PAGES: 949-956 DOI: 10.1590/S0074-02762010000800001 Review
Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation - A Review

Cristiana S de MacedoI; Ralph T SchwarzII; Adriane R TodeschiniI; Jose O PreviatoI; Lucia Mendonça-PreviatoI, +

IInstituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
IIInstitut fur Virologie, Universitat Marburg, Marburg, Germany and Unité de Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille, Villeneuve-d'Ascq, France


Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.

Malaria is a major human parasitic disease. According to the World Malaria Report (WHO 2008), 3.3 billion people live at risk for malaria infection. About 247 million episodes are reported every year, leading to 881,000 deaths, of which 85% are children under five years old.

Malaria is caused by parasites from the Plasmodium genus. There are 156 different species that infect vertebrates, four of which infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae (Warrell & Gilles 2002). Recently, Plasmodium knowlesi, which causes malaria in monkeys, was detected in many cases of human malaria in Asia using molecular techniques (Singh et al. 2004).

P. vivax, P. ovale and P. malariae cause infections that are seldom deadly, known as benign tertian malaria (P. vivax and P. ovale) and quartan malaria (P. malariae). These names refer to the periodicity of the fever attacks, which occur at the end of the intraerythrocytic cycle when infected erythrocytes break up (every 48 h in the case of P. vivax and P. ovale or every 72 h for P. malariae) and this event releases merozoites and stimulates the secretion of inflammatory cytokines by the host (Clark et al. 2006). P. falciparum causes infections that can lead to death if they are not treated appropriately. Because it has an intraerythrocytic cycle of 48 h and thus results in fever every three days, P. falciparum infection is known as malignant tertian malaria. Malaria caused by P. knowlesi infection is fatal in about one in 10 patients (Daneshvar et al. 2009).

The life cycle of Plasmodium parasites alternates between a definitive host (a mosquito from the genus Anopheles), in which sexual reproduction takes plac, and an intermediary host (a vertebrate), in which parasite reproduction is asexual. Plasmodium species that infect humans (P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi) have similar life cycles with slight variations.

The Plasmodium life cycle begins in humans after an infected Anopheles female draws blood from the human during the blood meal and the mosquito injects saliva with sporozoites into the skin (Sinnis & Zavala 2008). A few hours after infection, the sporozoites reach the liver by traversing the Kupffer cells, where they begin to invade hepatocytes (Vaughan et al. 2008, Ejigiri & Sinnis 2009). This phase is known as the first asexual reproduction phase, or exoerythrocytic schizogony. This phase of the cycle varies between species: P. falciparum and P. malariae begin asexual replication immediately while P. vivax and P. ovale experience a dormant period inside hepatocytes. This latent stage is called the hypnozoite stage (Krotoski et al. 1982) and this stage can persist for long periods of time before the parasites undergo asexual replication to cause a primary infection. Hypnozoites are responsible for malaria relapses (Krotoski 1989) and they have not been detected in P. knowlesi infections in monkeys (Krotoski & Collins 1982). The asexual hepatic schizogony is a phase of significant parasite growth and division, during which hepatic schizonts are observed. These hepatic schizonts contain millions of merozoites, which are released into the blood stream and invade erythrocytes.

These merozoites begin the intraerythrocytic schizogony phase of the cycle, during which symptoms of the malaria infection are presented. Giemsa staining has been used to detect infected erythrocytes in thick and thin blood smears of infected patients and blood smear microscopy is still the standard method for malaria diagnosis (Ndao 2009). After invasion, the merozoite undergoes differentiation into early trophozoite (0-20 h), known as the ring stage due to its morphology. Within 20-40 h, the mature trophozoite develops and exhibits an increased metabolism, during which erythrocyte cytoplasm is ingested and haemoglobin is digested. The toxic free haem moieties that are released are inactivated by the parasite through polymerization, giving rise to hemozoin, also called malarial pigment (Pisciotta & Sullivan 2008). After these events, nuclear division takes place to give rise to the schizont. Following maturation (40-48 h), schizonts rupture leading to the release merozoites into the blood stream. Newly released merozoites will either invade other erythrocytes, thereby beginning a new cycle of infection (Bannister & Mitchell 2009), or they will differentiate into sexual forms called gametocytes, which are either macrogametocytes (female) or microgametocytes (male) (Alano 2007). In the case of P. falciparum, merozoites appear 7-15 days after the onset of the intraerythrocytic cycle and they may circulate in the blood anywhere from 2 days-4 weeks (Talman et al. 2004).

The Anopheles mosquito ingests circulating gametocytes with the blood meal. Inside the mosquito, gametocytes undergo a temperature drop (from 37-32ºC) as well as pH changes (from 7.4-8.2). These factors, together with exposure to xanthurenic acid, a gametocyte-activating factor present in the mosquito (Billiker et al. 1998, Hirai et al. 2001, Kuehn & Pradel 2010), induce gametogenesis, the sexual phase of the Plasmodium life cycle. The male and female gametes are formed during this stage: the microgamete (male) is formed through a process known as exflagellation and it fertilises the macrogamete (female) to form a zygote (Alano 2007). After zygote development, the resulting ookinete will migrate through gut epithelial cells (Baton & Ranford-Cartwright 2005) where it forms an oocyst, which then undergoes multiple asexual replications to form sporozoites. The sporozoites are released by oocyst rupture into the body cavity of the mosquito and migrate to and infect the salivary glands (Matuschewski 2006). Thus, sporozoites are able to invade two different target cell types: liver cells in the vertebrate host and salivary gland cells in the mosquito vector. When the infected mosquito bites another vertebrate host, the infection is spread.

