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Originally published In Press as doi:10.1074/mcp.M700004-MCP200 on June 4, 2007.
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Molecular & Cellular Proteomics 6:1485-1499, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Glycomics Analysis of Schistosoma mansoni Egg and Cercarial Secretions*,S

Jihye Jang-Lee{ddagger}, Rachel S. Curwen§, Peter D. Ashton§, Bérangère Tissot{ddagger}, William Mathieson§, Maria Panico{ddagger}, Anne Dell{ddagger},, R. Alan Wilson§ and Stuart M. Haslam{ddagger},||

From the {ddagger} Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom and § Department of Biology, University of York, York YO10 5YW, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries. Glycoconjugates, and in particular the carbohydrate component of these products, represent the main immunogenic challenge to the host and could therefore represent one of the crucial determinants for successful parasite establishment. Here we report a comparative glycomics analysis of the N- and O-glycans derived from glycoproteins present in S. mansoni egg (egg-secreted protein) and cercarial (0–3-h released protein) secretions by a combination of mass spectrometric techniques. Our results show that S. mansoni secrete glycoproteins with glycosylation patterns that are complex and stage-specific. Cercarial stage secretions were dominated by N-glycans that were core-xylosylated, whereas N-glycans from egg secretions were predominantly core-difucosylated. O-Glycan core structures from cercarial secretions primarily consisted of the core sequence Galß1->3(Galß1->6)GalNAc, whereas egg-secreted O-glycans carried the mucin-type core 1 (Galß1->3GalNAc) and 2 (Galß1->3(GlcNAcß1->6)GalNAc) structures. Additionally we identified a novel O-glycan core in both secretions in which a Gal residue is linked to the protein. Terminal structures of N- and O-glycans contained high levels of fucose and include stage-specific structures. These glycan structures identified in S. mansoni secretions are potentially antigenic motifs and ligands for carbohydrate-binding proteins of the host immune system.

The blood fluke Schistosoma mansoni is a cause of significant human morbidity and mortality throughout Africa and parts of South America (1, 2). Infection is acquired when aquatic cercariae directly penetrate through the skin, transforming into schistosomula as they enter their host. Schistosomula migrate from the skin to the portal system, the ultimate site of parasitization, where maturation and pairing of adult worms occurs. Each female deposits approximately ~300 eggs per day in the blood vessels of the gut wall; these either cross the tissues to reach the lumen and are voided in the feces or are carried down the portal vein to embolize in the liver. Passage of both parasite stages across epithelial barriers involves the release of secretions; thus there is symmetry in the way that the parasite enters and leaves its mammalian host.

The cercarial secretions (termed 0–3-h released proteins (RPs1)) originate from pre- and postacetabular gland cells (3), whereas those of the eggs (termed ESPs) are derived from the syncytial subshell envelope (4). Cercarial secretions appear to be weakly immunogenic, and only with repeated exposure do they elicit marked immune responses. The function of cercarial secretions is to facilitate skin penetration, and they are the first parasite components encountered by the mammalian host's immune surveillance mechanisms. In contrast, the eggs are highly immunogenic and are involved in interactions with the gut to facilitate the passage of eggs through these tissues and in the liver, driving granuloma formation that initiates the pathology of schistosomiasis (4, 5). However, there could be a dose effect at work because the total of 45,000 attenuated cercariae that are required to induce protective immunity in a baboon is only equivalent to the biomass of eggs deposited daily in the tissues of an animal with 500 worm pairs. Furthermore much of the antibody response elicited by eggs is directed against glycan epitopes and is cross-reactive with glycans expressed on the cercarial surface and cercarial secretions (6–8). Recent studies have described detailed proteomics characterization of cercarial secretions identifying novel proteases and immunomodulators (9, 10). When mammalian hosts are exposed to radiation-attenuated cercariae, delivering a very much larger antigenic load than the maximum feasible dose of normal cercariae, virtually the entire antibody response is directed against glycan epitopes common to both cercarial and egg secretions (6). These facts led us to suggest that highly immunogenic glycans could represent a smoke screen to subvert the immune system away from more vulnerable peptide epitopes (6). It was also shown that vaccination of mice with live schistosome eggs elicited high titers of anti-glycan antibodies but afforded no protection against cercarial challenge.2

It has long been recognized that schistosome glycoconjugates may play an important role in host-parasite interactions enhancing their survival within the host (11). Thus, a large number of studies have been carried out to characterize the schistosome glycome. Structural studies have demonstrated that S. mansoni parasites express a complex set of glycans throughout their life cycle (12–18). However, most structural studies have focused on the glycans of whole stage extracts (12–14), and studies on schistosome secretions have been largely limited to the adult stage gut-associated antigens present in the host circulation, the circulating cathodic antigen and circulating anodic antigen (19–21), with the exception of a single purified glycoprotein, interleukin-4-inducing principle of S. mansoni eggs (IPSE)/alpha-1 (22). Although the data generated from such studies on whole extracts have been informative, analysis of S. mansoni secretions will provide a more accurate representation of the molecules that directly interact with the host.

