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Molecular & Cellular Proteomics 7:891-910, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions^*,S

Sylvain Fauquenoy, Willy Morelle, Agnès Hovasse, Audrey Bednarczyk, Christian Slomianny^¶, Christine Schaeffer, Alain Van Dorsselaer and Stanislas Tomavo^,||

From the Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France, Laboratoire de Spectrométrie de Masse Bioorganique, CNRS UMR 7178, Université Louis Pasteur, 67087 Strasbourg, France, and ^¶ Laboratoire de Physiologie Cellulaire, INSERM U 800, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man_5–8(GlcNAc)₂) and paucimannosidic (Man_3–4(GlcNAc)₂) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

View larger version (29K): [in this window] [in a new window]   FIG. 9. Effect of tunicamycin on T. gondii gliding motility and host cell invasion. A, effect of tunicamycin on gliding motility of T. gondii. Tachyzoites treated with tunicamycin or with DMSO alone (Control) for 48 h during the first cycle of infection were released and allowed to glide on serum-coated slides. Trails were visualized by staining with anti-SAG1 antibodies followed by a fluorescent secondary antibody. B, effect of tunicamycin on host cell entry. The tunicamycin-treated and untreated tachyzoites during the first cycle of infection described above were also used to infect new monolayer HFF cells and grown for 24 h. The intracellular tachyzoites were paraformaldehyde-fixed and stained with anti-SAG1 or anti-GRA1 antibodies. 500 vacuoles were counted, and three different experiments were performed. The data are expressed as mean ± S.D. C, schematic drawing illustrating the host cell entry by T. gondii, the basic elements (ER, Golgi apparatus, and plasma membrane), and parasite-specific organelles (dense granules, micronemes, rhoptries, and apicoplast). The current accepted model of host cell entry or invasion proposes contributions from both the glideosome and the moving junction. The glideosome is composed of TgMyoA, actin filaments, and membrane anchor myosin XIV or GAP50 and GAP45, which are located between the plasma membrane and the inner membrane complex, whereas the moving junction contains rhoptry proteins that are secreted during host cell entry. These moving junction proteins include RON2, RON4, and the microneme AMA1. In light of the observations related in this study, we describe that several components of both glideosome and moving junction are potentially N-glycosylated or contain a number of consensus sites suggesting them to be N-glycosylated. We believe that this post-translational modification may be required for their proper intracellular transport to the pellicle and for protein-protein interactions.

Although studies have clearly highlighted N-glycosylation as an essential modification for protein folding, stability, half-life, secretion, and other relevant biological functions in most eukaryotic cells, very little is known concerning this issue in T. gondii. The presence of N-glycoproteins is indeed still controversial (15–19). In general, N-glycosylation is considered a rare post-translational modification in apicomplexan parasites. For example, the causative agent of malaria, Plasmodium falciparum, lacks most genes involved in lipid (dolichylpyrophosphate)-linked oligosaccharide donors (20). We applied proteomics and glycomics analyses as well as pharmacological approaches to elucidate the parasite's N-glycosylation biosynthetic pathway. This led to the elucidation of the major structures of protein N-linked glycans that suggests the presence of an almost complete early N-glycosylation biosynthetic pathway in T. gondii. In particular, we showed that several key proteins involved in gliding motility, moving junction, and other functions that are essential for host cell invasion by T. gondii are modified by N-glycosylation. Consistently inhibition of this post-translational modification dramatically impaired the parasite's motility, host cell invasion, and intracellular growth.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Growth and Isolation of Parasites—
Human foreskin fibroblasts (HFFs) were maintained in Dulbecco's modified Eagle's medium (DMEM, BioWhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum, 2 mM glutamine (Sigma), and 0.05% gentamycin (Sigma). Tachyzoites from RH or 76K strains were grown in monolayers of HFF cells, harvested, and purified as described previously (21). Encysted bradyzoites were purified from brains of mice chronically infected by T. gondii 76K for 2 months (22). These in vivo bradyzoites were freed by pepsin digestion (0.05 mg·ml^–1 pepsin in 170 mM NaCl, 60 mM HCl) for 5–10 min at 37 °C. For some experiments, we generated transgenic lines of T. gondii 76K expressing β-galactosidase using the plasmid containing the promoter 5'GRA1 and untranslated 3'SAG1 that drives β-galactosidase expression as described previously (23). This plasmid also contains TUB1-CAT-3'SAG1, which allowed the expression of chloramphenicol acetyltransferase for selection of T. gondii stable transgenic β-galactosidase-expressing clones. One of the β-galactosidase-expressing clones of T. gondii was used to quantify parasite numbers during host cell infection and growth.

Detergent Extraction of Toxoplasma Proteins, Reduction, and Carboxyamidomethylation—
Proteins were extracted on ice in a buffer of 0.5% (w/v) SDS in 100 mM Tris·HCl (pH 7.4). Insoluble material was removed by centrifugation at 3000 rpm for 15 min. Detergent was removed by extensive dialysis against 50 mM ammonium bicarbonate at 4 °C. The protein extracts were dissolved in 500 µl of 600 mM Tris·HCl (pH 8.3) and denatured by guanidine hydrochloride (6 M final concentration). The sample was incubated at 45 °C for 90 min, and the number of disulfide bridges was reduced using 300 µg of DTT. The sample was flushed with argon and incubated at 45 °C for 5 h. After addition of 1.8 mg of iodoacetamide, the sample was flushed with argon and incubated at room temperature overnight in the dark. The sample was then extensively dialyzed against 50 mM ammonium bicarbonate at 4 °C and lyophilized.

Tryptic, PNGase F, PNGase A, or -Mannosidase Digestions—
The reduced carboxymethylated proteins were digested with L-1-tosylamide-2-phenylethylchloromethylketone bovine pancreas trypsin (EC 3.4.21.4; Sigma) with an enzyme-to-substrate ratio of 1:20 (by mass), and the mixture was incubated for 24 h at 37 °C in 50 mM ammonium bicarbonate. The reaction was terminated by boiling for 5 min before lyophilization.

