Glycobiology Advance Access originally published online on September 15, 2006
Glycobiology 2007 17(1):56-67; doi:10.1093/glycob/cwl050
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Major O-glycans in the spores of two microsporidian parasites are represented by unbranched manno-oligosaccharides containing 
-1,2 linkages
2 Equipe Parasitologie Moléculaire et Cellulaire, LBP, CNRS UMR6023, Université Blaise Pascal, 24 Avenue des Landais, 63177 Aubière Cedex, France
3 Laboratoire de Glycobiologie Structurale et Fonctionnelle, CNRS UMR8576, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq Cedex, France
1 To whom correspondence should be addressed; Tel: +33 4 73 40 74 57; Fax: +33 4 73 40 76 70; e-mail: christian.vivares{at}univ-bpclermont.fr
Received on July 12, 2006; revised on September 8, 2006; accepted on September 11, 2006
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Abstract |
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Protein glycosylation in microsporidia, a fungi-related group comprising exclusively obligate intracellular parasitic species, is still poorly documented. Here, we have studied glycoconjugate localization and glycan structures in spores of Encephalitozoon cuniculi and Antonospora locustae, two distantly related microsporidians invading mammalian and insect hosts, respectively. The polar sac-anchoring disc complex or polar cap, an apical element of the sporal invasion apparatus, was strongly periodic acid-thiocarbohydrazide-Ag proteinate-positive. Mannose-binding lectins reacted with the polar cap and recognized several bands (from 20 to 160 kDa) on blots of E. cuniculi protein extracts. Physicochemical analyses provided the first determination of major glycostructures in microsporidia. O-linked glycans were demonstrated to be linear manno-oligosaccharides containing up to eight
1, 2-linked mannose residues, thus resembling those reported in some fungi such as Candida albicans. No N-linked glycans were detected. The data are in accordance with gene-based prediction of a minimal O-mannosylation pathway. Further identification of individual mannoproteins should help in the understanding of spore germination mechanism and host–microsporidia interactions. Key words: glycan analysis / microsporidia / O-mannosylation / polar cap / ultracytochemistry
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Introduction |
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The identification of glycans and glycoconjugates in eukaryotic parasites is of interest for the knowledge of host–parasite interactions and pathogenic determinants (Guha-Niyogi et al. 2001
). It is worth noting that the capacity of classical O-linked and N-linked glycosylations can be highly reduced in some species exhibiting an obligate intracellular lifestyle. This is the case of the apicomplexan parasite Plasmodium falciparum (malaria agent) in which the most abundant glycoconjugate structures are glycosylphosphatidylinositol (GPI) anchors (Gowda et al. 1997). Bioinformatic analysis of the P. falciparum genome sequence revealed only four potential enzymes of the N-glycosylation pathway and no enzyme characteristic of the synthesis of complex O-linked glycans (Aravind et al. 2003).
Relatively little is known about carbohydrate diversity and glycosylation processes in microsporidia, an assemblage of over 1200 unicellular eukaryotic species that are all obligate intracellular parasites. These organisms are viewed as highly derived fungi having undergone rapid reductive evolution (Keeling 2003; Thomarat et al. 2004). Several species are human pathogens and may cause severe diseases in immune-deficient patients (Weiss 2001). Their development inside host cells comprises a proliferation phase (merogony) followed by a differentiation phase (sporogony) producing small spores that can be released in the environment. Surrounded by a resistant cell wall, the microsporidian spore contains a very long coiled organelle (polar tube) that plays an essential role in the onset of cell invasion. Indeed, the polar tube can be quickly extruded at the apical pole of the spore in order to inject the sporoplasm into a new host cell (Xu and Weiss 2005).
Chitin appears as the unique microsporidial polysaccharide and is associated with the thick inner layer of the spore wall, named the endospore (Bigliardi et al. 1996; Vavra and Larson 1999). Glycoconjugates should be abundant in a periodic acid-Schiff (PAS)-positive apical region of the spore, called the "polar sac-anchoring disc complex" or "polar cap", that comprises a dome-shaped vesicular structure closely associated with a discoid element capping the anterior end of the polar tube (Vavra and Larson 1999). Current biochemical information about microsporidial glycoproteins mainly derives from lectin-binding experiments. Blotting of Glugea plecoglossi spore extracts with eight different lectins showed that only concanavalin A (ConA) and wheat germ agglutinin (WGA) react with some protein bands (Kim et al. 1999). In Encephalitozoon intestinalis, two proteins of the outer spore wall layer (exospore) were assumed to be N-glycosylated on the only basis of their detection with specific antibodies in the fractions of infected cell lysates that were immobilized on either ConA or WGA-coated agarose beads (Hayman et al. 2001). In contrast, purified E. hellem polar tube protein (PTP) 1 should be O-mannosylated because of its lack of reactivity with an antibody to O-GlcNAc, its binding to only ConA among 10 different lectins, and elimination of this binding by NaOH treatment (Xu et al. 2004). The occurrence of O-mannosylation is more in agreement with annotation data for the genome sequence of the closely related species E. cuniculi (Katinka et al. 2001; Vivarès and Méténier 2004).
