Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum

Sanz, Sílvia; Bandini, Giulia; Ospina, Diego; Bernabeu, Maria; Mariño, Karina; Fernández-Becerra, Carmen; Izquierdo, Luís

doi:10.1074/jbc.m112.439828

Cited by 38 publications

(56 citation statements)

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“…Glycosyltransferase activity and sugar nucleotides donors have long been linked to protein folding and thermotolerance in many organisms, including different parasites353637. It has been suggested that Protein O -fucosyltransferase 2 (PoFUT2), of which a homolog is conserved and expressed in the P. falciparum genome18, may be involved in a novel quality control mechanism for the proper folding of TSR-domain containing proteins in the endoplasmic reticulum (ER)3738. Proteins with TSR-domains have been shown to be expressed in asexual stages and to be involved in host-cell invasion3940.…”

Section: Resultsmentioning

confidence: 99%

“…A C18 reversed-phase column was used for sugar nucleotide separation with a gradient from 0.5% to 4% acetonitrile in 20 mM triethylammonium acetate buffer over 20 min at a flow rate of 25 µl/min. Sugar nucleotides were identified by their diagnostic MRM transitions18. The peak areas for each sugar nucleotide, along with their empirically determined molar relative response factors and the known amount of internal GDP-Glc, were used to quantify sugar nucleotides.…”

Section: Methodsmentioning

confidence: 99%

“…These precursors feed the biosynthesis of glycans by acting as donors for glycosylation reactions17. We recently analysed the sugar nucleotide pool in asexuals and found that P. falciparum synthesises five different sugar nucleotides including GDP-fucose (GDP-Fuc) or UDP-galactose (UDP-Gal), both of which are not involved in the biosynthesis of the aforementioned GPI-anchors or N -glycans18. Therefore, the presence of these metabolites suggests that they may be involved in the biosynthesis of glycans not yet characterised in the parasite19.…”

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The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages

Sanz

López-Gutiérrez

Bandini

et al. 2016

Sci Rep

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Glycosylation is an important posttranslational protein modification in all eukaryotes. Besides glycosylphosphatidylinositol (GPI) anchors and N-glycosylation, O-fucosylation has been recently reported in key sporozoite proteins of the malaria parasite. Previous analyses showed the presence of GDP-fucose (GDP-Fuc), the precursor for all fucosylation reactions, in the blood stages of Plasmodium falciparum. The GDP-Fuc de novo pathway, which requires the action of GDP-mannose 4,6-dehydratase (GMD) and GDP-L-fucose synthase (FS), is conserved in the parasite genome, but the importance of fucose metabolism for the parasite is unknown. To functionally characterize the pathway we generated a PfGMD mutant and analyzed its phenotype. Although the labelling by the fucose-binding Ulex europaeus agglutinin I (UEA-I) was completely abrogated, GDP-Fuc was still detected in the mutant. This unexpected result suggests the presence of an alternative mechanism for maintaining GDP-Fuc in the parasite. Furthermore, PfGMD null mutant exhibited normal growth and invasion rates, revealing that the GDP-Fuc de novo metabolic pathway is not essential for the development in culture of the malaria parasite during the asexual blood stages. Nonetheless, the function of this metabolic route and the GDP-Fuc pool that is generated during this stage may be important for gametocytogenesis and sporogonic development in the mosquito.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages

Sanz

López-Gutiérrez

Bandini

et al. 2016

Sci Rep

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“…gondii Nuclear Fucosylation Is Dependent on the de Novo GDPFucose Biosynthetic Pathway. Because T. gondii and other apicomplexans have no salvage pathway for fucose (25,26), GDPfucose is synthesized from GDP-mannose in a three-step process catalyzed by GDP-mannose dehydratase (GMD, TGGT1_238940) and GDP-fucose synthase (Fig. S2D) (21).…”

