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• W2063399247 abstract "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. 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 D-mannosamine D-glucosamine phosphatidylinositol high performance thin-layer chromatography GlcN mannose L-methionine ethanolamine hydrofluoric acid polyacrylamide gel electrophoresis Glycosylphosphatidylinositol (GPI)1 anchors represent a distinct class of glycolipids found covalently attached to proteins in almost all eukaryotic cells (1McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (805) Google Scholar, 2Englund P.T. Annu. Rev. Biochem. 1993; 62: 121-138Crossref PubMed Google Scholar); they are particularly abundant in parasites, in which they occur as free lipids or linked to proteins (3Ferguson M.A.J. Masterson W.J. Homans S.W. McConville M.J. Cell Biol. Int. Rep. 1991; 15: 991-1005Crossref PubMed Scopus (8) Google Scholar,4Ferguson M.A.J. Parasitol. Today. 1994; 10: 48-52Abstract Full Text PDF PubMed Scopus (62) Google Scholar). 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 (1McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (805) Google Scholar,5Ralton J.E. Milne K.G. Günther M.L.S. Field R.A. Ferguson M.A.J. J. Biol. Chem. 1993; 268: 24183-24189Abstract Full Text PDF PubMed Google Scholar, 6Field M.C. Glycobiology. 1997; 7: 161-168Crossref PubMed Scopus (42) Google Scholar). 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 (7Triggs P.I. Kondrachine A.V. Sherman I.W. Malaria: Parasite Biology, Pathogenesis, and Protection. ASM Press, Washington, D. C.1998: 11-22Google Scholar). 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 (7Triggs P.I. Kondrachine A.V. Sherman I.W. Malaria: Parasite Biology, Pathogenesis, and Protection. ASM Press, Washington, D. C.1998: 11-22Google Scholar). 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 (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,9Gowda D.C. Davidson E.A. Parasitol. Today. 1999; 15: 147-152Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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 (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 10Braun-Breton C. Rosenberry T.L. Pereria da Silva L.H. Res. Immunol. 1990; 141: 743-755Crossref PubMed Scopus (34) Google Scholar, 11Marshall V.M. Silva A. Foley M. Cranmer S. Wang L. McColl D.J. Kemp D.J. Coppel R.L Infect. Immun. 1997; 65: 4460-4467Crossref PubMed Google Scholar). These observations suggest that GPI biosynthesis is vital to the parasite. The P. falciparum GPIs contain a conserved core structure, ethanolamine-phosphate-6Manα1–2Manα1–6Manα1–4GlcN, that is α-(1–6)-linked to the inositol residue of PI (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 13Gerold P. Dieckmann-Schuppert A. Schwarz R.T. J. Biol. Chem. 1994; 269: 2597-2606Abstract Full Text PDF PubMed Google Scholar,14Gerold P. Schofield L. Blackman M.J. Holder A.A. Schwarz R.T. Mol. Biochem. Parasitol. 1996; 75: 131-143Crossref PubMed Scopus (180) Google Scholar). 2R. 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. 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 (15Ferguson M.A.J. Brimacombe J.S. Brown J.R. Crossman A. Dix A. Field R.A. Güther M.L. Milne K.G. Sharma D.K. Smith T.K. Biochim. Biophys. Acta. 1999; 1455: 327-340Crossref PubMed Scopus (125) Google Scholar).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 cells (16Lisanti M. Field M.C. Caras I. Menon A.K. Rodriguez-Boulan E. EMBO J. 1991; 10: 1969-1977Crossref PubMed Scopus (65) Google Scholar, 17Pan Y.-T. De Gespari R. Warren C.D. Elbein A.D. J. Biol. Chem. 1992; 267: 8991-8999Abstract Full Text PDF PubMed Google Scholar, 18Pan Y.-T. Elbein A.D. Arch. Biochem. Biophys. 1985; 242: 447-456Crossref PubMed Scopus (17) Google Scholar, 19Sevlever D. Rosenberry T.L. J. Biol. Chem. 1993; 268: 10938-10945Abstract Full Text PDF PubMed Google Scholar, 20Pan Y.