P. falciparum remains the best studied Plasmodium species due to the establishment of adequate culture methods (Trager & Jensen 1976). Although genome sequencing (Gardner et al. 2002) together with investigations of the biochemistry, molecular biology, cell biology and host-parasite interactions have been published for P. falciparum, protein glycosylation remains controversial because there is little information about the function of P. falciparum glycosylated molecules and because the few studies that are available are inconsistent due to differing methodologies. This review mainly focuses on N- and O-glycosylation in P. falciparum because glycosylphosphatidylinositol (GPI) anchors are already well established in this parasite (Gerold et al. 1994, 1996a, Hoessli et al. 1996, Davidson & Gowda 2001, Guha-Niyogi et al. 2001, Mendonça-Previato et al. 2005, von Itzstein et al. 2008, Chung et al. 2009, Shams-Eldin et al. 2009a, b, Debierre-Grockiego & Schwarz 2010).

Glycosylated proteins in P. falciparum

The first studies on glycosylation of Plasmodium proteins started with work by Trigg et al. (1977). These authors reported labelling of P. knowlesi-infected erythrocytes with galactose oxidase and tritiated sodium borohydride, which specifically label terminal galactose and galactosamine residues in glycolipids and glycoproteins (Gahmberg & Hakomori 1973). Infected erythrocytes exhibited lower levels of labelled glycoproteins than uninfected erythrocytes, possibly as a result of infection-association changes in the host-derived components of the erythrocyte membrane. Galactose oxidase binds to terminal and penultimate galactose residues in sialoglycoproteins and since Plasmodium organisms do not synthesise sialic acids (Schauer et al. 1984), they probably lack the structures that are typically labelled by this method.

The presence of glycoproteins in P. falciparum has been demonstrated in several studies. Kilejian and Olson (1979) demonstrated the presence of P. falciparum proteins that were able to bind concanavalin A (Con A), which suggested the presence of mannosylated glycans. Later, Kilejian (1980) demonstrated the presence of glycoproteins, mainly in the late trophozoite and schizont stages, by radioactive labelling with glucosamine. Glycoproteins were also observed in merozoites in this study. There were 12 labelled proteins observed in the schizont stage; of those proteins, five remained in the merozoites and one was identified on the infected erythrocyte surface. Udeinya and Van Dyke (1980) treated P. falciparum-infected erythrocytes with radioactive sugars (glucosamine, fucose and mannose) to identify glycoproteins in these samples. In agreement with Kilejian (1980), the incorporation of radioactive glucosamine was stronger for erythrocytes infected with parasites in later stages (trophozoites and schizonts). A more detailed study identified eight labelled glycoproteins, ranging from 19-90 kDa, that were incorporated into the membrane (Udeinya & Van Dyke 1981a). These results might reflect the biosynthesis of GPI anchors, which are known to be upregulated in trophozoites and schizonts (Schmidt et al. 1998). In the same year, David et al. (1981) demonstrated that erythrocytes infected with later stages of P. falciparum preferentially bound to agarose beads coated with lectins (Con A, Ricinus communis lectin, soybean lectin and peanut agglutinin), which was in contrast with ring-infected and uninfected erythrocytes. These observations suggested that P. falciparum infection might result in increased mannosyl and/or glucosyl residues as well as increased galactosyl residues on the erythrocyte membrane. This observation could be a result of glycoprotein production by the parasite or infection-induced modifications of erythrocyte proteins. Ramasamy (1987) later confirmed the presence of P. falciparum glycoproteins that could bind to Con A, wheat germ agglutinin (WGA), R. communis lectin 120 and Bandeirea simplicifolia lectin (BS). Binding to WGA exhibited the highest affinity, thereby suggesting the presence of N-acetyl glucosamine (GlcNAc) residues.

Perkins (1982) investigated the incorporation of radioactive methionine, glucosamine and mannose into the surface of schizont-infected erythrocytes as well as in the merozoite stage by adding proteolytic (trypsin and pronase) and glycolytic (neuraminidase) enzymes. In this study, glycoproteins were released from schizont-infected cells upon pronase treatment but not after neuraminidase treatment, suggesting the absence of sialic acids in P. falciparum. In contrast to the conclusions of Kilejian (1980), this study did not identify glycoproteins in merozoites.

Further studies demonstrated the importance of glycosylated proteins in the immunogenicity of P. falciparum. Ramasamy and Reese (1985) observed that treating P. falciparum antigens (obtained from schizont-infected erythrocytes) with mixed glycosidases from Charonia lampas (sea snail) decreased their affinity to antibodies from infected hosts, such as owl monkeys that were immunised with P. falciparum and Nigerian subjects living in malaria endemic areas. Specifically, two parasite proteins at 65 and 100 kDa exhibited decreased reactivity after enzyme treatment, and the carbohydrate modification of the epitopes was confirmed by their insensitivity to protease activity and their heat stability. Endoglycosidase F (PNGase F) treatment had little effect on the reactivity of the antibodies, which suggested that N-linked glycans exhibit little contribution to this effect. A more detailed study (Ramasamy & Reese 1986) demonstrated that antibody binding was abolished by treatment with ?-galactosidase obtained from Aspergillus niger and green coffee beans. A mixture of ?-galactosidase, fucosidase, mannosidase and N-acetyl hexosaminidase also abolished antibody binding. Several antigens were not recognised by the antibodies after treatment with these enzymes, particularly, antigens at 185, 135, 120 and 75 kDa. Notably, the enzyme mixture was ineffective without ?-galactosidase, suggesting an important role for terminal ?-galactosyl residues in the antigenicity of P. falciparum glycoproteins. Supporting these results, a fluorescent derivative of BS that specifically binds to ?-galactosidase demonstrated binding to acetone-fixed parasites (Jakobsen et al. 1987). In addition, antibody reactivity to soluble P. falciparum molecules was sensitive to ?-galactosidase treatment. Later, Ravindran et al. (1988) reported that sera from subjects living in endemic areas, as well as from patients with acute P. falciparum infection, exhibited high titres of antibodies specific for ?-galactose, which was in contrast to sera from patients with acute cerebral malaria. Panton et al. (1989) demonstrated that the histidine-rich protein II (HRP-II) from P. falciparum, an extracellular protein found in sera from infected patients and in the supernatant of cultured parasites, could be labelled with radioactive galactose. However, intracellular HRP-II was not labelled, suggesting that glycosylation might act as a signal for secretion. In 1990, Jackobsen et al. described a soluble 60 kDa glycoprotein that was present on the surface of trophozoites and schizonts, but not merozoites, and this protein was able to bind to Erythrina christagalli agglutinin, but not to Con A or Griffonia simplicifolia agglutinin, suggesting the presence of ?-D-galactose(1-4)-D-N-acetylglucosamine. This protein was recognised by human sera from different endemic regions, as well as by lymphocytes from malaria-immune individuals. Other studies on P. falciparum immunogenic proteins, specifically the merozoite surface protein (MSP), demonstrated that both MSP-1 (195 kDa) and MSP-2 (43 kDa) could be galactosylated after metabolic labelling with tritiated N-acetylglucosamine, and both proteins were digested with ? and ?-galactosidases. However, the incorporation of galactose into the GPI moiety of this protein could not be ruled out (Qazi et al. 1994). Furthermore, Klabunde et al. (2002) identified P. falciparum glycoproteins that bound to mannan-binding protein during innate immunity.