PNGase F (peptide-N⁴-(N-acetyl-β-glucosaminyl)asparagine amidase, EC 3.5.1.52; Roche Applied Science) digestion was carried out in ammonium bicarbonate buffer for 16 h at 37 °C. The reaction was terminated by lyophilization, and the products were purified on a Sep-Pak C₁₈ (Waters Ltd.) to separate the N-glycans from the peptides. After conditioning the Sep-Pak C₁₈ by sequential washing with methanol (5 ml) and 5% acetic acid (2 x 5 ml), the sample was loaded onto the Sep-Pak, and the glycans were eluted with 3 ml of 5% acetic acid. Peptides were eluted with 3 ml of 80% acetonitrile containing 5% acetic acid. Acetonitrile was evaporated under a stream of nitrogen, and the samples were freeze-dried. Glycopeptides remaining after PNGase F digestion were further digested with PNGase A (peptide-N⁴-(N-acetyl-β-glucosaminyl)asparagine amidase, EC 3.5.1.52; Roche Applied Science) in ammonium acetate buffer (50 mM, pH 5.0) for 16 h at 37 °C using 0.5 milliunit of the enzyme. The reaction was terminated by lyophilization, and the products were purified on a Sep-Pak C₁₈ (Waters Ltd.) as described above.

-Mannosidase digestion was carried out on PNGase F-released glycans using the following conditions: 0.5 unit of enzyme (jack bean -mannosidase, EC 3.2.1.24; Sigma) in 200 µl of 50 mM ammonium acetate buffer, pH 4.5. The enzyme digestion was incubated at 37 °C for 48 h with a fresh aliquot of enzyme added after 24 h and terminated by boiling for 10 min before lyophilization. An aliquot corresponding to 25% of the total sample was taken after the digestion and permethylated for MALDI-TOF-MS and gas chromatography (GC)-MS analysis.

Permethylation of Glycans—
Permethylation of the freeze-dried glycans was performed according to the procedure developed by Ciucanu and Kerek (24). The reaction was terminated by adding 1 ml of ultrapure water followed by three extractions with 500 µl of chloroform. The pooled chloroform phases (1.5 ml) were then washed eight times with ultrapure water. The methylated derivative-containing chloroform phase was finally dried under a stream of nitrogen, and the extracted products were further purified on a Sep-Pak C₁₈. The Sep-Pack C₁₈ was sequentially conditioned with methanol (5 ml) and water (2 x 5 ml). The derivatized glycans dissolved in methanol were applied on the cartridge, washed with 3 x 5 ml of water and 2 ml of 10% (v/v) acetonitrile in water, and eluted with 3 ml of 80% (v/v) acetonitrile in water. Acetonitrile was evaporated under a stream of nitrogen, and the samples were freeze-dried.

MALDI-TOF-MS—
MALDI-TOF-MS experiments were carried out on a Voyager Elite DE-STR Pro instrument (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm) and a gridless delayed extraction ion source. The spectrometer was operated in the positive reflectron mode by delayed extraction with an accelerating voltage of 20 kV, a pulse delay time of 200 ns, and a grid voltage of 66%. All spectra shown represent accumulated spectra obtained by 400–500 laser shots. Permethylated glycans were co-crystallized with 2,5-dihydroxybenzoic acid as matrix (10 mg/ml 2,5-dihydroxybenzoic acid in methanol/water solution (50:50)). The instrument was externally calibrated with a maltose ladder encompassing the m/z values of the analyzed samples.

Linkage Analysis—
The permethylated native N-glycans were hydrolyzed in 300 µl of 4 M TFA at 100 °C for 4 h. After removing TFA by drying in vacuo, the permethylated compounds were then reduced at room temperature overnight by adding 200 µl of 2 M ammonia solution containing sodium borodeuteride (4 mg/ml). The reduction was terminated by adding acetic acid, and borates were eliminated under a stream of nitrogen in the presence of methanol containing 5% (v/v) acetic acid. After adding 20 µl of pyridine and 200 µl of acetic anhydride, peracetylation was carried out at 100 °C for 2 h. After evaporation under a stream of nitrogen, the partially methylated alditol acetates (PMAAs) were dissolved in chloroform, and the chloroform phase was washed 10 times with water. This PMAA-containing phase was finally dried under a stream of nitrogen, and the PMAAs were dissolved in methanol before GC-MS analysis. GC separation of PMAAs was performed using a Carbo Erba GC 8000 gas chromatograph fitted with a 25-m x 0.32-mm CP-Sil5 CB low bleed capillary column, 0.25-µm film phase (Chrompack France, Les Ullis, France). The temperature of the Ross injector was 260 °C. Samples were analyzed using a temperature program starting by a gradient of 2 °C/min from 130 to 180 °C after 2 min at 130 °C followed by a gradient of 4 °C/min until 240 °C. The column was coupled to a Finnigan Automass II mass spectrometer. PMAA analyses were performed in the electron impact mode using an ionization energy of 70 eV. Quantification of the various PMAA derivatives was carried out using total ion current of the MS detector in the positive ion mode.

Affinity Chromatography Using Concanavalin A-linked Agarose—
All purification steps were carried out at 4 °C. Purified tachyzoites of RH strain (10⁹ parasites) were lysed with 1 ml of buffer A containing 10 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM CaCl₂, 1 mM MnCl₂, 1% Triton X-100, and protease inhibitor mixture (Roche Diagnostics). After 1 h at 4 °C, insoluble material was removed by centrifugation at 10,000 x g for 15 min. The supernatant was incubated with concanavalin A (ConA)-agarose beads (from Canavalin ensiformis, jack bean, Type VA, Sigma) for 3 h and washed five times with buffer A without protease inhibitors. After a final wash with 62.5 mM Tris·HCl (pH 6.8), the bound proteins were eluted with 2 volumes of 2x SDS sample buffer and boiled at 95 °C for 5 min or with 0.5 M -methyl-D-mannoside. Competition assays were also performed using ConA beads incubated with protein extract in the presence of 0.5 M -methyl-D-mannoside. Large scale purification of glycoproteins for proteomics analyses was also performed according to the protocol described above except that protein extracts from 10¹⁰ tachyzoites were used. The eluted samples were analyzed by SDS-PAGE followed by Coomassie Blue staining.