There is a wealth of evidence suggesting that glycans from parasitic helminths are involved in host immune evasion and modulation (for reviews, see Refs. 11 and 23). Therefore, we postulate that glycans of S. mansoni-secreted glycoproteins are likely to be key players in the parasite-host interactions associated with infection and onward transmission. We report here, in one of the most comprehensive studies of S. mansoni protein glycosylation, that 0–3-h RPs and ESPs contain a structurally diverse array of glycans. Glycan structures exhibited stage-specific as well as common structures with potential implications in terms of immunological recognition and host-parasite interactions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Cercarial Secretions (0–3-h RPs)—
The 0–3-h RPs were prepared as described previously (9).

Collection of Egg Secretions (ESPs)—
Egg secretions were prepared by extracting mature schistosome eggs from mouse livers 7 weeks postinfection, purifying them on a Percoll gradient, and culturing them aseptically for 72 h at 37 °C in RPMI 1640 medium as described previously (4).

Glycoprotein Analysis—
Samples of either 50 µg of 0–3-h RPs or 25 µg of ESPs were separated by 2DE on minigels as described previously (9). The distribution of glycans and proteins was determined by staining with ProQ Emerald (Invitrogen) and SYPRO Ruby (Bio-Rad), respectively, according to the manufacturers’ instructions. Images were captured using a VersaDoc Imaging System Model 3000 (Bio-Rad) and analyzed with Phoretix 2D Evolution software (Nonlinear Dynamics, Newcastle, UK). ESP spots were identified by tandem mass spectrometry using a 4700 Proteomics Analyzer with TOF-TOF optics (Applied Biosystems) as described previously (9).

Glycomics Analysis—
Analysis was performed on the secretions of ~0.5 million cercariae, which produced ~330 µg of 0–3-h RPs. Analysis was performed on the secretions of ~0.1 million eggs obtained from the livers of eight mice, which produced ~250 µg of ESPs.

Digestion and Reduction/Carboxymethylation of Parasite Material—
Parasite secretions were reduced, carboxymethylated, and digested with L-(tosylamido-2-phenyl)ethyl chloromethyl ketone bovine pancreas trypsin (EC 3.4.21.4) as described previously (24).

Release of N-Glycans—
N-Glycans from ~250 µg of ESPs and 330 µg of 0–3-h RPs were enzymatically released from the peptide backbone by sequential digestion with PNGase F and PNGase A. PNGase F (Roche Applied Science; peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase, EC 3.5.1.52) digestion was carried out in 50 mM ammonium hydrogen carbonate, pH 8.5, for 24 h at 37 °C with 3 units of enzyme. The reaction was terminated by lyophilization, and the products were purified using a propanol, 5% (v/v) acetic acid reverse-phase C18 Sep-Pak (Waters Corp.) system as described previously (24). Glycopeptides remaining after the PNGase F digestion were further digested with 0.2 milliunits of PNGase A (Roche Applied Science; peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase, EC 3.5.1.52) for 24 h at 37 °C, and products were purified on a C18 Sep-Pak (Waters Corp.) as described previously (24).

Reductive Elimination of O-Glycans—
O-Glycans were chemically released by reductive elimination (400 µl of 1 M KBH4 in 0.05 M KOH at 45 °C for 16 h) from the glycopeptides remaining after the release of the N-glycan populations and desalted through a Dowex 50W-X8(H) column. Excess borates were removed by coevaporation under a stream of nitrogen with 10% (v/v) acetic acid in methanol.

Permethylation of Glycans for Mass Spectrometric Analysis—
Prior to MS analyses, glycans were permethylated using the NaOH procedure as described previously (24). Permethylated glycans were then purified using an acetonitrile/water reverse-phase C18 Sep-Pak (Waters Corp.) system as described previously (24). A third of each of the released N- and O-glycan fractions was permethylated as required for MS experiments.

MALDI-MS(/MS) Analyses—
The permethylated glycans were dissolved in 10 µl of methanol, and a 1-µl aliquot of dissolved sample was mixed with 1 µl of matrix (2,5-dihydroxybenzoic acid, 20 mg/ml). The sample/matrix mixture was subsequently spotted onto a MALDI target plate and dried under vacuum. A Voyager DE® STR MALDI-TOF (Applied Biosystems) mass spectrometer in the reflectron mode with delayed extraction was used to obtain MS spectra. Peaks observed in the MS spectra were selected for further MS/MS. MS/MS data were acquired using a 4800 MALDI-TOF/TOF (Applied Biosystems) mass spectrometer. The collision energy was set to 1 kV, and air was used as the collision gas. All data are expressed as monoisotopic masses.

Collision-activated Dissociation ES-MS/MS Analyses—
The permethylated glycans were dissolved in 10 µl of methanol, and 2–3 µl of the samples were loaded into a NanoES spray capillary coated with a thin layer of gold/palladium (inner diameter, 2 µm (Proxeon, Odense, Denmark)). Glycans were sequenced by MS/MS using a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, UK) and/or a QSTAR Pulsar-i quadrupole TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada). MS and MS/MS spectra were collected in the positive ion mode. For the Q-TOF instrument, a potential of 1.5 kV was applied to the nanoflow tip to produce a flow rate of 120–130 nl/min. The drying and collision gases used were nitrogen and argon, respectively. Typical collision energies ranged from 30 to 80 eV depending on the size of the glycan, and the collision gas pressure was maintained at 10–4 millibar. MS/MS spectra were acquired and processed using MassLynx software (Micromass). For the QSTAR instrument, nitrogen was used as the collision gas with the pressure being maintained at 5.3 x 10–5 torr, and collision energies typically varied from 35 to 90 eV to analyze glycans. MS/MS spectra were acquired and processed using the Analyst QS data system (MDS Sciex, Toronto, Canada). Both instruments were precalibrated using [Glu1]fibrinopeptide B in acetonitrile in 5% aqueous acetic acid (1:3, v/v). All data are expressed as monoisotopic masses.