In this article, we report on the localization and structure of mannoconjugates in spores of the microsporidians E. cuniculi and Antonospora locustae. Labeling with mannose-specific lectins at the electron microscope (EM) level suggested that the polar cap is a major site for mannose-rich glycoproteins. Analyses of glycan fractions with mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques led to the first determination of oligosaccharide structures linked to microsporidial proteins.
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Results |
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Our primary choice of E. cuniculi, a monokaryotic (mononucleate) microsporidian that infects a wide range of mammals, including humans, was justified by the extreme reduction of its genome (2.9 Mbp) that was fully sequenced (Katinka et al. 2001
) and viewed as a model of "minimal genome" in eukaryotic cells (Méténier and Vivarès 2004). For comparison, analyses were also performed in the diplokaryotic (binucleate) species A. locustae that invades grasshoppers and locusts and has a 5.3-Mbp genome for which a sequencing project is in progress (http://gmod.mbl.edu/perl/site/antonospora01; Antonospora locustae Genome Project, Marine Biological Laboratory at Woods Hole, funded by NSF award number 0135272).
Reactivity of spore structures to PATAg andmannose-binding lectins
An adaptation of the PAS reaction to the EM localization of polysaccharides and glycoconjugates, known as the periodic acid-thiocarbohydrazide-Ag proteinate (PATAg) reaction (Thiéry 1967), was applied to ultrathin sections of epoxy resin-embedded E. cuniculi spores. A strongly labeled structure was a cup-shaped organelle, named the polar cap, close to the spore apex and abutting the anterior end of the polar tube (Figure 1A). Another PATAg-positive region was the lamellar polaroplast, a tightly folded membrane system that represents the precursor of the new plasma membrane surrounding the sporoplasm when transferred into a host cell. The reactivity of the spore envelope and polar tube was rather low. Interestingly, a section through a germinated spore (with extruded polar tube) revealed that the reactive material of the polar cap forms a collar-like structure around the aperture required for the passage of the polar tube (Figure 1B). Moreover, a significant labeling was associated with the surface of the extruded polar tube. The carbohydrate richness of the polar cap was also evident when applying the PATAg test to ultrathin frozen sections of spores from both A. locustae (Figure 1C) and E. cuniculi (data not shown).
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The EM localization of potential mannoconjugates in E. cuniculi cells was investigated by treatment of cryosections with two biotinylated mannose-binding lectins [ConA and Galanthus nivalis agglutinin (GNA)] followed by immunogold detection. ConA labeling was associated with polar tube coils located in the posterior region of the spore (Figure 2A). In the anterior region, both the polar tube straight part and the polar cap were ConA-reactive (Figure 2B). The main GNA-labeled spore structure was the polar cap (Figure 2C), as previously found with PATAg reaction. A few images were obtained for sporoblasts, the precursor cells in which the biogenesis of the extrusion apparatus occurs. Gold particles were clustered in a more or less central cytoplasmic area consisting of small vesicles and extending close to a tubular network characteristic of polar tube formation (Figure 2D). These vesicles possibly derive from the nondictyosomal Golgi apparatus that remains difficult to identify only on the basis of morphological criteria. The same labeling pattern was observed in A. locustae cells (data not shown).
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Detection of E. cuniculi glycoproteins on electrophoretic profiles
The spore proteome of E. cuniculi has been recently investigated using two-dimensional (2-D) electrophoresis and MS techniques, leading to sequence characterization of 177 protein spots (Brosson et al. 2006
). In a first attempt to detect glycoproteins in 2-D profiles, gels (pI range: 3–10) were either stained with Coomassie blue (Figure 3A) or blotted for further staining with a PAS-derived procedure involving sugar biotinylation (Figure 3B). A low number of reactive spots were observed. A poorly resolved acidic region was centered on 55 kDa and correlated with a group of 10 different proteins. Two of these proteins are known to be specifically secreted during sporogony: PTP1 (Delbac, Peyret, et al. 1998) and exospore protein spore wall protein (SWP) (Bohne et al. 2000). No sequence assignation was available for other glycoprotein spots, mainly seen between 30 and 35 kDa at neutral pI and in a 110-kDa basic region.
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The presence of mannose-containing glycoproteins was tested by ConA and GNA blotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of spore protein extracts. Because of their highly resistant cell wall, E. cuniculi spores were subjected to two different extraction procedures: one based on the combined action of urea and a disulfide-reducing agent [dithiothreitol (DTT)] and the other involving hot SDS treatment. Although protein patterns of the two kinds of extracts were significantly different, ConA and GNA reacted with some common bands in the two profiles: two bands at 38 and 55 kDa and least two other bands smearing between 100 and 160 kDa (Figure 4). In SDS extracts, a 65-kDa band and a doublet close to 20 kDa were also recognized by the two lectins. Ovalbumin preincubation prevented lectin binding for all bands (data not shown). Thus, in accordance with our previous ultracytochemical data, some microsporidial spore proteins should be mannosylated. Subsequent physicochemical studies were oriented toward the characterization of glycan structures.