Protein Identification by Mass Spectrometry—
In-gel digestion was performed with an automated protein digestion system, MassPREP station (Waters, Milford, MA). The gel plugs were washed twice with 50 µl of 25 mM NH₄HCO₃ and 50 µl of ACN. The cysteine residues were reduced by 50 µl of 10 mM DTT at 57 °C and alkylated by 50 µl of 55 mM iodoacetamide at room temperature. After dehydration of the gel bands with ACN, the proteins were digested overnight in gel with 50 µl of 12.5 ng/µl modified pig trypsin (Promega, Madison, WI) in 25 mM NH₄HCO₃ at room temperature. The peptides released were extracted twice with 60% ACN in 5% formic acid and 100% ACN followed by excess ACN removal. Tryptic digests were analyzed by nano-LC-MS/MS using an Agilent 1100 series HPLC-Chip/MS system with a 43-mm reversed phase C₁₈ column (Agilent Technologies, Palo Alto, CA) coupled to an HCTultra ion trap (Bruker Daltonics, Bremen, Germany). The voltage applied to the capillary cap was optimized to –1750 V. For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The three most abundant peptides and preferentially doubly charged ions were selected on each MS spectrum for further isolation and fragmentation. The MS/MS scanning was performed in the ultrascan resolution mode at a scan rate of 26.000 m/z per second. A total of six scans were averaged to obtain an MS/MS spectrum. The complete system was fully controlled by ChemStation (Agilent Technologies) and EsquireControl (Bruker Daltonics) softwares.

Mass data collected during nano-LC-MS/MS analysis were processed, converted into *.mgf files, and searched first against the National Center for Biotechnology Information non-redundant (NCBInr) database using a local Mascot^TM server (Matrix Science, London, UK). In addition, an in-house genome database was constructed using the complete genome sequence, downloaded as a single text file from The Institute for Genomic Research, and segmented into regular segments of well chosen length (7500 bp) with a determined overlap length (2500 bp). This nucleic acid database was imported into the local Mascot server, and the segments were translated in all possible reading frames.² Once expressed coding regions were identified thanks to the matching peptides on the genome sequence, the protein functions were determined by MS-BLAST sequence similarity searches. Searches were also performed using the annotated genome sequence available at ToxoDB. In both cases, protein identification was validated according to the established guidelines for proteomics data publication (25–27).

N-Glycosylated Protein Identification by Mass Spectrometry—
Tryptic digests were analyzed by nano-LC-MS/MS using an Agilent 1100 series HPLC-Chip/MS system with a 43-mm carbon graphite column (Agilent Technologies) coupled to an HCTplus ion trap (Bruker Daltonics). The voltage applied to the capillary cap was optimized to –1750 V. The mass spectrometer ion transmission parameters were optimized for high m/z (1000 m/z). For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The three most abundant peptides and preferentially doubly charged ions were selected on each MS spectrum for further isolation and fragmentation. The MS/MS scanning was performed in the ultrascan resolution mode at a scan rate of 26.000 m/z per second. A total of six scans were averaged to obtain an MS/MS spectrum. The complete system was fully controlled by ChemStation (Agilent Technologies) and EsquireControl (Bruker Daltonics) softwares. For each protein identified, potential N-glycosylation sites were predicted using the NetNGlyc 1.0 Server, and in silico tryptic digestion was performed to generate the tryptic peptides containing a potential N-glycosylation site in their amino acid sequence. For each analysis, typical glycopeptide diagnostic ions were extracted from MS/MS ion current, and MS/MS spectra containing diagnostic ions were manually interpreted. Results were validated taking into account the following three types of fragment ions classically generated for glycopeptides: sugar neutral losses, non-glycosylated peptide fragments, and glycan backbone internal fragments.

Western Blotting—
Glycoproteins, affinity-purified using a ConA column, resuspended in 2x SDS sample buffer and freshly isolated tachyzoites lysed with the same sample buffer were separated on SDS-polyacrylamide gels under non-reducing conditions and blotted to nitrocellulose by an electrophoretic transfer system (Bio-Rad system). Blots were blocked in TNT buffer (15 mM Tris·HCl (pH 8), 140 mM NaCl, 0.05% Tween 20) with 5% dry milk and incubated with primary antibodies (monoclonal or rabbit polyclonal antibodies) followed by goat anti-rabbit IgG or goat anti-mouse IgG conjugated to peroxidase (Sigma), and the signal was detected by chemiluminescence using the ECL kit (Amersham Biosciences). T. gondii-specific antibodies were generously provided by Drs. M. Lebrun (anti-rhoptry neck protein 2 (RON2) and anti-apical membrane antigen 1 (AMA1), D. Boothroyd (anti-AMA1), P. Bradley (anti-RON1 and anti-apicoplast), D. Soldati (anti-myosin A (MyoA), anti-MLC1, and anti-gliding-associated protein 45 (GAP45) and J.-F. Dubremetz (anti-ROP2/3/4, anti-MIC, and anti-GRA).

Lectin and Immunofluorescence Localizations—
Freshly isolated and purified tachyzoites were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min on ice. For the localization of glycoproteins, fixed slides were permeabilized with 0.1% Triton X-100 prepared in buffer B containing 10 mM Tris·HCl (pH 7.5), 0.15 M NaCl, 1 mM CaCl₂, 1 MnCl₂ at room temperature. Free PFA was blocked by 0.1 M glycine in buffer C (similar to buffer B except that the Triton X-100 was at 0.01%) for 10 min at room temperature. Then parasites were incubated either with 10 µg/ml biotinylated concanavalin A (Type IV, Sigma) or 100 µg/ml FITC-coupled Pisum sativum agglutinin (PSA; Sigma) diluted in buffer C for 30 min at 37 °C. After three washes with buffer C, the ConA-treated slides were incubated with FITC-coupled streptavidin (Sigma) for 30 min at 37 °C and washed three times. For co-localization, slides containing intracellular parasites were incubated with lectins followed by incubation with monoclonal or polyclonal antibodies in PBS with 0.01% Triton X-100. Fluorescently conjugated secondary antibodies were added and incubated for 30 min at 37 °C. After three washes, slides were mounted in ImmunO (immunofluor mounting medium, MP Biochemicals Inc.), examined, and photographed on a Zeiss confocal microscope.

In Vitro Gliding Assays—
Intracellular tachyzoites of the RH strain were treated with 5 µg/ml tunicamycin (Sigma) prepared at an initial concentration of 1 mg/ml in DMSO for 40 h at 37 °C. The untreated intracellular parasites incubated in DMSO only or tunicamycin-treated parasites were filtered as described above; chased with DMEM containing 10 mM HEPES, 1 mM EGTA; and centrifuged at 1,000 rpm for 10 min at room temperature. The parasite pellet was resuspended in medium as above, and 5 x 10⁸ tunicamycin-treated or untreated tachyzoites were inoculated into a chamber containing a slide that had previously been coated overnight with 50% fetal calf serum in PBS. The slides were then incubated with the parasites at 37 °C for 15 min, rinsed in PBS, and fixed in 4% PFA in PBS. Trails left by gliding parasites were visualized by staining with the monoclonal antibody anti-surface antigen (SAG) 1 and photographed using a Zeiss Axiophot microscope.