GC-MS—
The permethylated glycans were dissolved in 10 µl of methanol, and 5-µl aliquots of the sample were processed for GC-MS linkage analyses. Permethylated glycans were hydrolyzed with 2 M trifluoroacetic acid for 2 h at 121 °C and reduced with 10 mg/ml sodium borodeuteride in 2 M aqueous ammonium hydroxide at room temperature for 2 h, and the reaction was terminated by the addition of acetic acid. Samples were dried and then acetylated with acetic anhydride at 100 °C for 1 h. The reagent was removed under a stream of nitrogen. Samples were then dissolved in chloroform and washed several times with water before drying under nitrogen. Samples were dissolved in 20 µl of hexanes, and 1-µl aliquots were analyzed by GC-MS using a PerkinElmer Clarus 500 instrument fitted with an RTX-5 column (30 m x 0.25-mm internal diameter, Restek Corp.). The sample was injected into the column at 60 °C, and the temperature was steadily increased at a rate of 8 °C/min to a temperature of 300 °C.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Evaluation of Glycosylation in ESPs and 0–3-h RPs—
ESPs were separated by 2DE and subsequently stained with ProQ Emerald and SYPRO Ruby (Fig. 1, A and B, respectively). A combined image was generated using Phoretix 2D Evolution software, which revealed three spots reacting strongly to both protein and glycans (Fig. 1C). The spots were identified as hepatotoxic ribonuclease omega-1 (Omega-1; spot 1) and IPSE (spots 2 and 3) (25). Supplemental Table 1 contains all protein identification data.


Figure 1
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  		FIG. 1. A, 2DE gel of ESPs stained with SYPRO Ruby. B, ProQ Emerald-stained ESP 2DE gel. C, combined images of SYPRO Ruby- and ProQ Emerald-stained ESP 2DE gel. Spot 1, Omega-1; spots 2 and 3, IPSE. D, 0–3-h RP 2DE gel stained with SYPRO Ruby. E, 0–3-h RP 2DE gel stained with ProQ Emerald. F, combined images of SYPRO Ruby- and ProQ Emerald-stained 0–3-h RP 2DE gel. Spot 1, SmPepM8; spot 2, SmCE; spot 3, SmSerpin_c; spot 4, SmDPP IV; spot 5, SmSCP_c; spot 6, SmSCP_b. The combined images were obtained using Phoretix 2D Evolution software.

 
Similarly 0–3-h RPs were separated by 2DE and stained with ProQ Emerald and SYPRO Ruby (Fig. 1, D and E, respectively). Staining with SYPRO Ruby produced a spot pattern similar to that described previously (9). All gel spot identities were assigned on the basis of their comparative position in the gel images presented in Ref. 9. Only a minority of constituents were glycoproteins, namely the protease inhibitor serpin_c (Fig. 1F, spot 3), dipeptidyl-peptidase IV (Fig. 1F, spot 4), two proteins with sperm-coating protein (SCP) domains (SmSCP_c and SmSCP_b; Fig. 1F, spots 5 and 6), whereas the unnumbered spots correspond to glycoproteins whose identities are unknown. Notably the most abundant constituents in 0–3-h RPs corresponded to the proteases metalloproteinase (SmPepM8) and cercarial elastase (SmCE) (Fig. 1F, spots 1 and 2, respectively), which were not glycosylated.

General Strategy for Glycomics Analysis of S. mansoni Secretions—
Collected secretions were reduced/carboxymethylated and digested with trypsin to facilitate the subsequent release of N- and O-glycans. N-Glycans were released using two different enzymes, PNGase F and PNGase A. Digestion with PNGase F releases all N-glycans apart from those containing {alpha}1->3-linked fucose to the reducing end GlcNAc of the core; such glycans are released by PNGase A. Sequential digestion with PNGase F and PNGase A allowed the analysis of two populations of N-glycans based on their core fucosylation (26). O-Glycans were released by reductive elimination from the remaining glycopeptides after the removal of the two different N-glycan populations. Purified glycans were permethylated to increase the sensitivity of detection and to direct the subsequent MS/MS fragmentation. Consistent and reproducible data were obtained in multiple repeats of experiments.

Initially MALDI-MS was used to obtain a profile of the molecular ions from each of the glycan pools. This gave singly charged sodiated molecular ions ([M + Na]+) and allowed the assignment of the composition of molecular ions in terms of the number of Hex, deoxyhexose, HexNAc, and pentose residues. Based on their molecular compositions, knowledge of the biosynthetic pathway, and previously published structural data, putative structures could be proposed. To confirm putative structures, molecular ions observed in the MS spectrum were subjected to MS/MS analysis using two different ionization methods, ESI and MALDI, which afforded sequence-informative fragment ions that provided vital structural information such as the non-reducing end sequences, i.e. antennae structures, branching patterns, and sometimes linkage positions. GC-MS linkage analyses were performed to define monosaccharide compositions, glycosidic bond positions, and relative abundance of each sugar component. Results from the various experiments were combined to assign detailed glycan structures.