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N-glycans are virtually absent in peptide N-glycosidase-digested fractions
Potential N- and O-linked glycans were purified from total delipidated extracts by sequential enzymatic digestions with peptide N-glycosidase F (PNGase F) and peptide N-glycosidase A (PNGase A), followed by reductive ß-elimination. Separation of released glycans from remaining glycopeptides was achieved between each step by passage through C-18 columns, which was shown to permit collection of N- and O-linked glycans from a single sample (Dell et al. 1994
). Both PNGase F and A, with different substrate specificities depending on the presence of fucose residue on chitobiose core, were used to release both possible types of N-glycans. Each of the three glycan fractions was tested for its monosaccharide composition in gas chromatography coupled to mass spectrometry (GC–MS) and MS-analyzed as permethylated derivatives. This strategy repetitively failed to conclusively demonstrate the presence of N-linked glycans in several batches of spores from both E. cuniculi and A. locustae. Composition analysis of PNGase-digested products did not show the presence of GlcNAc and Man residues in significant amounts to be attributed to N-linked glycans. Accordingly, MS analyses of these fractions in native forms, or after permethylation, did not show any signal due to potential N-glycans. We conclude that either endogenous N-glycans were absent or, if present, the quantities were too low to be detected by physicochemical means. As expected, N-glycans were found in mammalian Madin–Darby canine kidney (MDCK) cells used for our E. cuniculi cultures after application of the same purification protocol and matrix assisted-laser desorption/ionization time of flight (MALDI-TOF) MS.
O-glycans are linear mannosylated oligosaccharides
Composition analyses established that mannose is the major monosaccharide component of O-linked glycan fractions (at least 70%). In order to facilitate further analysis, O-glycan fractions were separated into two subfractions (flow through subfraction and included subfraction) by gel filtration on a Bio-Gel P2 column. These subfractions were then permethylated and subjected to MALDI-TOF and electro-spray (ES)-ion trap MS analyses. In all experiments involving permethylated products, CD3I was used as primary permethylation reagent to detect natural monosaccharides modified by methyl groups, as found in various organisms (Kocharova et al. 2000; Guerardel et al. 2001). The MALDI-TOF MS spectrum of permethylated flow through O-glycan fraction from E. cuniculi revealed a set of ions ranging from m/z 947 to m/z 1799 (Figure 5A). Their calculated compositions correspond to sodium adducts of permethylated-reduced tetra-hexosides (Hex4-ol) to octa-hexosides (Hex8-ol). The absence of [M-3+Na]+ or [M-6+Na]+ clearly demonstrated the absence of natural mono- or di-methylated hexose residues within the detected oligosaccharides. MS analysis of the gel filtration included fraction of O-glycans and showed only two [M+Na]+ ions, at m/z 520 and 733, attributed to smaller reduced di- and tri-hexosides (data not shown).
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The nature of all oligosaccharides was confirmed by collision-induced decay tandem mass spectrometry (CID-MS/MS) analysis of all [M+Na]+ ions detected by MALDI-TOF MS. For ions at m/z 1373 and 1586, fragmentation patterns of all compounds were consistent with the presence of linear-reduced oligomers of hexoses (Figure 5A and B). All fragmentation spectra were dominated by a series of Y-type (m/z at 503, 716, 929, 1143, 1356) and B-type (m/z at 466, 679, 892, 1105, 1319) ions resulting from the cleavage of glycosidic bonds from terminal nonreducing and reducing ends of oligomers, respectively. In addition, two sets of ions tentatively attributed to internal 1,3A or 2,4A fragment ions (m/z at 756, 969, 1182) and to internal 1,5A fragment ions (m/z at 651, 864, 1077, 1291) were observed as minor fragments. The nature of the 1,5A fragment ions was confirmed by MS3 analysis. As expected, MS3 fragmentation spectra were all characterized by the presence of a set of B-type [M-185-(213)n+Na]+ ions indicating the release of CH2OCD3-CH2-(CHOCD3)2-CH2O fragment and a set of Y-type ions resulting from the release of nonreducing hexose residues (data not shown). It is noteworthy that no hydroxyl group containing secondary fragment ion, indicative of the presence of branched structures, was observed in any of the CID-MS/MS spectra. Taken together, these data strongly suggested the presence of a family of mannosylated linear oligosaccharides with a degree of polymerization (DP) ranging from two to at least eight.
