Developmental Stage-specific Biosynthesis of Glycosylphosphatidylinositol Anchors in IntraerythrocyticPlasmodium falciparum and Its Inhibition in a Novel Manner by Mannosamine*

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 Manα1–6Manα1–4GlcN-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.

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 abun-dant 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␣1-2Man␣1-6Man␣1-4GlcN, 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 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 * 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ 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. 1 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. 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. Culturing of the Parasite-P. falciparum (FCR-3 strain) were cultured in RPMI 1640 medium supplemented with 22 mM HEPES, 29 mM NaHCO 3 , 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% N 2 , 5% O 2 , and 5% CO 2 .

Materials
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 [ 3 H]GlcN (50 Ci/ml) for 6 h in a medium containing 5 mM glucose. Labeling with [ 35 S]Met (50 Ci/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 NaH 2 PO 4 , 10 mM K 2 HPO 4 , 11 mM NaHCO 3 , 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 Ϫ80°C.
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 Ϫ20°C.
Characterization of GPI Glycan Core-The [ 3 H]GlcN-labeled GPIs or GPI intermediates (50,000 -100,000 cpm) were treated with HF and then with HNO 2 (25,26). The samples were reduced with 100 l of 1 M NaBH 4 in 100 mM NaOH at room temperature for 6 h. Excess NaBH 4 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 ϩ ) and AG 4-X4 (base) resins, dried, and analyzed by HPTLC.

SDS-PAGE and Fluorography-The [ 3 H]GlcN-or [ 35 S]
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 Ϫ80°C.

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 nor- mally 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 (ϳ90 and ϳ75%, respectively) compared with that of the control culture. The survival of parasites treated with Ͻ5 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 [ 3 H]GlcN into GPIs. Synchronous cultures of the parasites were treated with [ 3 H]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 [ 3 H]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 ␣-mannosidase. The results are summarized in Table II. All the major [ 3 H]GlcN-labeled bands on HPTLC corresponded to GPIs and their intermediates; glycolipids other than GPI were not present at significant amounts.
In P. falciparum, EtN-P-Man 4 -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 Man 4 -GlcN-PI that lacks the ethanolamine phosphate substituent (see also Ref. 13), in addition to EtN-P-Man 3 -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-Man 4 -GlcN-PI, occurs through two different pathways: (i) by transfer of a mannose residue to EtN-P-Man 3 -GlcN-PI, and (ii) by the addition of mannose to Man 3 -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 [ 3 H]GlcN-labeled intermediate to the matured GPI when [ 3 H]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 ex-3 R. S. Naik and D. C. Gowda, unpublished data.  (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 [ 3 H]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 [ 3 H]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 ϳ30, 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, inositolacylated GlcN-PI was the major intermediate in ManN-treated parasites (Fig. 4). However, whereas untreated parasites contain a small amount of Man 1 -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 Man 2 -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   (8), and ␣-mannosidase treatment, by comparison of R F values (see Ref. 29), and by HPTLC identification of the glycan cores. *, GPI species without an acyl substituent on the inositol residue. Note: M 1 GnPI and GnAcPI* were not separated by TLC; however, they were identified by analysis of products formed by treatment with ␣-mannosidase and HNO 2 . Abbreviations are listed in the legend to Fig. 2  other organisms, where either the accumulation of Man 2 -GlcN-PI or the formation of ManN-Man-GlcN-PI was observed (5,(17)(18)(19)(20)(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 [ 35 S]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).

DISCUSSION
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 inositolacylated GlcN-PI intermediate, a mechanism that is distinctively different from those in other organisms, including man; and 4) The matured GPI, EtN-P-Man 4 -GlcN-PI, is formed by two different reaction sequences: transfer of a mannose residue to EtN-P-Man 3 -GlcN-PI and to Man 3 -GlcN-PI; Man 4 -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.  Fig. 4 and harvested. Parasites from the infected erythrocytes were released with 0.015% saponin in 56 mM NaCl, 59 mM KCl, 1 mM NaH 2 PO 4 , 14 mM K 2 HPO 4 , 11 mM NaHCO 3 , and 14 mM glucose, pH 7.4. The parasites were recovered by centrifugation, washed, suspended in 100 l of nonreducing electrophoresis buffer, and heated for 5 min in a boiling water bath. Lysates corresponding to 3 ϫ 10 7 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.
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 [ 3 H]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 [ 3 H]GlcN through erythrocyte membranes and its entry into the parasites, because trophozoites synthesize significant amounts of GPIs within 30 min after the addition of [ 3 H]GlcN to the culture medium.
Although GPI structures of all eukaryotes contain EtN-P-Man 3 -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-Man 4 -GlcN-PI occurs through the transfer of a mannose residue to EtN-P-Man 3 -GlcN-PI. However, the results of this study suggest that, in P. falciparum, EtN-P-Man 4 -GlcN-PI is formed via two distinct reaction sequences: 1) by transfer of a mannose residue to EtN-P-Man 3 -GlcN-PI, and 2) by the addition of mannose to Man 3 -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 ␣-(1-2)-mannosyltransferase responsible for the transfer of the third Man residue, causing the accumulation of the Man␣1-6Man␣1-4GlcN-PI intermediate (17)(18)(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.
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.