-T. Kamitani T. Bhuvaneswaran C. Hallaq Y. Warren C.D. Yeh E.T.H. Elbein A.D. J. Biol. Chem. 1992; 267: 21250-21255Abstract Full Text PDF PubMed Google Scholar, 21Field M.C. Medina-Acosta E. Cross G.A.M. J. Biol. Chem. 1993; 268: 9570-9577Abstract Full Text PDF PubMed Google Scholar). In eukaryotes, ManN can also inhibit the assembly of N-linked oligosaccharides (17Pan Y.-T. De Gespari R. Warren C.D. Elbein A.D. J. Biol. Chem. 1992; 267: 8991-8999Abstract Full Text PDF PubMed Google Scholar), and it is a precursor for the synthesis of sialic acid (22Schauer R. Adv. Carbohydr. Chem. Biochem. 1982; 40: 131-234Crossref PubMed Scopus (939) Google Scholar). BecauseP. falciparum contains little or noN-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. 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 α-mannosidase (400 milliunits/mg) and jack bean α-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). En3HanceTMfluorographic 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). 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 (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Cultures were maintained at 37 °C in an atmosphere of 90% N2, 5% O2, and 5% CO2. The parasites were synchronized 4–6 h after invasion using 5% sorbitol (23Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2854) Google Scholar). 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. 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 mmNaHCO3, 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 (24Udeinya I.J. Van Dyke K. Bull. W. H. O. 1980; 58: 445-448PubMed Google Scholar), and the parasites were washed with the buffer and stored at −80 °C. The parasites were lyophilized and extracted four times with 5 volumes of chloroform/methanol/water (10:10:3, v/v/v) (25Ferguson M.A.J. Hooper N.M. Turner A.J. Lipid Modification of Proteins: A Practical Approach. IRL Press, New York1992: 191-231Google Scholar, 26Menon A.K. Methods Enzymol. 1994; 230: 418-442Crossref PubMed Scopus (37) Google Scholar). 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 −20 °C. 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 mNaNO2 (25Ferguson M.A.J. Hooper N.M. Turner A.J. Lipid Modification of Proteins: A Practical Approach. IRL Press, New York1992: 191-231Google Scholar, 26Menon A.K. Methods Enzymol. 1994; 230: 418-442Crossref PubMed Scopus (37) Google Scholar). 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 [3H]GlcN-labeled GPIs (30,000–50,000 cpm) were treated with 50% aqueous HF (50 μl) in an ice bath for 48 h (25Ferguson M.A.J. Hooper N.M. Turner A.J. Lipid Modification of Proteins: A Practical Approach. IRL Press, New York1992: 191-231Google Scholar, 26Menon A.K. Methods Enzymol. 1994; 230: 418-442Crossref PubMed Scopus (37) Google Scholar). The reaction mixture was neutralized with frozen saturated LiOH, extracted with water-saturated 1-butanol, dried, and analyzed by HPTLC. The [3H]GlcN-labeled GPIs or GPI intermediates (50,000–100,000 cpm) were treated with HF and then with HNO2 (25Ferguson M.A.J. Hooper N.M. Turner A.J. Lipid Modification of Proteins: A Practical Approach. IRL Press, New York1992: 191-231Google Scholar, 26Menon A.K. Methods Enzymol. 1994; 230: 418-442Crossref PubMed Scopus (37) Google Scholar). The samples were reduced with 100 μl of 1m 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. The [3H]GlcN-labeled GPIs (25,000–50,000 cpm) were treated with jack bean α-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 (27Milne K.G. Ferguson M.A.J. Englund P.T. J. Biol. Chem. 1999; 274: 1465-1471Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). 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 α-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 α-mannosidase (0. 5 milliunits/ml) in 100 mm NaOAc, pH 5.0, at 37 °C for 20 h (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), deionized, and lyophilized. 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 (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). 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 (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). 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 (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207522) Google Scholar). 