O-mannosyl glycans consist of -1,2 linked mannose residues
In order to identify the linkage position of Man residues within the detected oligomers, permethylated O-glycan fractions from E. cuniculi were submitted to methanolysis and acetylation prior to analysis by GC–MS. Total ion chromatogram (TIC) showed three major peaks labeled 1 to 3 (Figure 6A). By comparison of their retention times with standard molecules, peaks 1 and 2 were identified as 2,3,4,6-Me4 Man(Me) (terminal nonreducing mannose) and 3,4,6-Me3-2-Ac Man(Me) (internal 2-linked mannose), respectively. The positions of methyl groups were confirmed by electronic impact (EI)-MS (Figure 6B and C). Similarly, peak 3 was first identified as 1,3,4,5,6-Me5-2-Ac Hex-ol residue according to its fragmentation pattern (Figure 6D). Subsequent comparison of its retention time with standards produced from acetolysis and reduction of commercial Saccharomyces cerevisiae mannan definitely typified it as a 2-linked Man-ol residue. This residue results from the reduction of the reducing mannose following release of O-glycans by reductive ß-elimination. In accordance with the ES-MS/MS analysis, no di-substituted mannose residue indicative of branched structures was observed in GC–MS analysis. Differential integration of peak areas established that t-Man, 2-Man, and 2-Man-ol were present in a ratio of 0.8/2.9/1, establishing an average DP of five for the high molecular mass fraction. Similar results were obtained from low molecular mass O-glycan fraction, which differs from high molecular mass fraction only by a lower average DP of 2.5 (data not shown). It is, therefore, clear that these O-mannosyl glycans are made of linear stretches of 2-linked Man residues.
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Anomery of mannose residues was determined by 800 MHz 1H NMR analyses. One-dimensional (1-D) 1H (Figure 7A) and 2-D COSY 90 (Figure 7B) NMR spectra revealed two individual anomeric protons at
5.224 and 5.045 ppm that correlated with their respective H-2 protons at 3.98 and 4.07 ppm. It also revealed a group of at least four H-1 protons (5.286–5.312 ppm), correlating with H-2 protons between 4.08 and 4.10 ppm. Chemical shifts of anomeric protons superior to 5.04 ppm as well as J1,2 coupling constant of approximately 1.8 Hz clearly established that all residues were anomers (Cohen and Ballou 1980; Faille et al. 1992). According to published 1H NMR parameters, signals at 5.045 and 5.286–5.312 ppm were attributed to terminal nonreducing -Man residue and internal 1,2-linked Man residues, whereas signal at 5.224 was attributed to the single -Man residue substituting Man-ol in C-2 position (Hayette et al. 1992; Trimble et al. 2004). The multiplicity of signals associated with internal (1-2)Man residues results from the polydispersity of the oligomannosides, as revealed by MS analysis, that influences individual NMR parameters (Kobayashi et al. 1994).
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Altogether, the data collected from composition, linkage, MS, MS/MS, and NMR analyses established the presence in E. cuniculi of a family of O-linked glycans exclusively composed of
1,2-linked mannose residues, with a size ranging from two to at least eight residues. Identical strategy conducted in A. locustae showed very similar results. As the two species belong to two different genera that have been shown to be distantly related in rRNA phylogenetic analyses (Slamovits et al. 2004), such glycan structures might be a general feature in the microsporidian phylum. |
Discussion |
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As first observed in Stempellia spores (Vavra 1972
), the application of the PATAg procedure to ultrathin sections of E. cuniculi and A. locustae spores indicated glycoconjugate richness of the polar cap. This organelle was also labeled with biotinylated ConA and GNA, which supports the presence of mannose-containing glycoconjugates. After extrusion, the polar tube must be firmly attached to the spore apex in order to maintain the integrity of the sporoplasm flowing toward a host cell. A critical role of the polar cap in this attachment is conceivable because of its positioning between apical plasma membrane and polar tube domains and its conversion into a collar-like structure during polar tube extrusion (Lom 1972). Some PATAg-positive material seen at the junction between extruded polar tube and spore body (Figure 1B) likely reflects controlled exocytosis of polar cap contents. An abundance of hydrophilic sugars associated with polar cap proteins may facilitate polar tube exit than sliding through the apical aperture. The IgG response of immunocompetent humans against Encephalitozoon spp. was found to be directed against carbohydrate moieties of PTP1 and of some proteins migrated as a smear in SDS–PAGE between 100 and 250 kDa (Peek et al. 2005). Immunofluorescence data supported a localization of the last antigenic glycoproteins in the anchoring region, and a role in the adherence of the spore to the host cell surface has been hypothesized by the authors. The adherence of E. intestinalis spores to host cells in vitro involved sulfated glycosaminoglycans of the host cell surface, and inhibition of spore adherence by chondroitin sulfate A caused a significant decrease in the percentage of infected host cells (Hayman et al. 2005). Exospore glycoproteins are the best candidates for interacting with host glycosaminoglycans. A putative contribution of polar cap glycoproteins to host cell–microsporidia interactions should occur only after polar tube extrusion.
