inhibit GPI anchor biosynthesis without affecting protein synthesis. In HeLa cells, ManN was shown to inhibit
the In conclusion, the data presented here show that intraerythrocytic
P. falciparum synthesizes GPIs in a developmental
stage-specific manner, exclusively at the trophozoite stage. The
inhibition of P. falciparum GPI biosynthesis by mannosamine
prevented the differentiation of trophozoites to schizonts. Mannosamine
inhibits the GPI biosynthesis by a novel mechanism, which suggests that
the enzymes of the parasite GPI biosynthetic pathway can be exploited
as parasite-specific targets for the development of antimalarial drugs.
We thank Manonmani Venkatesan for
assistance in cell culturing and Priyadarshan Gupta for preliminary studies.
*
The study was supported by Grant AI41139 from the NIAID,
National Institutes of Health.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.
Published, JBC Papers in Press, May 31, 2000, DOI 10.1074/jbc.M002151200
2
R. S. Naik, O. H. Branch, A. S. Woods, V. Matam, D. J. Perkins, B. L. Nahlen, A. A. Lal,
R. J. Cotter, C. F. Ockenhouse, E. A. Davidson, and
D. C. Gowda, submitted for publication.
3
R. S. Naik and D. C. Gowda,
unpublished data.
The abbreviations used are:
GPI, glycosylphosphatidylinositol;
ManN, D-mannosamine;
GlcN, D-glucosamine;
PI, phosphatidylinositol;
HPTLC, high performance thin-layer
chromatography;
Gn, GlcN;
Man, mannose;
Met, L-methionine;
EtN, ethanolamine;
HF, hydrofluoric acid;
PAGE, polyacrylamide gel
electrophoresis.
Developmental Stage-specific Biosynthesis of
Glycosylphosphatidylinositol Anchors in Intraerythrocytic
Plasmodium falciparum and Its Inhibition in a Novel Manner
by Mannosamine*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-6Man1-4GlcN-PI intermediate or the formation of ManN-Man-GlcN-PI, an aberrant GPI intermediate, which cannot be a substrate for further addition of mannose. Thus, the parasite GPI biosynthetic pathway could be a specific target for antimalarial drug development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-2Man1-6Man1-4GlcN, that is
-(1-6)-linked to the inositol residue of PI (8, 13,
14).2 The glycan core has an
additional Man residue linked to the third Man at O-2. The
parasite GPIs differ significantly from those of the human host,
particularly with respect to the absence of additional ethanolamine
substituents on the glycan core, the presence of a fourth Man residue,
the absence of an alkyl substituent at the sn-1 position,
and the type of acyl substituents on the glycerol and inositol moieties
(15).2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase (400 milliunits/mg) and jack bean -mannosidase (30 units/mg) were from
Oxford Glycosystems (Rosedale, NY). Gentamycin sulfate was from
Biofluids, Inc. (Rockville, MD). [6-3H]GlcN (23 Ci/mmol),
[35S]Met (1000 Ci/mmol), and 14C-labeled
protein molecular weight markers were from Amersham Pharmacia Biotech.
Silica gel 60 HPTLC plates were from either EM Science (Gibbstown, NJ)
or Whatman, Inc. (Clifton, NJ). En3HanceTM
fluorographic spray for TLC plates was from NEN Life Science Products. O-type human blood was obtained from the Georgetown University Hospital. O-type human serum was from Interstate Blood Bank,
Inc. (Memphis, TN).
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 room temperature for
2 h and then at 37 °C for 22 h (27). The solutions were heated in a boiling water bath for 5 min, extracted with
water-saturated 1-butanol, and analyzed by HPTLC. For digestion of the
GPI glycan cores (10,000 cpm) with jack bean -mannosidase, the above
buffer without the detergent was used; products were deionized with AG 50W-X16 (H+) and lyophilized. The glycan cores (5000 cpm)
were also digested with A. saitoi -mannosidase (0. 5 milliunits/ml) in 100 mM NaOAc, pH 5.0, at 37 °C for
20 h (8), deionized, and lyophilized.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of ManN on the growth and development
of P. falciparum. Parasites, 8 h after erythrocyte
invasion, were separately treated with 0.25-10 mM ManN in
complete medium; untreated parasites were cultured in parallel as
controls. Parasitemia was measured by counting the cells in
Giemsa-stained thin smears using light microscopy. Shown is the percent
decrease in parasitemia compared with untreated culture 50 h after
ManN treatment (second cell cycle).
The effect of ManN on parasitemia and development of intraerythrocytic
P. falciparum
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Fig. 2.
Analysis of [3H]GlcN-labeled
GPIs synthesized at different stages of P. falciparum
development. Parasite culture with 15% parasitemia was
incubated at 37 °C with [3H]GlcN (50 µCi/ml) in
complete medium 10 h after erythrocyte invasion. At 6-h intervals
the cultures were harvested, and the GPIs were isolated and analyzed.
A, [3H]GlcN-labeled free GPIs and their
intermediates synthesized by 7 × 106 parasites.