MALDI Profiling of ESP N-Glycans—
Typical MALDI-MS spectra of PNGase F- (Fig. 2A) and PNGase A (Fig. 2B)-released ESP N-glycans are shown in Fig. 2, and their assignments are compiled in Table I. Based on the compositions of each molecular ion the types of N-glycans were deduced. In the PNGase F-released ESP N-glycan profile hybrid structures comprised the major N-glycan family with dominant signals corresponding to compositions Fuc0–1Hex5–6HexNAc3 (m/z 1999, 2030, and 2204). The next most abundant were complex type structures (Fuc1–4Hex4–6HexNAc3–6) with dominant signals at m/z 2245 (Fuc1Hex5HexNAc4) and 2420 (Fuc2Hex5HexNAc4). Minor signals consistent with truncated type structures (Xyl0–1Fuc0–1Hex1–3HexNAc2–3) and high mannose structures (m/z 1580 and 1785, Hex5–6HexNAc2) were also present. The PNGase A-released ESP N-glycan profile revealed new highly fucosylated signals (m/z 2143, 2184, 2347, 2551, 2768, 2807, 2837, 2982, 3012, 3257, 3390, and 3431; see Table I).


Figure 2
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  		FIG. 2. MALDI-MS spectra of permethylated PNGase F- (A) and PNGase A (B)-released N-glycans from ESPs. ESP N-glycans were sequentially released by PNGase F and PNGase A, permethylated, and purified using an acetonitrile/water C18 Sep-Pak system. Signals observed correspond to singly charged sodiated molecular ions, [M + Na]+.

 

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  		TABLE I Compositional assignments of singly charged sodiated molecular ions, [M + Na]+, observed in MALDI-MS spectra of permethylated PNGase F- and PNGase A-released ESP N-glycans

 
Characterization of ESP N-Glycans by MS/MS Analysis—
To confirm the putative structures deduced from the MALDI-MS data, MS/MS analyses were performed on all ESP N-glycan molecular ions observed in Fig. 2. Molecular ions observed in the PNGase F-released N-glycan pool whose compositions indicated the presence of at least one fucose residue gave rise to a fragment ion at m/z 474, which is indicative of monofucosylated cores (Fuc{alpha}1->6GlcNAc), whereas molecular ions in the PNGase A-released N-glycan pool containing two or more fucose residues produced a fragment ion at m/z 648, which implies the presence of difucosylated cores (Fuc{alpha}1->6(Fuc{alpha}1->3)GlcNAc). Both signals result from a glycosidic cleavage between the GlcNAc residues of the N-glycan core (see examples in Supplemental Figs. 1–4).

Complex and hybrid type structures in both ESP N-glycan pools (m/z 1621, 1795, 1825, 1955, 1969, 1999, 2029, 2040, 2070, 2143, 2173, and 2203–3431 (see Fig. 2) produced diagnostic fragment ions at m/z 660 (Fuc1Hex1HexNAc1) with the concomitant elimination of Fuc giving rise to the signal at m/z 472, which is evidence for the Fuc residue being linked to the 3'-position of the GlcNAc. Therefore, this indicates the presence of Lex (Galß1->4(Fuc{alpha}1->3)GlcNAc) structures. Additionally the fragment ion at m/z 486 (Hex1HexNAc1) provides evidence of LacNAc structures (see example in Supplemental Figs. 1 and 2). Molecular ions above m/z 2693 also yielded fragment ions at m/z 935 (Hex2HexNAc2), 1109 (Fuc1Hex2HexNAc2), and 1283 (Fuc2Hex2HexNAc2), which are consistent with tandem repeats of LacNAc and Lex (see example in Supplemental Figs. 3 and 4). ESP N-glycans whose molecular compositions indicated a higher content of HexNAc than Hex residues and contained at least one fucose residue (m/z 2010, 2184, 2633, 2807, 2909, 2982, 3082, 3257, and 3431) produced signals at m/z 701 (Fuc1HexNAc2) and/or 875 (Fuc2HexNAc2) and fragment ions corresponding to the concomitant loss of these substituents from the molecular ion. These observations support monofucosylated and difucosylated LacdiNAc structures (see example in Supplemental Figs. 1 and 2) in ESP N-glycans. In addition, when molecular ions contained at least two fucose residues and difucosylated LacdiNAc structures (e.g. m/z 2633) MS/MS spectra also produced a fragment ion at m/z 403 (Fuc2) indicating that Fuc residues in difucosylated LacdiNAc structures can also be present in tandem (see example in Supplemental Figs. 1 and 2).

MS/MS analyses on ESP complex N-glycan structures whose compositions implied the possibility of triantennary or tetra-antennary structures (m/z 2663, 2693, 2807, 2837, 2909, 3012, 3042, 3082, 3216, 3257, 3390, and 3431) exhibited fragmentation patterns showing that such structures were predominantly biantennary N-glycans. These contained an extended antennae comprising of tandem repeats of LacNAc and/or Lex (see example in Supplemental Figs. 3 and 4). Only a minority of the components were multiantennary N-glycans.