The nature and structure of the glycans were not specifically addressed in these early studies, but later studies verified the presence of N- and O-glycosylation in P. falciparum.

N-glycosylation in P. falciparum

Early studies of N-glycosylation in P. falciparum made use of inhibitors, namely, tunicamycin and 2-deoxyglucose (Udeinya & Van Dyke 1980, 1981b, c). Both compounds arrested parasite development in a dose-dependent manner and both inhibited the incorporation of radiolabeled glucosamine into glycolipids, but not the incorporation of isoleucine into proteins. These findings suggested the specific inhibition of N-glycosylation, but the incorporation of glucosamine into O-glycans or GPI anchors was not considered. Tunicamycin blocks N-glycosylation by inhibiting UDP-GlcNAc:dolichol-pyrophosphate N-acetylglucosaminyltransferase (Schwarz & Datema 1982, McDowell & Schwarz 1988). However, tunicamycin also blocks ganglioside biosynthesis in the Golgi apparatus of rat liver (Yusuf et al. 1983a, b). Alternatively, 2-deoxyglucose blocks N-glycosylation through the formation of dolichyl-PP-(GlcNAc)2-2-deoxyglucose, thereby preventing the elongation of the mannose chain (Schwarz & Datema 1982, Elbein 1987, McDowell & Schwarz 1988). Notably, 2-deoxyglucose exhibits broader effects on P. falciparum metabolism and it blocks both glycolysis (van Schalkwyk et al. 2008) and GPI biosynthesis (Udeinya & Van Dyke 1981c, Dieckmann-Schuppert et al. 1992a, Santos de Macedo et al. 2001, de Macedo et al. 2003). Therefore, the arrest in parasite development that was observed after tunicamycin or 2-deoxyglucose treatment cannot be completely attributed to the inhibition of N-glycosylation.

Dieckmann-Schuppert et al. (1992a, b) reported that tunicamycin could not inhibit P. falciparum growth in the first life cycle (48 h). However, when parasites were subsequently treated with tunicamycin at concentrations that would not affect protein biosynthesis (12 ?M), parasites underwent arrested development, resulting in death. These observations were confirmed by Kimura et al. (1996). Furthermore, Dieckmann-Schuppert et al. (1992b, 1996) reported that lower concentrations of tunicamycin reduced the incorporation of radioactive methionine by 30% after 30 min of treatment, which was not observed by Kimura et al. (1996). Therefore, the exact biochemical effects of tunicamycin on P. falciparum metabolism remain unclear.

The first attempts to study N-glycosylation in P. falciparum protein investigated the sensitivity of the parasite proteins to peptide N-glycosidase F (also known as PNGase F and N-glycanase®), but these initial studies gave negative results (Ramasamy 1987, Dayal-Drager et al. 1991). Using metabolic labelling, Dieckmann-Schuppert et al. (1992a) demonstrated that PNGase-F treatment led to the release of a small percentage of tritiated glucosamine (7-12%) from proteins. While this finding supported the presence of some protein N-glycosylation, it was considered to be negligible at the time. Furthermore, dolichol-PP-linked intermediates, which would be indicative of dolichol-cycle intermediates, were not found in this study. In addition, P. falciparum lysates did not catalyse the transfer of radiolabeled sugars from dolichyl-pyrophosphoryl-oligosaccharides [Dol-PP-(GlcNAc)2, Dol-PP-(GlcNAc)2-Man9 or Dol-PP-(GlcNAc)2Man9Glc] to peptide acceptors of N-glycosylation.

In contrast, after radiolabeling with radioactive methionine for 18 h, Kimura et al. (1996) identified proteins that bound to Con A-Sepharose that were absent when parasites were pretreated with tunicamycin for 52 h before labelling. The same proteins lost their affinity to Con A-Sepharose after PNGase F treatment, which indicates that these proteins were N-glycosylated. Glycans released from these parasites were analysed by thin layer chromatography to identify oligosaccharides that migrated between GlcNAc2, Man3GlcNAc2 and Man9

GlcNAc2 standards. Because tunicamycin inhibited the transition of late trophozoites into schizonts and because N-glycosylated proteins were not observed in parasites that exhibited arrested development, these N-glycosylated proteins could play a role in the development of the intraerythrocytic stages of P. falciparum.