Parasite Attachment, Host Cell Invasion, and Growth—
To test parasite attachment and invasion, two assays were used to distinguish parasites that had attached and entered host cells from those that had entered and replicated in host cells. First, freshly purified tachyzoites of RH strain treated with tunicamycin or untreated parasites as described above were resuspended in DMEM containing 10% FCS, loaded onto slides containing confluent monolayer HFF cells, incubated at 37 °C for 10 min, then fixed with 4% PFA, and processed for immunofluorescence analysis. Second, tunicamycin-treated or untreated tachyzoites of RH strain were also loaded onto confluent monolayer HFF cells for 1 h, and intracellular growth was monitored for 24 h at 37 °C. Slides were rinsed with PBS, fixed with 4% PFA, washed with PBS, and stained with mAb anti-SAG1 followed by FITC-goat anti-mouse IgG. Attached/invaded parasites for 10 min-pulse infection or 1-h infection followed by growth for 24 h were quantified by single tachyzoites or replicated tachyzoites, and counting was performed for at least 500 host cells. Experiments were repeated three times, and data are expressed as the mean ± S.D. Third, parasite growth was alternatively quantified by using freshly isolated β-galactosidase-expressing 76K strain parasites to infect slides in 24-well plates containing HFF cells for 4 h at 37 °C. Four wells were individually treated with 5 µg/ml tunicamycin or DMSO alone. After 2 days of growth, one-fifth of each well was used to infect a new well containing monolayer HFF cells, and incubation was continued for 2 days under tunicamycin or DMSO treatment. The remaining parasites of the first cycle experiment were recovered, washed with PBS, and frozen. After the second cycle of treatment, one-fifth of each well was used to infect new monolayer HFF cells for the third cycle experiment. The parasite pellets were washed with PBS and lysed with buffer containing 100 mM HEPES (pH 8.0), 1 mM MgSO₄, 5 mM DTT, 1% Triton X-100. The β-galactosidase activity was quantified using a chlorophenol red-β-D-galactopyranoside (Roche Applied Science) colorimetric assay (23). Three independent experiments were performed, and data are expressed as mean ± S.D.

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
MALDI-TOF-MS of N-Glycans Released from Detergent Extracts—
To gain more insight into protein N-glycosylation by the parasite, we embarked on the determination of the major structures of N-glycans synthesized by T. gondii. Experimental strategies based on derivatization, chemical hydrolysis, exoglycosidase digestions, MALDI-TOF-MS, and GC-MS were used to characterize the glycans of Toxoplasma total protein extracts. Reduced and carboxamidomethylated Toxoplasma products were first digested with trypsin, and glycans were released from the resulting peptide/glycopeptide mixture by digestion with PNGase F. This enzyme is capable of releasing all known N-linked oligosaccharides except those with fucose attached to the 3-position of the Asn-linked GlcNAc residue. Such PNGase F-resistant oligosaccharides have been found to be sensitive to PNGase A. PNGase F-released glycans were separated from peptides and were analyzed by MALDI-TOF-MS after permethylation and purification on a Sep-Pak C₁₈ (Fig. 1). The permethylation derivatization of glycans increases the sensitivity of the detection of molecular ions. The peptide fraction was further digested with PNGase A, and the putative glycans released were analyzed by MALDI-TOF-MS after permethylation and purification on a Sep-Pak C₁₈. Fig. 1A shows the MALDI-TOF mass spectrum profile of the permethylated PNGase F-released glycans from Toxoplasma detergent extracts. Permethylated glycans give [M + Na]⁺ species as the major ion. Three major molecular ions that correspond to Hex₆(HexNAc)₂ + Na⁺ (m/z 1784), Hex₇(HexNAc)₂ + Na⁺ (m/z 1988), and Hex₈(HexNAc)₂ + Na⁺ (m/z 2192) were observed. Four minor molecular ions corresponding to Hex₃(HexNAc)₂ + Na⁺ (m/z 1171), Hex₄(HexNAc)₂ + Na⁺ (m/z 1375), Hex₅(HexNAc)₂ + Na⁺ (m/z 1580), and Hex₉(HexNAc)₂ + Na⁺ (m/z 2396) were also identified. Fig. 1, insets B and C, show enlargements of a spectrum that revealed these four minor species. Based on the MALDI-TOF-MS data and currently accepted models of N-glycan biosynthesis (10, 11), we conclude that the major N-glycans of T. gondii have compositions consistent with high mannose type structures (Hex_5–8(HexNAc)₂). Very minor N-glycans have compositions consistent with paucimannose type structures (Hex_3–4(HexNAc)₂). After PNGase F digestion, the peptide fraction was further digested with PNGase A. Putative PNGase A-released N-glycans were permethylated, purified on a Sep-Pak C₁₈ cartridge, and analyzed by MALDI-TOF-MS. No signals corresponding to N-glycans were observed (data not shown). Thus, the PNGase F digestion was complete, and we could find no evidence that N-linked glycans with fucose attached to the 3-position of the Asn-linked GlcNAc were an important component of Toxoplasma. In addition, also no modification of terminal galactose and sialic acid was identified, suggesting that T. gondii may lack a part of the endoplasmic reticulum and Golgi trimming and maturation pathways that are highly conserved in other eukaryotes (10, 11).