The mannose-binding lectins ConA and GNA recognized several bands in SDS–PAGE patterns of E. cuniculi spore proteins, and only mannose was clearly identified after GC–MS analysis of E. cuniculi and A. locustae oligosaccharide fractions subjected to methanolysis. ConA binding was reported for protein bands within a size range of 45–57 kDa in a fish-infecting microsporidian, the most labeled band being at 55 kDa (Kim et al. 1999), as for exospore proteins SWP1 and SWP2 in E. intestinalis (Hayman et al. 2001) and PTP1 in E. hellem (Xu et al. 2004), A. locustae, and Paranosema grylli (Polonais et al. 2005). SWP1 and PTP1 anomalously migrate in SDS–PAGE between 50 and 55 kDa and exist in different species, whereas SWP2 has a larger size (150 kDa) and is apparently restricted to E. intestinalis. In E. cuniculi, the ConA- and GNA-binding bands at 55 kDa may be partially correlated with a large PAS-reactive area that was also close to 55 kDa in 2-D gels and known to include both SWP1 and PTP1. Evidence for binding of PTP1 to GNA was previously obtained by lectin blotting in two insect-infecting microsporidia (Polonais et al. 2005). Whether at least one high molecular weight smeared band may contain a polar cap protein should deserve further investigations. The reactivity with GNA, a lectin that preferentially recognizes terminal 1,3-linked Man residues (Shibuya et al. 1988), may be somewhat surprising because only 1,2-linked Man residues were detected here and no microsporidial gene encoding 1,3-mannosyltransferase was identified. However, in the work of Shibuya et al. (1988), manno-oligosaccharides with an 1,2-linked linkage were found to also inhibit mannan-GNA precipitation, although less efficiently than oligosaccharides with terminal Man(1,3)Man unit. Moreover, the retardation of glycopeptides on GNA column was strongly dependent on the number and heterogeneity of disaccharide units. The binding of GNA to homogeneous 1,2-linked mannose chains seems therefore likely.
Neither GlcNAc and Man residues nor mass-specific N-glycan signal was identified in PNGase-treated glycan fractions of the studied microsporidia. Although this cannot formally exclude the possibility of a very low frequency of N-glycosylation, it should be stressed that a real loss of N-glycosylation is consistent with bioinformatic analyses of gene repertoires in E. cuniculi (Katinka et al. 2001) and A. locustae (Antonospora locustae Genome Project; http://gmod.mbl.edu/perl/site/antonospora01). Strikingly, no genes were found to encode subunits of the oligosaccharyltransferase complex needed for flipping of dolichol (Dol)-PP-GlcNAc2Man5 across the endoplasmic reticulum (ER) membrane and then linking the high-mannose oligosaccharide to asparagine residues on nascent peptides. The lack of key enzymes for protein N-glycosylation in E. cuniculi has been also verified through a recent inventory of Alg glycosyltransferases in several eukaryotic organisms; their comparison leading the authors to postulate that various secondary losses of enzymes from a common eukaryotic ancestor may have occurred (Samuelson et al. 2005). The potential E. cuniculi proteome also lacks critical factors for correct folding of N-glycosylated proteins in the ER, such as processing glycosidases and calnexin–calreticulin chaperone system (Katinka et al. 2001). In fungal organisms, the major fraction of N-linked glycans is incorporated within cell wall phosphomannoprotein complexes, and the importance of these glycans in host–fungal interactions is especially well illustrated by their involvement in the adherence of Candida albicans cells to host macrophages (Cutler 2001). It is noteworthy that, unlike typical fungi, microsporidia have no permanent cell wall. Proliferating intracellular stages (meronts) are indeed delimited only by plasma membrane, with cell wall formation occurring during sporogony. The low diversity of glycoprotein spots revealed after 2-D gel electrophoresis might be related to lacking N-glycosylated proteins.
Detailed analyses of microsporidial O-glycans have revealed linear oligosaccharides consisting of 1,2-linked Man residues. These structures are comparable with those identified in several fungi and share a common 1,2-linked mannotriose with -linkage of the reducing terminal Man residue to an hydroxy amino acid (Gemmill and Trimble 1999; Willer et al. 2003). Further processing varies according to the species under consideration. In S. cerevisiae, the mannotriose is capped with one or two 1,3-linked Man residues, whereas in Schizosaccharomyces pombe, up to two galactose residues can be attached via 1,2- and 1,3-linkages. Pichia pastoris and C. albicans have oligosaccharides with only 1,2-linked Man residues. Thus, E. cuniculi and A. locustae share with the two last yeasts very similar linear O-mannosyl glycans, except that maximum glycan length is higher in microsporidia (up to eight Man residues). The immobilization of E. intestinalis exospore proteins SWP1 and SWP2 on ConA-agarose columns (Hayman et al. 2001) might be re-interpreted as due to the presence of O-mannosylated chains. However, why the same proteins were found to be also immobilized on WGA-agarose columns is still unclear. The E. cuniculi coding sequence ECU08_1340 was initially annotated as having similarity with the 110-kDa subunit of O-GlcNAc transferase (OGT) (Katinka et al. 2001). In fact, it is improbable that ECU08_1340 encodes such an enzyme, the partial homology concerning only tetratricopeptide repeats, not the catalytic C-terminal domain of OGT. As the microsporidian spore wall contains chitin but no ß glucan, we tentatively suggest that binding of WGA to SWPs may be due to the presence of a GPI structure linked to chitin oligomers.