The inconsistent results obtained by Dieckmann-Schuppert et al. (1992a) and Kimura et al. (1996) could be attributed to different experimental approaches. While Dieckmann-Schuppert et al. (1992a) analysed late trophozoites, Kimura et al. (1996) used asynchronous cultures that contained three different stages (ring, trophozoite and schizont) after Percoll® gradient purification. In addition, Dieckmann-Schuppert et al. (1992a) used shorter pulse times (4 h) and glucose-free medium in their radiolabeling procedure. In contrast, Kimura et al. (1996) radiolabelled for both 8 h and 18 h and used glucose and methionine-deficient media because glucose-free medium had been reported to interfere with N-glycan biosynthesis in other systems (Elbein 1987). Gowda et al. (1997) confirmed the observations of Kimura et al. (1996) using similar radiolabeling conditions and they claimed to be the first to rule out GPI biosynthesis as an alternative explanation for the common experimental observations. A later study (Schmidt et al. 1998) demonstrated that GPI biosynthesis increases by 21-fold during intraerythrocytic development from the ring stages (6 h) to the late schizont stages (46 h). These data suggests that GPI biosynthesis is incipient during stages that also exhibit N-glycosylation, thereby supporting earlier conclusions from Kimura et al. (1996). N-glycosylation was also analysed for other P. falciparum proteins, such as MSP-1 and MSP-2. Neither MSP-1 nor MSP-2 exhibited lectin affinity or sensitivity to PNGase F, suggesting absence of N-linked glycans despite the presence of several potential N-glycosylation sites in both proteins (Berhe et al. 2000). Notably, when MSP-1 was expressed in mammalian cells (Murphy et al. 1990, Burghaus et al. 1999, Yang et al. 1999) and in the baculovirus system (Kedees et al. 2000), the proteins do become N-glycosylated.

The availability of the P. falciparum genome (Gardner et al. 2002) allowed for new studies on enzymes involved in N-glycosylation. P. falciparum lacks most of the enzymes involved in the assembly of N-glycans and phylogeny analyses suggested a secondary loss of Alg glycosyltransferases. The only two enzymes predicted to be present in P. falciparum were Alg7 and Alg14, which are responsible for the synthesis of Dol-PP-GlcNAc and Dol-PP-GlcNAc2, respectively, and STT3, which is responsible for the transfer of the Dol-PP-oligosaccharide to the Asn residue in the N-X-T/S sequon (Templeton et al. 2004, Samuelson et al. 2005, Anantharaman et al. 2007). Further biochemical studies confirmed that P. falciparum synthesises N-glycans that are shorter than their mammalian counterparts, namely, Dol-PP-GlcNAc and Dol-PP-GlcNAc2. The type of N-glycan that is produced is stage-dependent: early ring stages synthesise mainly Dol-PP-GlcNAc whereas schizonts synthesise both Dol-PP-GlcNAc and Dol-PP-GlcNAc2. These glycans are transferred to proteins, such as glucosamine-labelled parasite proteins, and they are recognised by the GlcNAc-binding G. simplicifolia lectin (GSL-II). This recognition is attenuated by PNGase F treatment, which decreases lectin binding (Bushkin et al. 2010). Although Kimura et al. (1996) did detect GlcNAc and GlcNAc2 among the glycans released by PNGase F treatment, their detection of larger glycans that were considered to be mannosylated is not fully understood. The N-glycan biosynthetic limitations exhibited by Plasmodium could explain why Dieckmann-Schuppert et al. (1992a, 1994) did not observe the transfer of radiolabeled Dol-PP-(GlcNAc)2Man9 or Dol-PP-(GlcNAc)2Man9Glc to acceptors in a cell-free system because, in this system, the oligosaccharyltransferase would not recognise these structures. However, when exogenous peptide acceptors were used, glycosylation by endogenous Dol-PP-GlcNAc or Dol-PP-GlcNAc2 was also not observed (Dieckmann-Schuppert et al. 1992a, 1994), which may have resulted from the limited size of the internal pool of Dol-PP-GlcNAc and/or Dol-PP-GlcNAc2, thus making the detection of the corresponding reaction products difficult. Bushkin et al. (2010) demonstrated that fluorescent GSL-II stained the endoplasmic reticulum, rhoptries and the parasite surface, but it did not stain the apicoplast, food vacuole, parasitophorous vacuole or the infected erythrocyte. This staining pattern may have resulted from the inability of GSL-II to label proteins decorated only with GlcNAc. In any case, GSL-II labelling of some but not all Plasmodium organelles suggests that N-glycosylation may have a role in protein targeting, which will need to be analysed in more detail in future experiments (Figure, Table).



O-glycosylation in P. falciparum

Dayal-Drager et al. (1991) investigated the nature of the sugar linkage to proteins in P. falciparum. Endo-O-glucosidase (O-glycanase®) released sugars from parasite proteins labelled with tritiated mannose and glucosamine and the presence of hexosamines linked to serine residues was confirmed by ?-elimination/reduction. Nasir-ud-Din et al. (1992) obtained similar results for a 195 kDa merozoite glycoprotein, probably MSP-1, thereby confirming the presence of O-GlcNAc, O-GalNAc and galactose in this protein. Dieckmann-Schuppert et al. (1992a) treated galactose, fucose and glucosamine-radiolabeled proteins with PNGase-F to remove possible N-glycans, purified the resulting material by gel filtration and subjected the void volume fraction to alkaline ?-elimination. About half of the radioactivity was shifted to the monosaccharide volume, suggesting the release of O-glycans. Dieckmann-Schuppert et al. (1993) characterised O-glycosylation reactions using exogenous galactosylation to identify O-GlcNAc in proteins. An analysis of the alditols released after ?-elimination demonstrated the presence of both reducing terminal and nonterminal GlcNAc, which could be elongated. O-GlcNAc transferase activity in parasite lysates was confirmed using exogenous peptide acceptors with a Pro-Tyr-Thr-Val-Val sequence and activity was higher in the later parasite stages. In contrast to the results of Nasir-ud-Din et al. (1992), mucin-type O-glycans were not detected in this study, which is consistent with later findings reporting that this parasite does not possess GalNAc-decorated proteins and is unable to synthesise GalNAc. Radiolabeling with GalN demonstrated that this precursor was mostly phosphorylated while GalNAc, GlcNAc and the corresponding sugar nucleotides were not detectable. These results are consistent with previous data that suggested that Plasmodium organisms are unable to epimerise GalN into GlcN and vice-versa, as shown by the absence of detectable GalN after labelling with GlcN (Dieckmann-Schuppert et al. 1992a, 1993, 1996). Kimura et al. (1996) confirmed the presence of O-linked glycoproteins that were mostly present in the schizont stage, which is in agreement with the identification of an O-GlcNAc modified protein in the same stage (Dieckmann-Schuppert et al. 1993). However, these authors suggested that the sensitivity to O-glycanase® could be attributed to the presence of Gal-GalNAc, which is in contrast to the observations of Dieckmann-Schuppert et al. (1992a, 1993). In contrast, Gowda et al. (1997) suggested that O-glycosylation does not take place in P. falciparum intraerythrocytic stages. Furthermore, in silico studies on O-glycosylation in P. falciparum suggested that this species lacks the genes that are necessary for the synthesis of mucin-type O-glycans (Templeton et al. 2004), which supports the findings from Dieckmann-Schuppert et al. (1992a, 1993).