View larger version (18K): [in this window] [in a new window]   FIG. 1. Positive MALDI-TOF mass spectrum of permethylated N-glycans from T. gondii glycoproteins. A, the N-glycans were released from tryptic glycopeptides by digestion with PNGase F, separated from peptides by Sep-Pak purification, and permethylated. The three major molecular ions identified are Hex6(HexNAc)2 + Na+ (m/z 1784), Hex7(HexNAc)2 + Na+ (m/z 1988), and Hex8(HexNAc)2 + Na+ (m/z 2192), which correspond to Man6(GlcNAc)2, Man7(GlcNAc)2, and Man8(GlcNAc)2, respectively. *, contaminants from the MALDI-TOF DHB solution used to resuspend the solution. B and C, four minor molecular ions, Hex3(HexNAc)2 + Na+ (m/z 1171), Hex4(HexNAc)2 + Na+ (m/z 1375), Hex5(HexNAc)2 + Na+ (m/z 1580), and Hex9(HexNAc)2 + Na+ (m/z 2396) corresponding, respectively, to Man3(GlcNAc)2, Man4(GlcNAc)2, Man5(GlcNAc)2, and Man9(GlcNAc)2 were also detected and are depicted in the insets B and C to view these minor peaks relative to the three major peaks. Ions that appear at m/z values slightly higher than those assigned to Hex5–8(HexNAc)2 in the spectrum correspond to species 30 Da larger than the fully methylated carbohydrate molecules (51). D, our proposed structures of N-glycans isolated from total extract glycoproteins of T. gondii. These fine N-glycan structures were determined twice in two independent productions of purified T. gondii materials by combining several enzymatic digestions, MALDI-TOF-MS, and GC-MS analyses. , N-acetylglucosamine; •, mannose.

View larger version (49K): [in this window] [in a new window]   FIG. 2. Confocal microscopy analysis of lectin-staining formaldehyde-fixed T. gondii. Extracellular tachyzoites were purified from infected human foreskin fibroblasts, and bradyzoites were isolated from brains of mice chronically infected by T. gondii. ConA that is specific to -D-mannose and -D-glucose and PSA that exclusively recognizes -D-mannose were used. The ConA signal was amplified by the FITC-conjugated streptavidin, whereas PSA was directly conjugated to FITC. Panels 1, phase-contrast. Panels 2, propidium iodide staining of nucleus (red). Note that mRNAs are also labeled by this dye, and the yellow signals around the nucleus (panel 4, PSA) indicate co-localization between the lectin signal and the endoplasmic reticulum-containing mannose-rich glycoproteins and lipid precursors and the propidium iodide staining. Panels 3, lectins. Panels 4, merged signal between propidium iodide staining and lectin labels.

View larger version (48K): [in this window] [in a new window]   FIG. 3. Colocalization of ConA fluorescence with different T. gondii organelle-specific markers. Double fluorescence assays were performed using ConA revealed with FITC-conjugated streptavidin followed by incubation with monoclonal or polyclonal antibodies specific for protein markers of rhoptries (Ron1), microneme (Mic1), dense granules (Gra1), apicoplast (Api), actin (Act), MyoA, gliding-associated protein (Gap45), and myosin light chain 1 (MLC). The signals corresponding to monoclonal and polyclonal antibodies were revealed by a secondary goat Alexa Fluor 594-conjugated antibody (red). Panel 1, monoclonal or polyclonal antibody; panel 2, ConA; panel 3, merged signals between antibodies and ConA. The images were examined and photographed on a Zeiss confocal microscope.

View larger version (59K): [in this window] [in a new window]   FIG. 4. Purification of T. gondii N-linked glycoproteins using a ConA column. A, the pilot experiment performed with detergent extract from 109 tachyzoites. Lane 1, total detergent extract used; lanes 2–4, efficacy of purity of glycoprotein-binding ConA column checked by taking three aliquots corresponding, respectively, to the first, third, and fifth/last washes; lane 5, eluate of ConA-binding glycoproteins using 2x SDS sample buffer; lane 6, competition assay performed by incubating the detergent protein extract in the presence of 0.5 M -methylmannoside inhibitor before elution as described in lane 5; lane 7, elution of ConA-binding glycoproteins with 0.5 M -methylmannoside; lane 8, elution of ConA beads alone by 2x SDS sample buffer. B, large scale purification of ConA-binding glycoproteins using detergent extract from 1010 tachyzoites. After elution with 2x SDS sample buffer and SDS-PAGE, 13 bands were excised and processed for proteomics analyses. The contaminated proteins corresponding to different ConA species eluted from the beads are indicated by arrows. MM, molecular mass in kilodaltons.

Deciphering the Precise Nature of N-Glycopeptides by Structural Analysis of Both N-Glycans and Peptides—
We established the structures of N-linked glycopeptides of a major T. gondii glycoprotein, GAP50, identified by proteomics analysis by determining the nature of both N-linked glycans and trypsin-generated peptides. Two MS/MS spectra show the presence of typical diagnostic ions, MH⁺ m/z 366.14 and 528.19 corresponding to glycan backbone internal fragments. Both are found in bands 1 and 3 (MS/MS spectra of doubly protonated parent ions at m/z 1248.10 and 1329.50). The MS/MS spectrum of the doubly protonated parent ion at m/z 1248.10 shows fragment ions, which correspond to sugar neutral losses on the glycopeptide. Therefore, one ion of mass MH⁺ m/z 953.47 was observed, corresponding to the peptide NYTSEALR that is a potential glycosylated tryptic peptide from the membrane anchor myosin XIV (GAP50), and was only identified in these two bands (Fig. 5). Combining the two data above allowed us to determine the glycan structure Man₇(GlcNAc)₂ at position Asn¹³⁶ of GAP50 (Fig. 5A). In addition, the MS/MS spectrum of the doubly protonated parent ion at m/z 1329.50 is clearly similar to the MS/MS spectrum of parent ion m/z 1248.10 (same fragment ions and presence of the ion of mass MH⁺ m/z 953.47). Only the doubly protonated parent ion masses show a mass difference of 162 Da corresponding to the mass of a mannose. These two data allowed us to define the glycan structure Man₈(GlcNAc)₂ at the same position, Asn¹³⁶, of the membrane anchor myosin XIV (GAP50) (Fig. 5B). Taken together, the data confirmed the presence of the following two high mannose structures, Man₇(GlcNAc)₂ or Man₈(GlcNAc)₂, on the peptide NYTSEALR with NYT as a consensus site in the membrane anchor myosin XIV. As mentioned, TgGAP50 is an integral membrane protein that anchors T. gondii myosin A (TgMyoA), its associated light chain (TgMLC1), actin, and TgGAP45 to the inner membrane complex, and these constitute the glideosome that is involved in motility required for host cell invasion (19). TgGAP50, which contains three Asn-X-(Ser/Thr) consensus sites (Fig. 5C, underlined and bold), has been reported previously as a genuine N-linked glycoprotein that can be cleaved off by PNGase F, and its mobility shift compared with PNGase F-untreated control suggested that the three consensus sites are likely occupied by N-glycans (19). We demonstrate here that the NYT sequon within the NYTSEALR peptide (boxed in TgGAP50 protein sequence in Fig. 5C) bears alternatively Man₈(GlcNAc)₂ and Man₇(GlcNAc)₂. The glycoprotein TgGAP50 represents a genuine pellicle component involved in the parasite's motile apparatus. However, the implication of N-glycosylation, not only in gliding motility but also in the biogenesis of the parasite's late secretory organelles, was further investigated in greater detail using both cell biology and pharmacological approaches.