B, HPTLC analysis of [3H]GlcN-labeled free
GPIs from 5 × 105 (lanes 1 and
2) and 2 × 105 (lanes
4-6) parasites. Lanes 1-6, GPIs from
cultures harvested at 16, 22, 28, 34, 40, and 46 h, respectively,
after erythrocyte invasion. Note: GPIs in lanes 1 and
2 were from 2.5 times more parasites compared with those in
lanes 3-6. C, HPTLC of [3H]GlcN-labeled GPIs
before (lane 1) and after (lane 2) treatment with
jack bean -mannosidase. The identity of the GPIs and their
intermediates are indicated. GnPI, GlcN-PI;
GnAcPI, GlcNAc-PI; M1GnPI,
Man1-GlcN-PI; M2GnPI,
Man2-GlcN-PI; M3GnPI,
Man3-GlcN-PI; M4GnPI,
Man4-GlcN-PI; EM3GnPI,
EtN-P-Man3-GlcN-PI; EM4GnPI,
EtN-P-Man4-GlcN-PI. *, GPI species without an acyl
substituent on the inositol residue.
-mannosidase. The results are summarized in Table
II. All the major
[3H]GlcN-labeled bands on HPTLC corresponded to GPIs and
their intermediates; glycolipids other than GPI were not present at
significant amounts.
The structures of [3H]GlcN-labeled GPIs and their
intermediates synthesized by P. falciparum
-mannosidase treatment, by
comparison of RF values (see Ref. 29), and by HPTLC
identification of the glycan cores. *, GPI species without an
acyl substituent on the inositol residue. Note: M1GnPI and
GnAcPI* were not separated by TLC; however, they were identified by
analysis of products formed by treatment with -mannosidase and
HNO2. Abbreviations are listed in the legend to Fig. 2.
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Fig. 3.
Analysis of the rate of GPI biosynthesis
during the development of P. falciparum
trophozoites. Parasite cultures with 15% parasitemia were
incubated at 37 °C with [3H]GlcN (50 µCi/ml) in
complete medium 30 h after erythrocyte invasion. At indicated time
intervals, the parasites were harvested, and the GPIs were isolated and
analyzed by HPTLC. A, [3H]GlcN-labeled free
GPIs and their intermediates synthesized by 12 × 106
parasites. B, HPTLC analysis of the free GPIs from 8 × 105 parasites. Lanes 1-8, GPIs from parasites
labeled with [3H]GlcN for 0.25, 0.5, 0.75, 1, 1.5, 2, 4, and 6 h, respectively. The identity of the GPIs and their
intermediates is indicated (abbreviated as in Fig. 2). *, GPI species
without an acyl substituent on the inositol residue.
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Fig. 4.
Analysis of GPI anchor biosynthesis in
ManN-treated P. falciparum. Parasite cultures with 15%
parasitemia were treated with 1.25, 2.5, 5, or 10 mM ManN
in complete medium 28 h after invasion; untreated parasites were
cultured in parallel as controls. After 2 h,
[3H]GlcN (50 µCi/ml) was added and cultured for 6 h, and GPIs were isolated. A, [3H]GlcN-labeled
free GPIs and their intermediates synthesized by 1.2 × 107 parasites. B, HPTLC analysis of free
[3H]GlcN-labeled GPIs from 7 × 105
parasites. Lane 1, GPIs from untreated parasites;
lanes 2-5, free GPIs from parasites treated with 1.25, 2.5, 5, and 10 mM ManN, respectively. The identity of the GPIs
and their intermediates is indicated (abbreviated as in Fig. 2). *, GPI
species without an acyl substituent on the inositol residue. Note:
Treatments with jack bean mannosidase and nitrous acid suggested the
absence of M1GnPI in ManN-treated parasites.
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Fig. 5.
SDS-PAGE analysis of GPI-anchored proteins in
ManN-treated P. falciparum. Parasite cultures
were treated with [3H]GlcN as in Fig. 4 and harvested.
Parasites from the infected erythrocytes were released with 0.015%
saponin in 56 mM NaCl, 59 mM KCl, 1 mM NaH2PO4, 14 mM
K2HPO4, 11 mM NaHCO3,
and 14 mM glucose, pH 7.4. The parasites were recovered by
centrifugation, washed, suspended in 100 µl of nonreducing
electrophoresis buffer, and heated for 5 min in a boiling water bath.
Lysates corresponding to 3 × 107 parasites were
analyzed on 6-20% gradient SDS-polyacrylamide gels, and the protein
bands on gels were viewed by fluorography. Lane 1, untreated
parasites; lanes 2-5, parasites treated with 1.25, 2.5, 5, and 10 mM ManN, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-(1-2)-mannosyltransferase responsible for the transfer of the
third Man residue, causing the accumulation of the
Man1-6Man1-4GlcN-PI intermediate (17-19). In Madin-Darby
canine kidney cells, Trypanosoma brucei, and
Leishmania mexicana, the inhibition of mature GPI formation
was shown to be because of the incorporation of ManN into Man-GlcN-PI.
This results in the formation of an aberrant GPI intermediate,
ManN-Man-GlcN-PI, which prevents further addition of Man residues
because of the lack of a hydroxyl group on C2 (5, 20, 21). In P. falciparum, however, ManN inhibits GPI biosynthesis by inhibiting
the addition of the first mannose to the inositol-acylated GlcN-PI,
suggesting significant differences in the specificity of the parasite
and mammalian enzymes.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd., NW, Washington, D. C. 20007. Tel.: 202-687-3840; Fax: 202-687-7186; E-mail: gowda@bc.georgetown.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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