The finding of O-mannosyl glycans fits with biochemical evidence for O-mannosylation of E. hellem PTP1 (Xu et al. 2004) and metabolic potentials inferred from E. cuniculi genome sequence (Vivarès and Méténier 2004). Starting from Dol-P-Man in the ER rather than from a nucleotide sugar in the Golgi apparatus, the O-mannosylation pathway in fungal organisms (Ernst and Prill 2001) should be minimally represented in microsporidia. Only one gene encodes 1,2 mannosyltransferase (KTR family) in E. cuniculi (locus ECU04_1130; UniProtKB entry: Q8SS28_ENCCU) and probably also in A. locustae (http://gmod.mbl.edu/perl/site/antonospora01). This excludes the presence of partially redundant enzymes involved in the addition of the second and third 1,2-linked Man residues, as demonstrated in both S. cerevisiae (Romero et al. 1999) and C. albicans (Munro et al. 2005). The conserved PMT family of protein O-mannosyltransferases is divided into PMT1, PMT2, and PMT4 subfamilies and contains up to seven members in S. cerevisiae. The unique member of the PTP4 subfamily forms homomeric complexes, whereas members of the PMT1 subfamily interact heterophilically with those of the PMT2 subfamily, the PMT1/PMT2 and PMT4 members differing in protein substrate specificity (Girrbach and Strahl 2003; Willer et al. 2003). In E. cuniculi, only two potential PMTs are present. The protein sequence ECU02_1300 (Q825D9) is clearly representative of the PMT2 subfamily (50–52% similarity with C. albicans and Aspergillus fumigatus PMT2s). The highly divergent character of the other PMT candidate (ECU06_0950; Q8SVA5) does not allow assignation to a known PMT subfamily. A clear homolog of ECU02_1300 has been found in A. locustae (ORF 1659; http://gmod.mbl.edu/perl/site/antonospora01). The formation of a PMT1/PMT2-like complex in microsporidia remains debatable.
Further isolation and characterization of individual O-mannosylated proteins should be useful for a better knowledge of the molecular organization of the microsporidian invasion apparatus, including the polar cap, and of possible ligands involved in host–microsporidia cell interactions. Moreover, as microsporidian gene potentialities exist for glypiation (Vivarès and Méténier 2004) and some recently studied endospore proteins are likely GPI-anchored (Brosson et al. 2005; Peuvel-Fanget et al. 2005; Xu et al. 2006), physicochemical analyses are still needed to elucidate the precise structure of these GPI anchors.
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Materials and methods |
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Isolation of microsporidian spores
E. cuniculi GB-M1, a mouse isolate that was used for genome sequencing (Katinka et al. 2001
), was produced on MDCK cells as previously described (Delbac, Duffieux, et al. 1998). Parasite cells were released in culture media by natural lysis of heavily infected MDCK cells. Culture media were harvested and then parasite cells were sedimented and repeatedly washed with phosphate-buffered saline (PBS), pH 7.4, through 10 successive centrifugations (20 000g, 3 min) to remove host cell contaminants of low density. The final pellet of E. cuniculi cells was represented by late sporogonial stages including a majority of mature spores (more than 70%). A. locustae spores, arising from infected grasshoppers, were commercially available from M&R Durango Insectary (Bayfield, CO). These spores were also washed in PBS, pH 7.4, prior to protein extraction.
EM cytochemistry
E. cuniculi and A. locustae spores were either epoxy resin-embedded or frozen. For resin embedding, spores were fixed for 1 h in 2% glutaralhehyde, 0.05% ruthenium red, and 0.07 M cacodylate buffer, pH 7.4. After washing for 30 min in 0.1 M cacodylate buffer, pH 7.4, they were postfixed for 1 h in 1% OsO4, dehydrated through a graded series of ethanol, infiltrated in propylene oxide, and embedded in Epikote 812 resin (Agar Scientific, Essex, UK). Ultrathin sections were obtained with a Leica Ultracut S ultramicrotome, then classically stained with uranyl acetate and lead citrate. For ultracryotomy, spores were fixed for 1 h with 4% paraformaldehyde–0.1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. After infusion for 1 h at room temperature in a 25% glycerol–5% dimethylsulfoxide mixture, the samples were rapidly frozen in slush nitrogen. Cryosections (90 nm) were obtained using a dry sectioning device at –110 °C and Ultracut S ultramicrotome fitted with the low-temperature sectioning system FC4. Sectioned material was mounted on collodion-coated nickel grids (150 meshes) and stored at 4 °C in PBS prior to cytochemical protocols.
The localization of glycoconjugates and polysaccharides was investigated using the PATAg procedure (Thiéry 1967). Unstained sections of resin-embedded or frozen cells were submitted to oxidation with 1% periodic acid for 30 min, washed in distilled water, and incubated in 0.2% thiocarbohydrazide for 4 h. After washing with 10% acetic acid and then with distilled water, grids were treated with 1% silver proteinate for 30 min.