View larger version (29K): [in this window] [in a new window]   FIG. 5. Identification of peptide and N-glycans from the membrane anchor myosin XIV also named GAP50. A, the MS/MS spectrum from parent ion at m/z = 1248.10 corresponding to the peptide NYTSEALR with the N-glycosylation Man7(GlcNAc)2 in position Asn136. B, the MS/MS spectrum from parent ion at m/z = 1329.50 corresponding to the peptide NYTSEALR with the glycosylation Man8(GlcNAc)2 in position Asn136. C, the position of the peptide NYTSEALR with the consensus NYT alternatively modified by Man7(GlcNAc)2 and Man8(GlcNAc)2 is indicated (boxed) in the GAP50 protein sequence. The two additional N-glycosylation sequons are underlined and bold.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Inhibitory effect of tunicamycin on T. gondii growth. A, the delayed effect of tunicamycin on T. gondii tachyzoites growth quantified using stable β-galactosidase-expressing 76K strain parasites. Four wells of 24-well plates containing HFF cells infected by tachyzoites were treated with 5 µg/ml tunicamycin or with DMSO alone. After 2 days of infection, one-fifth of each well of the first cycle treated or untreated parasites was used to infect new wells under tunicamycin or DMSO treatment. The remaining parasites of this first cycle experiment were recovered, washed with PBS, and frozen. After the second cycle of treatment, one-fifth of each well was used again to infect new monolayer HFF cells for the third cycle experiment. All parasite pellets were lysed and tested for β-galactosidase activity. Three independent experiments were performed, and data are expressed as mean ± S.D. B, comparison of the growth rate of tunicamycin-treated and untreated tachyzoites. Tachyzoites treated with tunicamycin or with DMSO alone during the first cycle of infection as described were loaded onto new monolayer of HFF cells and grown for 24 h in the absence of drug. The percent distribution of vacuole size (number of parasites per vacuole) was counted for 100 vacuoles in three independent experiments, and data are expressed as mean ± S.D.

View larger version (60K): [in this window] [in a new window]   FIG. 7. Tunicamycin alters the morphogenesis of T. gondii organelles. Tachyzoites treated with tunicamycin or with DMSO alone for 48 h during the first cycle of infection were released and loaded on new monolayer HFF cells and grown for 24 h without drug. The morphology of different organelles verified by markers such as the monoclonal antibody anti-surface protein SAG1, anti-dense granule protein GRA1, anti-MIC1, anti-rhoptries ROP2/3/4, anti-MLC, and anti-actin (ACT) were compared between tunicamycin-treated and the control DMSO-treated T. gondii tachyzoites. Note the pronounced effect of tunicamycin on the morphogenesis of inner membrane complex.

Inhibition of T. gondii N-Glycosylation by Tunicamycin—
At the molecular level, we also demonstrated that binding of T. gondii glycoproteins to ConA was affected when protein extracts were derived from tunicamycin-treated tachyzoites prior to lectin affinity purification. As shown in Fig. 8A, several N-glycosylated proteins ranging from 45 to >100 kDa that specifically bound to ConA, as reported in Fig. 4A, were no longer detected (see Fig. 8A, small arrows). Western blots in Fig. 8B further validated the absence of TgMyoA from the tunicamycin-treated material eluted from ConA (lane 4). The polyclonal antibodies specific to myosin light chain (MLC) cross-reacted with a novel T. gondii glycoprotein of 85 kDa that displayed a difference in electrophoretic mobility between tunicamycin-treated and untreated tachyzoites, respectively (Fig. 8C, lanes 1 and 2). As expected, when deglycosylated the 85-kDa protein was unable to bind ConA (lane 4). The precise nature of this novel 85-kDa glycoprotein remains to be determined. In addition, we demonstrated that the rhoptry neck protein RON1 was not capable of binding ConA when tunicamycin-treated (lane 4) compared with untreated lysate (lane 3). Because of the lower amount of rhoptry neck protein 2 detected by Western blots combined with our identification of only one peptide isolated by proteomics approaches, it was not surprising that we were unable to validate the N-glycosylated status of RON2 using total extract of tunicamycin-treated extracts (data not shown). In contrast, we were able to show that actin (Fig. 8E), ROP2/3/4 (Fig. 8F), and probably other cytoplasmic proteins can bind to ConA despite tunicamycin treatment, suggesting that these abundant proteins interact nonspecifically with the lectin. Nevertheless our proteomics data together with the direct evidence of the N-glycan structures Man₇(GlcNAc)₂ and Man₈(GlcNAc)₂ at position Asn¹³⁶ of the peptide NYTSEALR of GAP50 and the lack of ConA binding by TgMyoA after tunicamycin treatment also suggest that several crucial proteins involved in the parasite's motility and host-parasite interactions are likely N-glycosylated.

View larger version (58K): [in this window] [in a new window]   FIG. 8. Effect of tunicamycin on ConA binding and identification of novel N-glycoproteins of T. gondii. A, total detergent extract from the same number of tunicamycin-treated or untreated control (DMSO only) tachyzoites was incubated with ConA beads. After elution, the samples were analyzed by SDS-PAGE (7.5% polyacrylamide gel) followed by Coomassie Blue staining. The absence of glycoprotein binding to ConA is indicated by arrows. B–F, further verification of the tunicamycin effect on ConA binding by TgMyoA (B), TgMLC (C), TgRON1 (D), actin (TgACT; E), and TgROP2/3/4 (F) using lectin purification followed by Western blots. Lane 1, total detergent extract from the control tachyzoites; lane 2, total detergent extract from tunicamycin-treated tachyzoites; lane 3, presence of the parasite protein from the control tachyzoites detected in the ConA-binding material; lane 4, absence or presence of parasite protein from the material eluted from ConA beads when a lysate of tunicamycin-treated tachyzoites was used. Note the identification of a novel N-glycoprotein (TgGPX) that cross-reacted to anti-TgMLC antibodies. This TgGPX displayed an electrophoretic shift when the total lysate was prepared from tunicamycin-treated tachyzoites (C, lane 3) and could not bind to ConA (C, lane 4) confirming that TgGPX is N-glycosylated. The nature of this novel N-glycoprotein remains to be determined.