GC-MS Linkage Analysis of ESP N-Glycans—
The complexity of the glycan pools precluded the assignment of each detected component to an individual glycan. However, important conclusions could be drawn from the linkage data (Supplemental Table 2).

The PNGase F- and PNGase A-released N-glycan pools comprised mostly similar components with the exception of the presence of 3,4,6-linked GlcNAc in the PNGase A-released N-glycan pool that is an indication of core difucosylation. This result together with the MS/MS data unambiguously confirmed the presence of core difucosylated N-glycans in ESPs.

High levels of 3,6-linked mannose and 4-linked GlcNAc were in accordance with these residues being constituents of the core of the majority of N-glycans. Xylosylated cores were corroborated by terminal Xyl and 2,3,6-linked Man. However, these were present in relatively low amounts and were consistent with the MALDI-MS compositional assignments, indicating that xylosylation is not a dominant feature in ESP N-glycans. Monofucosylated cores in the PNGase F-released N-glycan pool were confirmed by the presence of 4,6-linked GlcNAc. The high abundance of 2-linked mannose indicated high levels of hybrid and complex structures. Minor quantities of 2,4- and 2,6-linked Man revealed the presence of tri- and possibly tetra-antennary complex N-glycans. This is consistent with observations made in MS/MS experiments where multiantennary complex N-glycans are minor components. Fuc, Man, and Gal are the major terminal sugars with lesser amounts of terminal GlcNAc and GalNAc. In addition, GlcNAc and GalNAc residues are substituted at their 3- and 4-positions; this together with MS/MS data (see previous section) supports the presence of Lex and fucosylated LacdiNAc (±Fuc{alpha}1->3GalNAcß1->4(±Fuc{alpha}1->3)GlcNAc) structures. The observation of 2-linked Fuc corroborates with the MS/MS data that some Fuc residues are further substituted.

Overall these data indicate that hybrid and complex N-glycans are abundant structures in ESP N-glycan pools. Mono- and difucosylated cores are notable features in the majority of ESP N-glycans, whereas core xylosylation occurs at low levels. Major terminal structures are Lex and LacNAc, which are also present as tandem repeats. Other terminal structures include LacdiNAc and mono- and difucosylated LacdiNAc, which are present in the less abundant high molecular weight structures. Complex N-glycans are predominantly biantennary structures with minor amounts of multiantennary structures. Taken together these data support the proposed structures summarized in Fig. 4.


Figure 4
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  		FIG. 4. Proposed structures of ESP and 0–3-h RP N-glycans.

 
MALDI Profiling of 0–3-h RP N-Glycans—
Fig. 3 shows typical MALDI-MS spectra of PNGase F- (Fig. 3A) and PNGase A (Fig. 3B)-released 0–3-h RP N-glycans, and assignments are shown in Table II. Their compositions revealed that the majority of the N-glycans carry xylose. The PNGase F- and PNGase A-released 0–3-h RP N-glycan spectra exhibited very similar profiles. Truncated type structures (Xyl0–1Fuc0–1Hex1–3HexNAc2) and complex type structures (Xyl0–1Fuc1–4Hex4–6HexNAc3–6) were the two most abundant N-glycan families in both N-glycan pools. New minor high molecular weight peaks were present in the PNGase A-released pool (m/z 1988, 2070, 2489, 2839, 2868, 3358, 3532, 3706, 3880, and 4054); some of these could potentially contain difucosylated cores according to their compositions.


Figure 3
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  		FIG. 3. MALDI-MS spectra of permethylated PNGase F- (A) and PNGase A (B)-released N-glycans from 0–3-h RPs. 0–3-h RP N-glycans were sequentially released by PNGase F and PNGase A, permethylated, and purified using an acetonitrile/water C18 Sep-Pak system. Signals in the spectrum correspond to singly charged sodiated molecular ions, [M + Na]+.

 

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  		TABLE II Compositional assignments of singly charged sodiated molecular ions, [M + Na]+, observed in MALDI-MS spectra of permethylated PNGase F- and PNGase A-released 0–3-h RP N-glycans

 
Characterization of 0–3-h RP N-Glycans by MS/MS Analysis—
MS/MS analyses were performed on the majority of the most abundant molecular ions. Molecular ions in the PNGase F-released N-glycan pool containing at least one fucose residue resulted in a diagnostic fragment ion at m/z 474 (see example in Supplemental Fig. 5), showing the presence of monofucosylated N-glycan cores. MS/MS analysis on molecular ions in the PNGase A-released N-glycan pool with at least two fucose residues did not yield fragment ions at m/z 648, suggesting that difucosylated N-glycan cores are not present in 0–3-h RPs. All signals in the MALDI-MS spectra (Fig. 4) whose compositions indicated the presence of a xylose (Table II) consistently produced fragment ions corresponding to the loss of a xylose residue (see example in Supplemental Fig. 5).