The presence of O-glycosylation in MSP from P. falciparum remains controversial. Nasir-ud-Din et al. (1992) observed that MSP-1 was modified by O-GlcNAc. Later, Khan et al. (1997) concluded that MSP-2 was O-glycosylated using asynchronous P. falciparum cultures. These authors employed ?-elimination, exogenous galactosylation assays, lectin binding assays and enzymatic treatments to investigate O-glycosylation, and they observed that GlcNAc?-Ser/Thr was the predominant structure whereas Gal-GalNAc?-Ser/Thr was also present to a minor degree. This second conclusion is incompatible with Dieckmann-Schuppert et al. (1992a, 1993), which reported the absence of GalNAc metabolism in P. falciparum. In contrast to the findings of Khan et al. (1997), Berhe et al. (2000) used a similar experimental approach to show that MSP-1 and MSP-2 obtained from merozoites were not modified with O-glycans because ?-elimination caused no effect on the migration of glycopeptides in a Bio-Gel P4 column. These conclusions were further supported using galactosylation and lectin binding assays. Neither MSP-1 nor MSP-2 incorporated labelled galactose or bound to the lectins in the assay, indicating that the sole post-translational modification of this protein was the GPI anchor (Gerold et al. 1996b). Hoessli et al. (2003) showed by immunoprecipitation that, in addition to the 19-kDa C-terminal fragment that contains the GPI anchor, MSP-1 fragments could be labelled with tritiated glucosamine. In addition, they also demonstrated that both N and C-terminal MSP-1 fragments, as well as the whole protein, could be exogalactosylated. Furthermore, five different MSP-1 sequences from various P. falciparum strains contained ?-GlcNAc (but not ?-GlcNAc) sites that were predicted in silico. Regarding nucleocytosolic O-glycosylation, which is the addition of O-GlcNAc to Ser/Thr residues, no O-linked N-acetylglucosaminyltransferase (OGT) related to other organisms was identified in the Plasmodium genome (Banerjee et al. 2009). However, recently, the presence of OGT has been investigated using antibodies against OGT, and these studies have suggested the presence of O-GlcNAcylation in P. falciparum (Perez-Cervera et al. 2010), supporting previous observations of GlcNAc?-Ser/Thr in MSP-2 (Khan et al. 1997).

Despite the progress made in elucidating the biology of malaria parasites and in the discovery of effective drugs and anti-vector measures, malaria still remains one of the most important diseases in the world. Treatment has become difficult due to the emergence of parasites that are resistant to chloroquine and pyrimethamine. The need for new antimalarial drugs has encouraged research to investigate the basic biochemistry of the malaria parasites. In the light of the results summarised in this review, it is clear that both N- and O-glycosylation occur in P. falciparum. However, the identity of the proteins that carry these modifications, as well as their biological significance in the parasite life cycle and their interactions with its vertebrate and invertebrate hosts remains to be elucidated.



Alano P 2007. Plasmodium falciparum gametocytes: still many secrets of a hidden life. Mol Microbiol 66: 291-302.

Anantharaman V, Iyer LM, Balaji S, Aravind L 2007. Adhesion molecules and other secreted host-interaction determinants in Apicomplexa: insights from comparative genomics. Int Rev Cytol 262: 1-74.

Banerjee S, Robbins PW, Samuelson J 2009. Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum. Glycobiology 19: 331-336.

Bannister LH, Mitchell GH 2009. The malaria merozoite, forty years on. Parasitology 136: 1435-1444.

Baton LA, Ranford-Cartwright LC 2005. How do malaria ookinetes cross the mosquito midgut wall? Trends Parasitol 21: 22-28.

Berhe S, Gerold P, Kedees MH, Holder AA, Schwarz RT 2000. Plasmodium falciparum: merozoite surface proteins 1 and 2 are not posttranslationally modified by classical N- or O-glycans. Exp Parasitol 94: 194-197.

Billker O, Lindo V, Panico M, Etienne AE, Paxton T, Dell A, Rogers M, Sinden RE, Morris HR 1998. Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature 392: 289-292.

Burghaus PA, Gerold P, Pan W, Schwarz RT, Lingelbach K, Bujard H 1999. Analysis of recombinant merozoite surface protein-1 of Plasmodium falciparum expressed in mammalian cells. Mol Biochem Parasitol 104: 171-183.

Bushkin GG, Ratner DM, Cui J, Banerjee S, Duraisingh MT, Jennings CV, Dvorin JD, Gubbels MJ, Robertson SD, Steffen M, O'Keefe BR, Robbins PW, Samuelson J 2010. Suggestive evidence for Darwinian selection against asparagine-linked glycans of Plasmodium falciparum and Toxoplasma gondii. Eukaryot Cell 9: 228-241.

Chung DW, Ponts N, Cervantes S, Le Roch KG 2009. Post-translational modifications in Plasmodium: more than you think! Mol Biochem Parasitol 168: 123-134.

Clark IA, Budd AC, Alleva LM, Cowden WB 2006. Human malarial disease: a consequence of inflammatory cytokine release. Malar J 5: 85.