To establish whether tunicamycin was blocking the attachment or invasion of host cells, we monitored host cell reinfection after the first cycle of intracellular parasite treatment. To do this, we examined and quantified the number of parasites attached to or invading host cells. Invasion was scored during a 1-h pulse infection followed by 24 h of intracellular growth. Fig. 9B shows an approximately 70% decrease in tachyzoite numbers within host cells, suggesting that tunicamycin treatment impaired host cell invasion.

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we have provided new evidence that T. gondii contains numerous N-glycosylated proteins that unexpectedly are components of the glideosome, moving junction, and other glycoproteins involved in host cell-parasite interactions. The cell wall or pellicle of apicomplexan parasites like T. gondii consists of the plasma membrane and the closely associated, flattened cisternae of the inner membrane complex. Both actin and myosin A homologues have been localized to the space between the plasma membrane and the inner membrane, and this glideosome is a key player in motility and host cell entry by T. gondii (see schematic model in Fig. 9C). Another essential element represents the moving junction, a structure built at the interface between the host cell and the parasite during its active entry into any kind of mammalian cell. The moving junction is a circumferential zone that forms at the apical tip of the parasite, moves backward, and pinches the vacuole from the host cell membrane (34, 36). Several components of the moving junction are secreted from late secretory organelles such as rhoptries (RON2) and micronemes (AMA1) (depicted in Fig. 9C). It is clear that the results presented here identified N-linked glycosylation on several key constituents involved in host-parasite interactions. The major Toxoplasma N-linked glycans released from total detergent extract proteins by PNGase F are oligomannosidic (Man_5–8(GlcNAc)₂) and paucimannosidic structures (Man_3–4(GlcNAc)₂), sugars that are rarely present on mature N-glycoproteins of higher eukaryotes. The three major N-glycans linked to proteins correspond to Man₆(GlcNAc)₂, Man₇(GlcNAc)₂, and Man₈(GlcNAc)₂, respectively. In addition, four other minor oligosaccharides, Man₃(GlcNAc)₂, Man₄(GlcNAc)₂, Man₅(GlcNAc)₂, and an extremely limited amount of Man₉(GlcNAc)₂, were also found. It has been shown that other eukaryotes can transfer structures other than the largest lipid-linked oligosaccharide precursor, Dol-PP-Glc₃Man₉(GlcNAc)₂ (12–14, 20). Thus, we postulate that the paucimannosidic N-linked glycan structures found on T. gondii glycoproteins could point to the transfer of Man₅(GlcNAc)₂ and its subsequent glucosylation as reported previously in Trypanosoma brucei (14). The genes encoding numerous endoplasmic reticulum-located glycosyltransferases, named ALG (asparagine-linked glycosylation), required for the biosynthesis of the lipid (dolichylpyrophosphate)-linked glycosyl donors and oligosaccharyltransferases needed for the N-glycan transfer to proteins, can be readily identified in the T. gondii genome (ToxoDB) using detailed bioinformatics and gene predictions.³ All of these genes involved in both synthesis of lipid precursor and transfer of N-glycan structures are transcribed in T. gondii tachyzoites and bradyzoites.⁴ In contrast, our biochemical analyses failed to detect sialylated, galactosyl, or fucosylated structures consistent with either the likely absence or the limited presence of complex glycan types containing sialic acid, galactose, and fucose residues in T. gondii. This also indicates that a part of the endoplasmic reticulum and Golgi trimming and maturation pathways that are highly conserved in other eukaryotes is absent in the parasite. However, only structures of major N-glycans were determined when the total detergent glycoprotein extracts were analyzed. We therefore cannot rule out the presence of other minor modifications and branching monosaccharides that escaped detection in this study.

We identified a number of known proteins that have not been suspected until now to be N-glycosylated. Conversely N-glycosylation is considered as a rare post-translational modification in apicomplexan parasites because most investigations have focused attention on surface and other antigens that are important for host cell-parasite interactions and future vaccine development. Our data confirmed that the key glideosome membrane anchor myosin XIV (also named GAP50), known to be involved in both parasite motility and host cell entry, contains N-glycosylated structures. From the mixed glycopeptide populations, we were fortunate to identify the trypsin-generated peptide NYTSRALR of the membrane anchor myosin XIV (GAP50) that is truly modified by two N-glycan structures at the same position, Asn¹³⁶. We report for the first time in T. gondii the fine N-glycan structures that are present on the GAP50 sequon NYT that is alternatively modified by Man₈(GlcNAc)₂ or Man₇(GlcNAc)₂. However, it was somewhat unexpected to find that TgMyoA, another component of the glideosome, was also capable of specifically binding to ConA. We have shown that TgMyoA failed to bind to ConA when the parasites were treated with the N-glycosylation poison tunicamycin. Neither the N-terminal signal peptide nor a transmembrane domain is present in TgMyoA. These data therefore suggest that an N-glycosylated binding partner might bring TgMyoA into interaction with ConA. As stated, no obvious domains of TgMyoA presently explain how it might be translocated across the ER membrane to the lumen where the addition of N-glycans is expected to occur co-translationally (10). However, almost nothing is known about ER translocation machinery in T. gondii, and previous work on Saccharomyces cerevisiae has described that carboxypeptidase Y and an acid phosphatase can be translocated to the ER, enter the secretory pathway, and be N-glycosylated without having their N-terminal signal sequences (44, 45). In addition, it has been shown that cytoplasmic and nuclear proteins are N-glycosylated, suggesting the existence of post-translational N-glycosylation mechanisms in higher eukaryotes (46, 47). It remains to be determined whether such a non-conventional N-glycosylated biosynthetic pathway operates in T. gondii. To our knowledge, almost nothing has been reported on N-glycosylation of myosins and components involved in the motile apparatus in other eukaryotic cells. Gliding is a unique form of apicomplexan parasite motility that manifests as either circular spirals or a series of helical turns revolving around the long axis of the body of the parasite (30–32). It has been suggested previously that the soluble protoglideosome and membrane-associated TgGAP50 are transported separately and are assembled into the glideosome in the inner membrane complex of mature parasites (19). The N-linked glycosylation of glideosome components such as GAP50 suggests that this post-translational modification may be involved in their intracellular trafficking mechanisms or in their transport to the parasite's space where the glideosome are assembled. It is also interesting to note that two key components of the moving junction (RON2 and AMA1) required for host cell invasion (Fig. 9C) are also present in material purified by ConA. These moving junction proteins also contain putative N-glycosylation sites. In addition, we showed that another rhoptry neck protein, RON1, with presently unknown function may be N-glycosylated. It should be mentioned that RON1 displayed the strongest fluorescence that co-localized with the ConA signal, and its deglycosylation by tunicamycin prevented lectin binding. However, we cannot rule out that RON1 may indirectly bind to ConA via other N-glycan-bearing RONs. We were not able to confirm that RON2 and AMA1 are N-glycosylated using Western blots because of the limited amount of protein detected after lectin purification. Their glycopeptides modified by N-glycan structures were also not identified using MALDI-MS/MS analyses because only a few peptides (only one peptide from RON2) from these proteins were obtained. Further biochemical analyses including purification of higher amounts of RON2 and AMA1 by affinity purification with their respective specific antibodies followed by direct determination of N-glycan structures using mass spectrometry are required. Many other proteins have been isolated by ConA affinity purification and identified by proteomics analyses. Among these are also proteins that do not have any predicted N-glycosylation sites (supplemental data), suggesting that these proteins may interact with other genuine N-glycosylated proteins. This may be the case for MIC1 (supplemental data) that has been reported previously as a parasite lectin (48) that might be retained on ConA by interacting with N-glycan structures carried by other N-glycoproteins that specifically bind to ConA. Even if some proteins without N-glycosylated sites (supplemental data) may behave like MIC1, we cannot exclude that these components may also interact with ConA nonspecifically as further demonstrated during this study for actin and other rhoptry proteins such as ROP2/3/4.