Major terminal structures in 0–3-h RPs were deduced from MS/MS experiments on hybrid and complex type components (m/z 1781 and above; see Fig. 3). MS/MS spectra of such molecular ions revealed signals at m/z 486 and 660 supporting LacNAc and Lex structures, respectively. Complex and hybrid type molecular ions at m/z 2663, 2782, 2837, 2867, 3202, 3357, 3376, 3406, 3447, 3621, and 3651 gave rise to fragment ions at m/z 935 (Hex2HexNAc2), 1109 (Fuc1Hex2HexNAc2), and 1283 (Fuc2Hex2HexNAc2), revealing tandem repeats of LacNAc and Lex. Moreover MS/MS spectra of the molecular ions whose HexNAc content was higher than Hex, e.g. m/z 2489, 2663, 2998, 3172, 3357, 3416, 3447, 3532, and 3591, produced fragment ions at m/z 731 (Hex1HexNAc2), 905 (Fuc2Hex1HexNAc2), and 1079 (Fuc2Hex1HexNAc2), which implicated the occurrence of the S. mansoni-specific terminal structure consisting of Galß1->4(±Fuc{alpha}1->3)GlcNAcß1->4(±Fuc{alpha}1->3)GlcNAc previously identified in whole cercarial extracts (14) (see example in Supplemental Fig. 5). In terms of branching patterns of 0–3-h RP complex N-glycans, major species correspond to monoantennary structures (e.g. m/z 1969 and 2129) with some biantennary structures (e.g. m/z 2244, 2418, 2578, 2592, and 2752) and minor amounts of multiantennary structures (molecular ions above m/z 2752).

GC-MS Linkage Analysis of 0–3-h RP N-Glycans—
The high relative abundance of 2,3,6-linked Man and terminal Xyl are indicative of xylosylated cores (Supplemental Table 3). This was in accordance with the MALDI-MS compositional data, which show that many N-glycans carried a xylose residue. 2-Linked Man was present in high levels indicating that complex mono- and biantennary N-glycans were dominant structures, and the lower abundance of 2,4- and 2,6-linked Man indicated that only a minority of the complex structures were multiantennary. The presence of LacNAc and Lex antennae were supported by terminal Fuc, terminal Gal, 4-linked GlcNAc, and 3,4-linked GlcNAc. Also the detection of 3-linked Gal suggested that these terminal structures may be present in tandem repeats as in ESP N-glycans. Analysis of the PNGase A-released N-glycans showed results similar to that obtained from PNGase F-released N-glycans (Supplemental Table 3). Moreover 3,4,6-linked GlcNAc was absent, and together with the MS/MS data this strongly indicates that 0–3-h RP N-glycans do not contain difucosylated cores.

Taken together, these results show that the 0–3-h RP N-glycan profile contain abundant truncated and complex structures. The dominant groups of 0–3-h RP N-glycans are core xylosylated and monofucosylated. Major terminal antennae structures include Lex and LacNAc, which are also present in tandem repeats, and the less abundant N-glycans carry the S. mansoni-specific terminal structure Galß1->4(±Fuc{alpha}1->3)GlcNAcß1->4(±Fuc{alpha}1->3)GlcNAc. The proposed 0–3-h RP N-glycan structures are shown in Fig. 4.

ESP O-Glycans—
A representative MALDI-MS spectrum of the ESP O-glycans is shown in Fig. 5, and compositional assignments are given in Table III. The most intense molecular ions are m/z 738 and 983, which correspond to glycans with the compositions Hex2HexNAc1-itol and Hex2HexNAc2-itol, respectively (the reducing ends of O-glycans were recovered as oligoglycosylalditols and therefore compositions are denoted as "-itols"). O-Glycan structures were further characterized by MS/MS experiments from which the core structures were confirmed. The MS/MS profile of the molecular ion m/z 738 is shown in Fig. 6 with fragmentation patterns shown as insets.


Figure 5
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  		FIG. 5. MALDI-MS spectrum of permethylated O-glycans from ESPs. Signals observed correspond to singly charged sodiated molecular ions, [M + Na]+.

 

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  		TABLE III Compositions of singly charged sodiated molecular ions, [M + Na]+, observed in the MALDI-MS spectrum of permethylated ESP O-glycans

The reducing ends of O-glycans were recovered as oligoglycosylalditols and therefore compositions are denoted as -itols.

 

Figure 6
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  		FIG. 6. Collision-activated dissociation ES-MS/MS spectrum for the singly charged [M + Na]+ molecular ion m/z 738 with the composition Hex2HexNAc1-itol. The spectrum was obtained using a Q-TOF instrument. Insets show fragmentation patterns from which two distinct isomers (A and B) can be assigned.

 
The fragment ions at m/z 486, 329, and 275 demonstrate the presence of a non-reducing end HexHexNAc unit implying the existence of a reducing end Hex-itol residue. In an attempt to identify this unknown Hex-itol residue, GC-MS linkage analysis on a range of sugar standards containing Hex-itols was carried out. A 4-linked Gal-itol sugar residue was demonstrated to co-elute and produced an electron impact spectrum consistent with the unknown Hex-itol. These results are indicative of a novel core consisting of a Gal residue linked to the protein backbone (Fig. 6, isomer A) rather than the more common GalNAc residue. In addition to this novel core, the molecular ion at m/z 738 also contained a second isomer, in which the non-reducing end consisted of two Hex units, supported by the fragment ions at m/z 259, 502, and 520 (Fig. 6, isomer B). The HexNAc-itol residue is likely to be a GalNAc-itol residue because a 3,6-linked GalNAc-itol residue is present in the linkage analysis (Supplemental Table 4). Therefore, these data supported the presence of two distinct isomers for this molecular ion: (i) isomer A (Fig. 6, isomer A), which contains the novel Gal core, and (ii) isomer B (Fig. 6, isomer B), which contains the previously identified S. mansoni-specific core sequence Galß1->3(Galß1->6)GalNAc (12).