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 −80 °C. We investigated the developmental stage-specific biosynthesis of GPIs in intraerythrocyticP. 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 mmManN 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. 1and 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).Table IThe effect of ManN on parasitemia and development of intraerythrocytic P. falciparumCulture treatmentCulture time1-aCulturing time after the addition of ManN (8 h after the erythrocyte invasion) to the cultures. and parasite development stage32 h56 h80 h104 hUntreatedLate trophozoitesRingsLate trophozoitesRings(3%)(12%)(12%)(4 × 12%)1.25 mm ManNLate trophozoitesRingsLate trophozoitesRings(3%)(12%)(11%)(4 × 11%)2.5 mm ManNLate trophozoitesRingsLate trophozoitesEarly Rings(3%)(12%)(9%)(4 × 9%)5 mm ManNLate trophozoitesRingsMidtrophozoitesUnhealthy trophozoites(2%)(5%)(5%)(5%)Early or midtrophozoites (1%)Schizonts (1%)10 mm ManNUnhealthy midtrophozoitesDead parasites(3%)Synchronous cultures of the parasites were treated with ManN, and at the indicated time the levels of parasitemia and development stage of parasites were assessed by examining Giemsa-stained thin smear of cultures under light microscope.1-a Culturing time after the addition of ManN (8 h after the erythrocyte invasion) to the cultures. Open table in a new tab Synchronous cultures of the parasites were treated with ManN, and at the indicated time the levels of parasitemia and development stage of parasites were assessed by examining Giemsa-stained thin smear of cultures under light microscope. 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). 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 α-mannosidase. The results are summarized in TableII. 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.Table IIThe structures of [3H]GlcN-labeled GPIs and their intermediates synthesized by P. falciparumGPI speciesStructureR F valuesGlycan core identifiedEM4GnPIEtN-P-Man4-GlcN-PI0.19Man4GlcNEM3GnPIEtN-P-Man3-GlcN-PI0.29Man3GlcNM4GnPIMan4-GlcN-PI0.61Man4GlcNM3GnPIMan3-GlcN-PI0.74Man3GlcNM2GnPIMan2-GlcN-PI0.84Man2GlcNGnPI*GlcN-PI*0.87GlcNGnAcPI*GlcNAc-PI*0.88GlcNAcM1GnPIMan1-GlcN-PI0.88Man1GlcNGnPIGlcN-PI0.90GlcNThe parasites were labeled with [3H]GlcN and GPIs and their intermediates synthesized by the parasites (see Figs. Figure 2, Figure 3, Figure 4) purified by preparative HPTLC. The structures of the GPI species were determined by analyzing their sensitivity to HNO2, HF,N-acetylation (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and α-mannosidase treatment, by comparison of R F values (see Ref. 29Gerold P. Jung N. Azzouz N. Freiberg N. Kobe S. Schwarz R. Biochem. J. 1999; 344: 731-738Crossref PubMed Scopus (32) Google Scholar), 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 α-mannosidase and HNO2. Abbreviations are listed in the legend to Fig. 2. Open table in a new tab The parasites were labeled with [3H]GlcN and GPIs and their intermediates synthesized by the parasites (see Figs. Figure 2, Figure 3, Figure 4) purified by preparative HPTLC. The structures of the GPI species were determined by analyzing their sensitivity to HNO2, HF,N-acetylation (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and α-mannosidase treatment, by comparison of R F values (see Ref. 29Gerold P. Jung N. Azzouz N. Freiberg N. Kobe S. Schwarz R. Biochem. J. 1999; 344: 731-738Crossref PubMed Scopus (32) Google Scholar), 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 α-mannosidase and HNO2. Abbreviations are listed in the legend to Fig. 2. In P. falciparum, EtN-P-Man4-GlcN-PI is the major GPI, and it is exclusively found in GPI-anchored parasite proteins (8Gowda D.C. Gupta P. Davidson E.A. J. Biol. Chem. 1997; 272: 6428-6439Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 14Gerold P. Schofield L. Blackman M.J. Holder A.A. Schwarz R.T. Mol. Biochem. Parasitol. 