Daneshvar C, Davis TM, Cox-Singh J, Rafa'ee MZ, Zakaria SK, Divis PC, Singh B 2009. Clinical and laboratory features of human Plasmodium knowlesi infection. Clin Infect Dis 49: 852-860.

David PH, Hommel M, Oligino LD 1981. Interactions of Plasmodium falciparum infected erythrocytes with ligand-coated agarose beads. Mol Biochem Parasitol 4: 195-204.

Davidson EA, Gowda DC 2001. Glycobiology of Plasmodium falciparum. Biochimie 83: 601-604.

Dayal-Drager R, Hoessli DC, Decrind C, Del Guidice G, Lambert PH, Nasir-ud-Din 1991. Presence of O-glycosylated glycoproteins in the Plasmodium falciparum parasite. Carbohydr Res 209: c5-8.

de Macedo CS, Shams-Eldin H, Smith TK, Schwarz RT, Azzouz N 2003. Inhibitors of glycosyl-phosphatidylinositol anchor biosynthesis. Biochimie 85: 465-472.

Debierre-Grockiego F, Schwarz RT 2010. Immunological reactions in response to apicomplexan glycosylphosphatidylinositols. Glycobiology 20: 801-811.

Dieckmann-Schuppert A, Bause E, Schwarz RT 1993. Studies on O-glycans of Plasmodium-falciparum-infected human erythrocytes. Evidence for O-GlcNAc and O-GlcNAc-transferase in malaria parasites. Eur J Biochem 216: 779-788.

Dieckmann-Schuppert A, Bause E, Schwarz RT 1994. Glycosylation reactions in Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma brucei brucei probed by the use of synthetic peptides. Biochim Biophys Acta 1199: 37-44.

Dieckmann-Schuppert A, Bender S, Odenthal-Schnittler M, Bause E, Schwarz RT 1992a. Apparent lack of N-glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum. Eur J Biochem 205: 815-825.

Dieckmann-Schuppert A, Gerold P, Schwarz RT 1996. Glycoproteins of parasites. Glycoproteins of malaria parasites. In J Montreuil, JFG Vliegenthart, H Schachter, Glycoproteins and disease, vol. 30, Elsevier Science, Amsterdam, p. 125-158.

Dieckmann-Schuppert A, Hensel J, Schwarz RT 1992b. Studies on the effect of tunicamycin on erythrocytic stages of Plasmodium falciparum. Biochem Soc Trans 20: 184S.

Ejigiri I, Sinnis P 2009. Plasmodium sporozoite-host interactions from the dermis to the hepatocyte. Curr Opin Microbiol 12: 401-407.

Elbein AD 1987. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu Rev Biochem 56: 497-534.

Gahmberg CG, Hakomori SI 1973. External labeling of cell surface galactose and galactosamine in glycolipid and glycoprotein of human erythrocytes. J Biol Chem 248: 4311-4317.

Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DM, Fairlamb AH, Fraunholz MJ, Roos DS, Ralph SA, McFadden GI, Cummings LM, Subramanian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C, Davis RW, Fraser CM, Barrell B 2002. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419: 498-511.

Gerold P, Eckert V, Schwarz RT 1996a. GPI-anchors: an overview. Trends Glycosci Glycobiol 8: 265-277.

Gerold P, Schofield L, Blackman MJ, Holder AA, Schwarz RT 1996b. Structural analysis of the glycosyl-phosphatidylinositol membrane anchor of the merozoite surface proteins-1 and -2 of Plasmodium falciparum. Mol Biochem Parasitol 75: 131-143.

Gerold P, Schuppert-Dieckmann A, Schwarz RT 1994. Glycosylphosphatidylinositols synthesized by asexual erythrocytic stages of the malaria parasite, Plasmodium falciparum. Candidates for plasmodial glycosylphosphatidylinositol membrane anchor precursors and pathogenicity factors. J Biol Chem 269: 2597-2606.

Gowda DC, Gupta P, Davidson EA 1997. Glycosylphosphatidylinositol anchors represent the major carbohydrate modification in proteins of intraerythrocytic stage Plasmodium falciparum. J Biol Chem 272: 6428-6439.

Guha-Niyogi A, Sullivan DR, Turco SJ 2001. Glycoconjugate structures of parasitic protozoa. Glycobiology 11: 45R-59R.

Hirai M, Wang J, Yoshida S, Ishii A, Matsuoka H 2001. Characterization and identification of exflagellation-inducing factor in the salivary gland of Anopheles stephensi (Diptera: Culicidae). Biochem Biophys Res Commun 287: 859-864.

Hoessli DC, Davidson EA, Schwarz RT, Nasir-ud-Din 1996. Glycobiology of Plasmodium falciparum: an emerging area of research. Glycoconj J 13: 1-3.

Hoessli DC, Poincelet M, Gupta R, Ilangumaran S, Nasir-ud-Din 2003. Plasmodium falciparum merozoite surface protein 1. Eur J Biochem 270: 366-375.

Jakobsen PH, Jepsen S, Riley EM, Theander TG, Grellier P, Lihme A, Hviid L, Dziegiel M, Schrevel J 1990. Biochemical characterization, localization and immunostimulating properties of a soluble glycoprotein, Ag1, isolated from in vitro cultures of Plasmodium falciparum. Parasitol Res 76: 657-661.

Jakobsen PH, Theander TG, Jensen JB, Mølbak K, Jepsen S 1987. Soluble Plasmodium falciparum antigens contain carbohydrate moieties important for immune reactivity. J Clin Microbiol 25: 2075-2079.

Kedees MH, Gerold P, Azzouz N, Blaschke T, Shams-Eldin H, Mühlberger E, Holder AA, Klenk HD, Schwarz RT, Eckert V 2000. Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system. Eur J Cell Biol 79: 52-61.