Section: Significancementioning

confidence: 99%

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

Bandini

Haserick

Motari

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Toxoplasma gondii is an intracellular parasite that causes disseminated infections in fetuses and immunocompromised individuals. Although gene regulation is important for parasite differentiation and pathogenesis, little is known about protein organization in the nucleus. Here we show that the fucose-binding Aleuria aurantia lectin (AAL) binds to numerous punctate structures in the nuclei of tachyzoites, bradyzoites, and sporozoites but not oocysts. AAL also binds to Hammondia and Neospora nuclei but not to more distantly related apicomplexans. Analyses of the AAL-enriched fraction indicate that AAL binds O-linked fucose added to Ser/Thr residues present in or adjacent to Ser-rich domains (SRDs). Sixty-nine Ser-rich proteins were reproducibly enriched with AAL, including nucleoporins, mRNA-processing enzymes, and cell-signaling proteins. Two endogenous SRDs-containing proteins and an SRD-YFP fusion localize with AAL to the nuclear membrane. Superresolution microscopy showed that the majority of the AAL signal localizes in proximity to nuclear pore complexes. Host cells modify secreted proteins with O-fucose; here we describe the O-fucosylation pathway in the nucleocytosol of a eukaryote. Furthermore, these results suggest O-fucosylation is a mechanism by which proteins involved in gene expression accumulate near the NPC.toxoplasma | fucose | nuclear glycosylation | nuclear pore complex T he apicomplexan parasite Toxoplasma gondii causes disseminated infections in humans, and these infections can lead to severe damage in immunocompromised individuals and fetuses (1, 2). There is no human vaccine against T. gondii, and recently the price of pyrimethamine, the drug used to treat toxoplasmosis in the United States, has increased more than 50-fold (2).T. gondii has a complex life cycle, and the parasite's ability to differentiate through its life stages in response to stresses and environmental conditions is fundamental for its pathogenicity and transmission (3). Transcriptome analyses have revealed that a large percentage of mRNAs show life stage-specific expression (4) and/or cell cycle regulation (5). Recent studies have increased our understanding of gene expression in T. gondii by identifying the AP2 family of transcription factors (6-8) and by describing posttranslational modifications (PTMs) of histones and some of the enzymes responsible for them (9-11). However, little is known about protein organization at the nuclear periphery, a subnuclear compartment that plays a critical role in transcriptional regulation in many eukaryotes. In particular, the gene-gating model (12) suggests that the nuclear pore complex (NPC) has a role in transcriptional regulation and chromatin organization as well as in protein and mRNA transport (13,14).In T. gondii chromodomain protein 1 localizes with heterochromatin at the nuclear periphery (15), and centromeres sequester to an apical nuclear region (16). Although the nuclear localization signal (NLS) and importin-α system are present, key nuclear import and export molecules are...

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“…Even so, homologs of several genes required for protein glycosylation are present and conserved in the genomes of Plasmodium spp. and metabolomic analyses of parasite material has demonstrated the presence of the nucleotide sugars required for protein glycosylation, which alluded to the existence of as yet undiscovered parasite glycans.With the aid of modern protein mass spectrometry methods, several research groups have recently tackled the issues of glycan lability and relatively small sample sizes to begin characterizing the true diversity of Plasmodium protein glycosylation [6][7][8][9][10][11][12]. Evidence for the synthesis of short N-linked glycans has been reported in asexual blood stages of Plasmodium falciparum [6], and there is every reason to expect that these modifications will be found in other life cycle stages as well.…”

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Implications of Plasmodium Glycosylation on Vaccine Efficacy and Design

Goddard‐Borger

Boddey

2018

Future Microbiol.

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Malaria remains a dire public health problem, despite significant efforts to control and eliminate the disease, and is caused by infection with any one of six species of Plasmodium parasite. Although focused control measures have halved the number of annual deaths from malaria to approximately 630,000 [1], the disease is still endemic in 91 countries and resistance to antimalarials continues to spread. Malaria eradication will require a combination of control measures including a vaccine that affords effective protection against different species and strains of Plasmodium parasites; a need that is currently unmet. Although much has been written about the design and evaluation of malaria vaccines, relatively little has been said about the impact that glycosylation has on the efficacy of existing or emerging vaccine technologies. This is probably because the exact nature of protein glycosylation in Plasmodium spp. has been contentious [2,3] and because glycopeptides were for a long time incorrectly thought to be T-independent antigens [4].Uncertainty surrounding protein glycosylation in Plasmodium parasites was driven in the past by an inability of common glycan-recognizing lectins to bind to parasite proteins and poor histological staining of parasites by periodic acid-Schiff methods. Indeed, it appeared that the installation of glycosylphosphatidylinositol anchors was the only type of glycosylation that Plasmodium parasites undertook [5]. Genome sequencing of Plasmodium spp. also revealed the absence of genes required to build and remodel common eukaryotic glycans [2,3]. Even so, homologs of several genes required for protein glycosylation are present and conserved in the genomes of Plasmodium spp. and metabolomic analyses of parasite material has demonstrated the presence of the nucleotide sugars required for protein glycosylation, which alluded to the existence of as yet undiscovered parasite glycans.With the aid of modern protein mass spectrometry methods, several research groups have recently tackled the issues of glycan lability and relatively small sample sizes to begin characterizing the true diversity of Plasmodium protein glycosylation [6][7][8][9][10][11][12]. Evidence for the synthesis of short N-linked glycans has been reported in asexual blood stages of Plasmodium falciparum [6], and there is every reason to expect that these modifications will be found in other life cycle stages as well. Cell surface proteomic studies of P. falciparum and P. vivax sporozoites, the two species responsible for the majority of malaria cases and deaths, have demonstrated that O-and C-linked glycans exist on the essential adhesive proteins, TRAP and CSP [8,10]. These abundant surface proteins are required for parasite infectivity and are prime vaccine candidates because sporozoites represent a population bottleneck in the life cycle as parasites pass from mosquito to human and establish an infection in the liver. Moreover, strain-transcending sterile immunity can be achieved through strong B-and T-cell-dependent respons...

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Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum

Cited by 38 publications

References 74 publications

The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages

The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

Implications of Plasmodium Glycosylation on Vaccine Efficacy and Design

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