1996; 75: 131-143Crossref PubMed Scopus (180) Google Scholar). Among various GPI intermediates formed during biosynthesis, the formation of Man4-GlcN-PI that lacks the ethanolamine phosphate substituent (see also Ref. 13Gerold P. Dieckmann-Schuppert A. Schwarz R.T. J. Biol. Chem. 1994; 269: 2597-2606Abstract Full Text PDF PubMed Google Scholar), 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. 3R. S. Naik and D. C. Gowda, unpublished data. 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. 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. 2 C 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 (5Ralton J.E. Milne K.G. Günther M.L.S. Field R.A. Ferguson M.A.J. J. Biol. Chem. 1993; 268: 24183-24189Abstract Full Text PDF PubMed Google Scholar, 17Pan Y.-T. De Gespari R. Warren C.D. Elbein A.D. J. Biol. Chem. 1992; 267: 8991-8999Abstract Full Text PDF PubMed Google Scholar, 18Pan Y.-T. Elbein A.D. Arch. Biochem. Biophys. 1985; 242: 447-456Crossref PubMed Scopus (17) Google Scholar, 19Sevlever D. Rosenberry T.L. J. Biol. Chem. 1993; 268: 10938-10945Abstract Full Text PDF PubMed Google Scholar, 20Pan Y.-T. Kamitani T. Bhuvaneswaran C. Hallaq Y. Warren C.D. Yeh E.T.H. Elbein A.D. J. Biol. Chem. 1992; 267: 21250-21255Abstract Full Text PDF PubMed Google Scholar, 21Field M.C. Medina-Acosta E. Cross G.A.M. J. Biol. Chem. 1993; 268: 9570-9577Abstract Full Text PDF PubMed Google Scholar). 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. 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 mmManN 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 (14Gerold P. Schofield L. Blackman M.J. Holder A.A. Schwarz R.T. Mol. Biochem. Parasitol. 1996; 75: 131-143Crossref PubMed Scopus (180) Google Scholar). Because intraerythrocytic P. falciparum has little or noN-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 inP. 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 (12Kinoshita T. Ohishi K. Takeda J. J. Biochem. (Tokyo). 1997; 122: 251-257Crossref PubMed Scopus (123) Google Scholar, 15Ferguson M.A.J. Brimacombe J.S. Brown J.R. Crossman A. Dix A. Field R.A. Güther M.L. Milne K.G. Sharma D.K. Smith T.K. Biochim. Biophys. Acta. 1999; 1455: 327-340Crossref PubMed Scopus (125) Google Scholar). Among known substituents, the presence of a fourth mannose residue appears to be the common feature of many eukaryotic GPIs (15Ferguson M.A.J. Brimacombe J.S. Brown J.R. Crossman A. Dix A. Field R.A. Güther M.L. Milne K.G. Sharma D.K. Smith T.K. Biochim. Biophys. Acta. 1999; 1455: 327-340Crossref PubMed Scopus (125) Google Scholar). 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 (11Marshall V.M. Silva A. Foley M. Cranmer S. Wang L. McColl D.J. Kemp D.J. Coppel R.L Infect. Immun. 1997; 65: 4460-4467Crossref PubMed Google Scholar), although they are present at markedly lower levels compared with those in parasites. Analysis of the GPI intermediates formed in P. falciparumtreated 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 α-(1–2)-mannosyltransferase responsible for the transfer of the third Man residue, causing the accumulation of the Manα1–6Manα1–4GlcN-PI intermediate (17Pan Y.-T. De Gespari R. Warren C.D. Elbein A.D. J. Biol. Chem. 1992; 267: 8991-8999Abstract Full Text PDF PubMed Google Scholar, 18Pan Y.-T. Elbein A.D. Arch. Biochem. Biophys. 1985; 242: 447-456Crossref PubMed Scopus (17) Google Scholar, 19Sevlever D. Rosenberry T.L. J. Biol. Chem. 1993; 268: 10938-10945Abstract Full Text PDF PubMed Google Scholar). In Madin-Darby canine kidney cells, Trypanosoma brucei, andLeishmania 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 (5Ralton J.E. Milne K.G. Günther M.L.S. Field R.A. Ferguson M.A.J. J. Biol. Chem. 1993; 268: 24183-24189Abstract Full Text PDF PubMed Google Scholar, 20Pan Y.-T. Kamitani T. Bhuvaneswaran C. Hallaq Y. Warren C.D. Yeh E.T.H. Elbein A.D. J. Biol. Chem. 1992; 267: 21250-21255Abstract Full Text PDF PubMed Google Scholar, 21Field M.C. Medina-Acosta E. Cross G.A.M. J. Biol. Chem. 1993; 268: 9570-9577Abstract Full Text PDF PubMed Google Scholar). 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 intraerythrocyticP. 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." @default.
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