It is intriguing that unlike all other eukaryotic cells, T. gondii tachyzoites treated with tunicamycin at conventional inhibitory concentrations did not display any obvious growth defect within its host cells during its first cycle of intracellular development that lasts 48 h. The growth defect appeared only during the second cycle of reinvasion of host cells with a very prominent effect during the third cycle. The effect of tunicamycin took place in the absence of drug during the second and third cycle of host cell infection, suggesting that this delayed effect, or unusual kinetics of tunicamycin on T. gondii, may reflect drastic differences in host cell re-entry by tunicamycin-treated relative to untreated tachyzoites. This conclusion is supported by the results that demonstrate the ability of tunicamycin to affect gliding motility of tachyzoites released after intracellular drug treatment. We cannot exclude that host cell attachment may also be affected by tunicamycin treatment. However, the lack of N-glycosylation of several SAGs and most MICs reported by lectin purification and proteomics analyses indicates that any interference of host cell attachment by tunicamycin may be marginal. Therefore, we suspect that it is the parasite's motility and new host cell invasion that are likely impaired in tunicamycin-treated tachyzoites. It remains to be determined whether moving junction formation is also inhibited by tunicamycin and absence of N-glycosylation. However, the delayed effect on intracellular replication of tunicamycin-treated parasites that have entered new host cells is highly reminiscent of the delayed death phenomenon seen with apicoplast inhibitors (49). It appears that ConA fluorescence co-localizes with the signal of apicoplast-specific monoclonal antibodies. Because it has been described that some apicoplast proteins transit through the ER (29, 50), it is likely that this plastid-like organelle may also contain N-glycosylated proteins and that the delayed effects seen with tunicamycin might be partially attributable to inhibition of apicoplast functions.

In summary, the current study illuminates a diverse repertoire of N-glycosylated proteins that contribute to parasite survival and pathogenesis during mammalian host cell infection. Novel parasite-specific N-glycoproteins identified here can significantly expand the number of potential targets for therapeutic intervention. We anticipate that ongoing in-depth exploration of biological functions of the identified N-linked glycans carried by components involved in gliding motility, moving junction, protein secretion, and protein-protein interaction would further unravel the invasive and pathogenic mechanisms developed by T. gondii and other apicomplexan parasites during infection.

	ACKNOWLEDGMENTS

 
We thank Drs. Dominique Soldati, Maryse Lebrun, Jean-François Dubremetz, Peter Bradley, and John Boothroyd for generous gifts of antibodies and Drs. Steven Ball and Gordon Langsley for a critical review of the manuscript. We acknowledge the Toxoplasma Genome Sequencing Consortium for making available the genome database. Preliminary genomic and/or cDNA sequence data were accessible via ToxoDB. The mass spectrometry facility used at the University of Lille was funded by the European Community (FEDER), the Région Nord-Pas de Calais (France), the CNRS, and the Université des Sciences et Technologies de Lille.

	FOOTNOTES

 
Received, August 17, 2007, and in revised form, January 9, 2008.

Published, MCP Papers in Press, January 9, 2008, DOI 10.1074/mcp.M700391-MCP200

¹ The abbreviations used are: Dol, dolichol; ER, endoplasmic reticulum; PP, pyrophosphate; HFF, human foreskin fibroblast; DMEM, Dulbecco's modified Eagle's medium; ConA, concanavalin A; PSA, P. sativum agglutinin; GC, gas chromatography; SAG, surface antigen; MIC, micronemal protein; GRA, dense granule protein; ROP, rhoptry protein; PNGase, peptide-N⁴-(N-acetyl-β-glucosaminyl)asparagine amidase; PMAA, partially methylated alditol acetate; BLAST, Basic Local Alignment Search Tool; PFA, paraformaldehyde; mAb, monoclonal antibody; Hex, hexose; HexNAc, N-acetylhexosamine; Tg, T. gondii; MyoA, myosin A; MLC, myosin light chain; GAP, gliding-associated protein; RON, rhoptry neck protein; AMA, apical membrane antigen.

² S. Fauquenoy, W. Morelle, A. Hovasse, A. Bednarczyk, C. Slomianny, C. Schaeffer, A. Van Dorsselaer, and S. Tomavo, unpublished data.

³ F. Dzierszinski, D. Ross, and S. Tomavo, unpublished data.

⁴ S. Fauquenoy and S. Tomavo, unpublished data.

^* This work was supported by the CNRS and INSERM. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

^|| To whom correspondence should be addressed: Equipe de Parasitologie Moléculaire, Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, Bâtiment C9, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq, France. Tel.: 33-3-20-43-69-41; Fax: 33-3-20-33-65-55; E-mail: Stan.Tomavo{at}univ-lille1.fr

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