Khan AH, Qazi AM, Hoessli DC, Torred-Duarte AP, Senaldi G, Qazi MH, Walker-Nasir E, Nasir-ud-Din 1997. Carbohydrate moiety of Plasmodium falciparum glycoproteins: the nature of the carbohydrate-peptide linkage in the MSP-2 glycoprotein. Biochem Mol Biol Int 43: 655-668.

Kilejian A 1980. Stage-specific proteins and glycoproteins of Plasmodium falciparum: identification of antigens unique to schizonts and merozoites. Proc Natl Acad Sci USA 77: 3695-3699.

Kilejian A, Olson J 1979. Proteins and glycoproteins from human erythrocytes infected with Plasmodium falciparum. Bull World Health Organ 57 (Suppl 1): 101-107.

Kimura EA, Couto AS, Peres VJ, Casal OL, Katzin AM 1996. N-linked glycoproteins are related to schizogony of the intraerythrocytic stage in Plasmodium falciparum. J Biol Chem 271: 14452-14461.

Klabunde J, Uhlemann AC, Tebo AE, Kimmel J, Schwarz RT, Kremsner PG, Kun JF 2002. Recognition of Plasmodium falciparum proteins by mannan-binding lectin, a component of the human innate immune system. Parasitol Res 88: 113-117.

Krotoski WA 1989. The hypnozoite and malarial relapse. Prog Clin Parasitol 1: 1-19.

Krotoski WA, Collins WE 1982. Failure to detect hypnozoites in hepatic tissue containing exoerythrocytic schizonts of Plasmodium knowlesi. Am J Trop Med Hyg 31: 854-856.

Krotoski WA, Collins WE, Bray RS, Garnham PC, Cogswell FB, Gwadz RW, Killick-Kendrick R, Wolf R, Sinden R, Koontz LC, Stanfill PS 1982. Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection. Am J Trop Med Hyg 31: 1291-1293.

Kuehn A, Pradel G 2010. The coming-out of malaria gametocytes. J Biomed Biotechnol 2010: 976827.

Matuschewski K 2006. Getting infectious: formation and maturation of Plasmodium sporozoites in the Anopheles vector. Cell Microbiol 8: 1547-1556.

McDowell W, Schwarz RT 1988. Dissecting glycoprotein biosynthesis by the use of specific inhibitors. Biochimie 70: 1535-1549.

Mendonça-Previato L, Todeschini AR, Heise N, Previato JO 2005. Protozoan parasite-specific carbohydrate structures. Curr Opin Struct Biol 15: 499-505.

Murphy VF, Rowan WC, Page MJ, Holder AA 1990. Expression of hybrid malaria antigens in insect cells and their engineering for correct folding and secretion. Parasitology 100: 177-183.

Nasir-ud-Din, Drager-Dayal R, Decrind C, Hu BH, Del Giudice G, Hoessli D 1992. Plasmodium falciparum synthesizes O-glycosylated glycoproteins containing O-linked N-acetylglucosamine. Biochem Int 27: 55-64.

Ndao M 2009. Diagnosis of parasitic diseases: old and new approaches. Interdiscip Perspect Infect Dis 2009: 278246.

Panton LJ, McPhie P, Maloy WL, Wellems TE, Taylor DW, Howard RJ 1989. Purification and partial characterization of an unusual protein of Plasmodium falciparum: histidine-rich protein II. Mol Biochem Parasitol 35: 149-160.

Perez-Cervera Y, Harichaux G, Schmidt J, Debierre-Grockiego F, Dehennaut V, Bieker U, Meurice E, Lefebvre T, Schwarz RT 2010. Direct evidence of O-GlcNAcylation in the apicomplexan Toxoplasma gondii: a biochemical and bioinformatic study. Amino Acids, in press.

Perkins M 1982. Surface proteins of schizont-infected erythrocytes and merozoites of Plasmodium falciparum. Mol Biochem Parasitol 5: 55-64.

Pisciotta JM, Sullivan D 2008. Hemozoin: oil versus water. Parasitol Int 57: 89-96.

Qazi MA, Senaldi G, Tacchini-Cottier F, Qazi MH, Fayyaz-ud-Din, Hoessli DC, Walker-Nasir E, Nasir-ud-Din 1994. Galactose residues in Plasmodium falciparum glycoproteins: incorporation and elimination. Biochem Soc Trans 22: 362S.

Ramasamy R 1987. Studies on glycoproteins in the human malaria parasite Plasmodium falciparum-lectin binding properties and the possible carbohydrate-protein linkage. Immunol Cell Biol 65: 147-152.

Ramasamy R, Reese RT 1985. A role for carbohydrate moieties in the immune response to malaria. J Immunol 134: 1952-1955.

Ramasamy R, Reese RT 1986. Terminal galactose residues and the antigenicity of Plasmodium falciparum glycoproteins. Mol Biochem Parasitol 19: 91-101.

Ravindran B, Satapathy AK, Das MK 1988. Naturally-occurring anti-alpha-galactosyl antibodies in human Plasmodium falciparum infections: a possible role for autoantibodies in malaria. Immunol Lett 19: 137-141.

Samuelson J, Banerjee S, Magnelli P, Cui J, Kelleher DJ, Gilmore R, Robbins PW 2005. The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci USA 102: 1548-1553.

Santos de Macedo C, Gerold P, Jung N, Azzouz N, Kimmel J, Schwarz RT 2001. Inhibition of glycosyl-phosphatidylinositol biosynthesis in Plasmodium falciparum by C-2 substituted mannose analogues. Eur J Biochem 268: 6221-6228.

Schauer R, Wember M, Howard RJ 1984. Malaria parasites do not contain or synthesize sialic acid. Hoppe Seylers Z Physiol Chem 365: 185-194.

Schmidt A, Schwarz RT, Gerold P 1998. Plasmodium falciparum: asexual erythrocytic stages synthesize two structurally distinct free and protein-bound glycosylphosphatidylinositols in a maturation-dependent manner. Exp Parasitol 88: 95-102.