For the localization of lectin-binding sites, cryosections were saturated for 1 h with PBS-1% ovalbumin and then incubated for 1 h with a 1:50 dilution of biotinylated ConA or GNA lectins (EY Laboratories, San Mateo, CA). Grids were subsequently reacted for 1 h with goat antibiotin antibody (Sigma, St. Louis, MO) at 1:100, then for 1 h with 5 nm gold-conjugated antigoat IgG (Sigma) at 1:100. Cryosections were finally contrasted and protected with a 0.8% uranyl acetate–1.6% methylcellulose mixture. All specimens were examined under a JEOL 1200EX transmission EM.
Protein extractions
For 2-D electrophoresis, E. cuniculi spore proteins were extracted through repeated cycles of freezing–thawing in liquid nitrogen and sonication (15x1 min on ice) in the presence of 7 M urea, 2 M thiourea, 100 mM DTT, 4% CHAPS, and 0.2% SDS. For SDS–PAGE followed by lectin blotting, proteins were solubilized in either Laemmli buffer ("SDS extract") or a solution containing 4 M urea and 100 mM DTT ("DTT–urea extract").
For MS and NMR analyses, E. cuniculi or A. locustae spores (total cell number: 109–1010) were disrupted in a lysis buffer containing 1% (v/v) Triton X-100 and 100 mM DTT, by repeated cycles of freezing–thawing and sonication, then incubated in the extraction solution (7 M urea, 2 M thiourea, 100 mM DTT, 1% Triton X-100) under agitation for 3 days. The entire sample was finally dialyzed (6000–8000 cutoff) for further 3 days.
2-D gel electrophoresis and glycoprotein detection
Isoelectrofocalization (IEF) of E. cuniculi protein samples (50 µg) was performed along linear immobilized pH gradient strips of 7 cm, pH 3–10 (GE Healthcare, Piscataway, NJ) in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 2 mM tributyl phosphine, and 0.5% ampholytes), with the IPGPhor apparatus (GE Healthcare). The program of voltage increase was 30 V for 12 h, 400 V for 30 min, 500 V for 30 min, 800 V for 30 min, 1000 V for 1 h, 4000 V for 1 h, and 8000 V for 2 h. After equilibration with 50 mM Tris–HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, 100 mM DTT, and then 135 mM iodoacetamide, strips were deposited on 12% polyacrylamide slab gels. After SDS–PAGE for 1 h at 25 mA, proteins were either stained with Coomassie brilliant blue or electrophoretically transferred onto poly vinylidenedifluoride PVDF membranes (Millipore, Billerica, MA). Glycoprotein detection on these membranes was carried out with the BioRad Immun-Blot® kit, involving successively periodate oxidation of carbohydrate groups, biotinylation, incubation with streptavidin-alkaline phosphatase conjugate, and color development with 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium chloride.
Lectin overlay
SDS and DTT–urea extracts of E. cuniculi were analyzed by SDS–PAGE (12% polyacrylamide) and separated proteins were transferred onto PVDF membranes. Blots were saturated in Tris buffer saline (TBS) (50 mM Tris–HCl, pH 7.4, 150 mM NaCl)–5% skimmed milk and washed in TBS and then in lectin reaction buffer (20 mM Tris–HCl, pH 7.4, 0.2 M NaCl, 1 mM MgCl2, 1 mM MnCl2, and 1 mM CaCl2). They were subsequently incubated for 1 h with one of the two following biotin-labeled lectins (EY Laboratories): ConA diluted at 1:1500 and GNA at 1:1000. After washing in TBS, the membranes were reacted with a goat antibiotin antibody (Sigma) diluted at 1:1000 and finally with a peroxidase-conjugated antigoat IgG (Sigma) at 1:10 000. Lectin binding was visualized with a chemoluminescent system (ECL+Western blot detection kit, Amersham). Specificity was tested by preincubation of lectin conjugates with an ovalbumin blot at 4 °C overnight.
Isolation of glycan fractions
E. cuniculi and A. locustae extracts were delipidated by chloroform–methanol (2:1). The separated pellet was reduced with 6 M guanidine chloride, 25 mM DTT in 0.6 M Tris for 8 h at 50 °C, alkylated with 50 mM iodoacetamide overnight, and dialyzed. The product (3 mL) was then digested with 1 mg of trypsin in 50 mM ammonium bicarbonate pH 8.4 at 37 °C for 1 day and with additional 0.2 mg of chymotrypsin at 37 °C under agitation for a night. A C18 Sep-Pak (Waters, Milford, CT) was used to purify the resulting supernatant. Putative N-linked glycans were released by digestion with PNGase F and PNGase A. Oligosaccharides were separated from peptides and glycopeptides using a C18 Sep-Pak. Putative O-linked glycans were released by alkaline reductive degradation in 1 M NaBH4 and 0.1 M NaOH at 37 °C for 72 h. The reaction was stopped by the addition of Dowex 50x8, 25–50 mesh, H+ form (Bio-Rad) at 4 °C until pH reaches 6.5, and after evaporation to dryness, borate salts were removed by repeated evaporation with methanol. Total material was subjected to cationic exchange chromatography on Dowex 50x2, 200–400 mesh, H+ form (Bio-Rad) to remove residual peptides. The oligosaccharide fraction was then purified on a Bio-Gel P2 column (Bio-Rad) and C18 Sep Pak.