All major molecular ions observed in the ESP O-glycan MALDI-MS spectrum were subjected to MS/MS analyses. These MS/MS analyses together with GC-MS linkage analysis showed that the major ESP O-glycans (e.g. m/z 534, 779, 983, 1157, 1228, 1432, 1607, and 1822) contained the more conventional mucin-type core 1 and 2 structures (Galß1->3(±GlcNAcß1->6)GalNAc). Only a minor proportion also expressed the Galß1->3(Galß1->6)GalNAc core (m/z 738 and 1607) (see example in Supplemental Figs. 6 and 7).

Dominant terminal structures in ESP O-glycans were deduced from MS/MS spectra given by molecular ions such as e.g. m/z 983, 1157, 1228, 1433, 1607, and 1822. These produced fragment ions at 486 (Hex1HexNAc1) and 660 (Fuc1Hex1HexNAc1) consistent with LacNAc and Lex (see example in Supplemental Figs. 6 and 7). Signals such as m/z 1228 and 1432 also produced fragment ions corresponding to the loss of Hex1HexNAc2 from the molecular ion that is consistent with Galß1->3GalNAcß1->4GlcNAc. These observations were also in accordance with the presence of terminal Gal, 4-linked GlcNAc, 3-linked GalNAc, and 3,4-linked GlcNAc in the GC-MS linkage analysis (Supplemental Table 4).

MS/MS spectra of molecular ions such as m/z 1822, whose composition indicated the presence of at least one fucose residue and whose HexNAc content is higher than that of Hex, yielded fragment ions at m/z 456 (Fuc1HexNAc1), 527 (HexNAc2), and 701 (Fuc1HexNAc2) (data not shown). These signals together with the presence of 3-linked GalNAc in the GC-MS linkage analyses (Supplemental Table 4) are consistent with minor components containing LacdiNAc and fucosylated LacdiNAc structures. In addition, the detection of 2-linked fucose in the linkage data (Supplemental Table 4) and the fragment ion at m/z 403 indicates that some fucose residues are further substituted in molecular ions with difucosylated LacdiNAc structures.

In summary, the major ESP O-glycans contain the more conventional mucin-type core 1 and 2 structures. Minor O-glycans carry the S. mansoni-specific Galß1->3(Galß1->6)GalNAc core and the novel type of core in which a Gal residue is attached to the protein backbone. The most abundant terminal structures are LacNAc and Lex, and fucosylated terminal LacdiNAc structures are found in minor components. Fig. 8 illustrates the proposed ESP O-glycan structures.


Figure 8
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  		FIG. 8. Proposed structures of O-glycans from ESPs and 0–3-h RPs.

 
0–3-h RP O-Glycans—
A typical MALDI-MS O-glycan profile for the 0–3-h RP O-glycans is shown in Fig. 7, and compositional assignments are shown in Table 4. O-Glycan sequences were confirmed by MS/MS analyses of molecular ions observed in the MALDI-MS spectrum.


Figure 7
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  		FIG. 7. MALDI-MS spectrum of permethylated O-glycans from 0–3-h RPs. Signals observed correspond to singly charged sodiated molecular ions, [M + Na]+.

 

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  		TABLE IV Compositions of singly charged sodiated molecular ions, [M + Na]+, observed in MALDI spectrum of permethylated 0–3-h RP O-glycans

The reducing ends of O-glycans were recovered as oligoglycosylalditols.

 
MS/MS experiments on the molecular ion at m/z 738 showed that 0–3-h RP O-glycans also contained the novel core identified in ESPs (Supplemental Fig. 10); this is further supported by a 4-linked Gal-itol in the linkage data (Supplemental Table 4). Fragmentation patterns of molecular ions at m/z 534, 738, 779, 983, 1187, 1361, 1391, 1565, 1595, 1607, 1637, 1811, 1841, 1985, 2015, 2189, 2219, and 2464 established that the predominant O-glycan core in 0–3-h RPs is Galß1->3(±Galß1->6)GalNAc (see example in Supplemental Figs. 8 and 9). This is further corroborated by 3- and 3,6-linked GalNAc-itol in the linkage analyses (Supplemental Table 4). Minor 0–3-h RP O-glycans (m/z 1157 and those above 2464), whose HexNAc residue content is higher than that of Hex residues, contained the mucin-type 2 core structures (data not shown).

All molecular ions that were subjected to MS/MS analyses gave rise to fragment ions from which the terminal structures were also deduced. MS/MS spectra of the major molecular ions with relative intensities in the MALDI-MS (Fig. 7) spectrum above 10% relative intensity, e.g. m/z 1187, 1361, 1391, 1565, 1595, 1607, 1637, 1811, 1841, 1985, 2015, and 2189, established that 0–3-h RP O-glycan antennae mostly consisted of LacNAc (m/z 486) and Lex (m/z 660 and 472) (see example in Supplemental Figs. 8 and 9). Fragmentation patterns of higher molecular weight components present at below 5% relative intensity in the MALDI-MS spectrum (Fig. 7) and whose masses were above m/z 2519 produced fragment ions at m/z 403 (Fuc2), 456 (Fuc1HexNAc1), 577 (Fuc3), and 630 (Fuc2HexNAc1) indicating multifucosylated LacdiNAc structures. These results are consistent with the detection of 3-linked GalNAc, 4-linked and 3,4-linked GlcNAc, and 2-linked Fuc in the GC-MS linkage data.