Schwarz RT, Datema R 1982. The lipid pathway of protein glycosylation and its inhibitors: the biological significance of protein-bound carbohydrates. Adv Carbohydr Chem Biochem 40: 287-379.

Shams-Eldin H, Debierre-Grockiego F, Kimmel J, Schwarz RT 2009a. Glycosylphosphatidylinositol (GPI) anchoring of proteins. GPIs of apicomplexan protozoa. In AK Menon, T Kinoshita, P Orlean, F Tamanoi, The Enzymes, vol. XXVI, Academic Press, Burlington, p. 159-180.

Shams-Eldin H, Debierre-Grockiego F, Schwarz RT 2009b. Glycosylphosphatidylinositol anchors: structure, biosynthesis and functions. In HJ Gabius, The sugar code: fundamentals of glycosciences, Wiley VCH Verlag, Weinheim, p. 155-173.

Singh B, Kim Sung L, Matusop A, Radhakrishnan A, Shamsul SS, Cox-Singh J, Thomas A, Conway DJ 2004. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 363: 1017-1024.

Sinnis P, Zavala F 2008. The skin stage of malaria infection: biology and relevance to the malaria vaccine effort. Future Microbiol 3: 275-278.

Talman AM, Domarle O, McKenzie FE, Ariey F, Robert V 2004. Gametocytogenesis: the puberty of Plasmodium falciparum. Malar J 3: 24.

Templeton TJ, Iyer LM, Anantharaman V, Enomoto S, Abrahante JE, Subramanian GM, Hoffman SL, Abrahamsen MS, Aravind L 2004. Comparative analysis of apicomplexa and genomic diversity in eukaryotes. Genome Res 14: 1686-1695.

Trager W, Jensen JB 1976. Human malaria parasites in continuous culture. Science. 193: 673-675.

Trigg PI, Hirst SI, Shakespeare PG, Tappenden L 1977. Labelling of membrane glycoprotein in erythrocytes infected with Plasmodium knowlesi. Bull World Health Organ 55: 205-209.

Udeinya IJ, Van Dyke K 1980. Labelling of membrane glycoproteins of cultivated Plasmodium falciparum. Bull World Health Organ 58: 445-448.

Udeinya IJ, Van Dyke K 1981a. Plasmodium falciparum: synthesis of glycoprotein by cultured erythrocytic stages. Exp Parasitol 52: 297-302.

Udeinya IJ, Van Dyke K 1981b. Concurrent inhibition by tunicamycin of glycosylation and parasitemia in malarial parasites (Plasmodium falciparum) cultured in human erythrocytes. Pharmacology 23: 165-170.

Udeinya IJ, Van Dyke K 1981c. 2-deoxyglucose: inhibition of parasitemia and of glucosamine incorporation into glycosylated macromolecules in malarial parasites (Plasmodium falciparum). Pharmacology 23: 171-175.

van Schalkwyk DA, Priebe W, Saliba KJ 2008. The inhibitory effect of 2-halo derivatives of D-glucose on glycolysis and on the proliferation of the human malaria parasite Plasmodium falciparum. J Pharmacol Exp Ther 327: 511-517.

Vaughan AM, Aly AS, Kappe SH 2008. Malaria parasite pre-erythrocytic stage infection: gliding and hiding. Cell Host Microbe 4: 209-218.

von Itzstein M, Plebanski M, Cooke BM, Coppel RL 2008. Hot, sweet and sticky: the glycobiology of Plasmodium falciparum. Trends Parasitol 24: 210-218.

Warrell DA, Gilles HM 2002. Essential malariology, 4th ed., A Hodder Arnold Publication, London, 384 pp.

WHO - World Health Organization 2008. World Malaria Report 2008. World Health Organization, Geneva, 190 pp.

Yang S, Nikodem D, Davidson EA, Gowda DC 1999. Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells. Glycobiology 9: 1347-1356.

Yusuf HK, Pohlentz G, Sandhoff K 1983a. Tunicamycin inhibits ganglioside biosynthesis in rat liver Golgi apparatus by blocking sugar nucleotide transport across the membrane vesicles. Proc Natl Acad Sci USA 80: 7075-7079.

Yusuf HK, Pohlentz G, Schwarzmann G, Sandhoff K 1983b. Ganglioside biosynthesis in Golgi apparatus of rat liver. Stimulation by phosphatidylglycerol and inhibition by tunicamycin. Eur J Biochem 134: 47-54.

Received 25 September 2010
Accepted 16 November 2010
Financial support: CNPq, FAPERJ, CAPES, INCT-V
Work in the laboratory of RTS was supported by Deutsche Forschungsgemeinschaft and the Human Frontier Science Program.

+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.


Memórias do Instituto Oswaldo Cruz

Av. Brasil 4365, Castelo Mourisco
sala 201, Manguinhos, 21040-900
Rio de Janeiro, RJ, Brazil

Tel.: +55-21-2562-1222

This e-mail address is being protected from spambots. You need JavaScript enabled to view it.



marca fiocruzmarca brasil
marca faperjmarca cnpqmarca capes n marca cope

and diabetes. Erection dysfunction or ED is certainly one of mens most usual problem. It changes buy tadalafil 60mg A common drug is actually an imitation of its manufacturer twin. Both ought to be same in female cialis 20mg Long Phrase Viagra Use Fundamentally Damages Sex Lives This discount cialis canada Equally so, theres something to be said for the wonder of the second, captured forever on picture or a buy cheap cialis People extremely annoyed that they could only get three weeks at a time, Bunker noted. Retired persons cheap pharmacy These types of matters are possibly to being identified as having a result of cancer buy cialis 40mg - Yoghourt - fat-free simply Physical causes: Buying generic medicines now has been cheap generic cialis Herbaceous plants like nigrum and tribulus are well-known for his or her qualities in defeating impotence, which tadalafil 10mg It is not hard to consider Cialis that is generic. Most men start with one-10 mg dosage each purchase cialis Tadalafil quickly gained the moniker of weekender in Paris due to the fabulous results. The bash freaks buy female cialis