Monosaccharide composition
Monosaccharides were analyzed by GC–MS as perheptafluorobutyryl derivatives (Zanetta et al. 1999). Shortly, N- and O-oligosaccharides were subjected to methanolysis in 500 µL of 0.5 M HCl in anhydrous methanol at 80 °C for 20 h and incubated in 200 (L of anhydrous acetonitrile (ACN) and heptafluorobutyric acid (HFB) at 180 °C for 10 min. The reagents were evaporated, and the sample was dissolved in ACN prior to GC–MS analysis.
Permethylation and linkage analysis
Permethylation was performed according to the procedure of Ciucanu and Kerek (1984). Briefly, compounds were incubated overnight in a suspension of 200 mg/mL NaOH in dry dimethylsulfoxide (300 µL) and iodomethane (200 µL). The methylated products were extracted in chloroform and washed with water. After methanolysis, they were dried and then peracetylated in 200 µL of acetic anhydride and 50 µL of pyridine overnight at room temperature. The reagents were evaporated, and the sample was dissolved in chloroform before analysis in GC–MS.
MALDI-TOF and ES-MSn
The molecular masses of N- and O-oligosaccharides were measured by MALDI-TOF on a Voyager Elite reflectron mass spectrometer (PerSeptive Biosystems, Framingham, MA), equipped with a 337 nm UV laser. Native and permethylated samples were prepared by mixing directly on the target 1 µL of water (native) or ACN (permethylated) diluted oligosaccharide solution and 1 µL of 2.5-dihydroxybenzoic acid matrix solution (10 mg/mL dissolved in ACN–H2O). For electro-spray ionization multistage mass spectrometry (ES-MSn), permethylated samples were reconstituted in methanol and analyzed by mass spectrometry on a LCQ DK XP+ion trap (Thermo Finnigan, Waltham, MA) instrument. After mixing with an equal volume of methanol–0.1 M aqueous formic acid, samples were directly infused at 50 nL/min, using the nanoflow probe option for MS and MSn analyses.
1H NMR spectroscopy
One-dimensional and 2-D 1H NMR spectra were recorded on a Bruker Avance 800 spectrometer (Université des Sciences et Technologies de Lille), equipped with a TXI probe-head. Prior to 1H NMR analyses, oligosaccharides were twice exchanged with 99.97% 2H2O and finally solubilized in 250 µL of 2H2O in 5-mm Shigemi tube matched for 2H2O. The spectrometer operated at 300 K without solvent presaturation. The chemical shifts were expressed relative to residual acetate salts ( 1.909 ppm). Spectral width was 8012 Hz with 16 k points for a spectral resolution of 0.49 Hz/pt. The 2-D 1H–1H COSY (correlation spectroscopy) spectrum was acquired with z-gradient pulse from cosygp pulse program available in Bruker software. Spectral width was 8012 Hz for both dimensions with 4016 points for F2 and 256 points for F1 giving spectral resolution of 1.96 and 31.3 Hz/pt, respectively.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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We thank Yves Leroy (CNRS UMR 8576) and Philippe Timmerman (CNRS UMR 8576) for technical assistance on carbohydrate composition. We are grateful to Dr Catherine Texier for her help in the interpretation of two-dimensional protein profiles.
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None declared.
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Abbreviations |
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ACN, acetonitrile; CID-MS/MS, collision-induced decay tandem mass spectrometry; ConA, concanavalin A; COSY, correlation spectroscopy; Dol, dolichol; DP, degree of polymerization; DTT, dithiothreitol; EI, electronic impact; EM, electron microscopy; ER, endoplasmic reticulum; ES, electro-spray; GC–MS, gas chromatography coupled to mass spectrometry; GNA, Galanthus nivalis agglutinin; GPI, glycosylphosphatidylinositol; HFB, heptafluorobutyric acid; IEF, isoelectrofocalization; MALDI-TOF, matrix assisted-laser desorption/ionization time of flight; MDCK, Madin–Darby canine kidney; NMR, nuclear magnetic resonance; OGT, O-GlcNAc transferase; PAS, periodic acid-Schiff; PATAg, periodic acid-thiocarbohydrazide-Ag proteinate; PBS, phosphate-buffered saline; PNGase A, peptide N-glycosidase A; PNGase F, peptide N-glycosidase F; PTP, polar tube protein; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SWP, spore wall protein; TBS, Tris buffer saline; TIC, total ion chromatogram; WGA, wheat germ agglutinin; 1-D, one-dimensional; 2-D, two-dimensional.
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