Overall the major 0–3-h RP O-glycans are based on the Galß1->3(Galß1->6)GalNAc core. The lower abundance components carry mucin-type core 2 structures, and an O-glycan with Gal as its reducing end residue is present in trace amounts. The main terminal structures found are LacNAc and Lex, whereas the less abundant O-glycans express highly fucosylated terminal epitopes. A summary of proposed O-glycan structures from 0–3-h RPs is shown in Fig. 8.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here using a combination of mass spectrometric techniques, we have identified the major glycoproteins from cercarial (0–3-h RP) and egg (ESP) secretions and elucidated their N- and O-glycan structures. Our results show that ESPs only contained two major glycoproteins, Omega-1 and IPSE. The former has recently been characterized and linked to a previously described egg protein with hepatotoxic properties consistent with its ribonuclease T2 domain (27). Its in vivo role remains to be established, but possibilities include modulation of host cell-mediated immune responses. Extracellular ribonucleases have been implicated in inhibiting the proliferation of and inducing apoptosis of activated lymphocytes thus causing immunosuppressive activity (28–30). IPSE, also known as alpha-1, has been identified previously to be a major immunogenic secreted glycoprotein of S. mansoni eggs and was the subject of recent functional studies (25, 31, 32). The main glycoproteins present in 0–3-h RPs are inhibitor serpin_c, two glycoproteins with SCP domains (SmSCP_b and SmSCP_c) that are related to venom allergens, a dipeptidyl-peptidase IV, and several unknown glycoproteins. The 0–3-h RPs have been demonstrated to drive Th2 responses (33) and stimulate macrophage cytokine production through toll-like receptor 4-dependent and -independent pathways (34). The specific constituents responsible for these effects are unknown, but glycan components are required for optimal cytokine production (34). Serpins are serine protease inhibitors and could therefore be active against proteins of the host immune system. The structural determination of a protein containing an SCP domain in the human hookworm Necator americanus revealed similarities to chemokines, which could explains its immunomodulatory properties (35).

The comparative glycomics analyses in this study have clearly demonstrated that ESPs and 0–3-h RPs express a complex and vast array of glycans. Detailed structural analyses revealed both common and unique stage-specific structures. The major terminal structures found in both secretions are LacNAc, Lex, and tandem Lex/LacNAc in both the N- and O-glycans. Many functional roles have been attributed to such terminal structures, and thus these epitopes could contribute to the immunomodulatory properties of the secreted glycoproteins and also explain the immunological cross-reactivity between the two secretions.2

Lex has been demonstrated to induce the production of immune mediators such as interleukin-10 and prostaglandin E2 that promote Th2-type responses; it can also act as a Th2-inducing adjuvant (36, 37). Lex-containing glycoconjugates have also been shown to drive the maturation of naïve dendritic cells (DCs) to a DC2 phenotype via the toll-like receptor 4-dependent mechanisms, consequently leading to a Th2-type response (38). The lectin dendritic cell-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) binds Lex and could therefore be involved in the glycan-driven DC2 phenotype maturation (39). In vitro, it has been demonstrated that antibodies against Lex mediate a complement-dependent lysis of granulocytes (40), which may explain the mild neutropenia observed in patients suffering from schistosomiasis (41). Here we show that Lex can also be present in tandem repeats in both secretions. Studies investigating the effects of the multimericity of Lex demonstrated that IgM antibody responses were higher for poly(Lex) than monomeric Lex, and the different presentation of Lex also evokes different classes of antibodies (42). Granulocytes are known to express poly(Lex) on their surface (43); therefore anti-poly(Lex) antibodies in infected individuals might bind more strongly to granulocytes subsequently leading to neutropenia. Also the schistosome-specific terminal structure Galß1->4(±Fuc{alpha}1->3)GlcNAcß1->4(±Fuc{alpha}1->3)GlcNAc present in 0–3-h RP N-glycans is likely to induce immune responses similar to those induced by Lex.

ESP N- and O-glycans and 0–3-h RP O-glycans express LacdiNAc and its fucosylated counterparts. Studies with schistosomes have shown that these epitopes stimulate the production in monocytes of cytokines that induce antibody responses. This capacity is enhanced by the presence of fucosylated LacdiNAc structures (44). LacdiNAc binds to rat macrophage galectin-3, which plays a role in mediating recognition and phagocytosis of LacdiNAc-containing neoglycoconjugates by macrophages (45). The macrophage galactose-type lectin expressed by dendritic cells binds both LacdiNAc and fucosylated LacdiNAc (46) and thus potentially mediates the uptake of secretions by phagocytic cells. The multifucosylated LacdiNAc structures identified in ESP and 0–3-h RP O-glycans resemble those found in the cercarial glycocalyx (15). Studies have shown that immunization with the cercarial glycocalyx enhanced maturation of a secondary infection with cercariae (47).

A major difference between ESP and 0–3-h RP N- and O-glycans is core modifications. Difucosylated cores are prominent in ESP N-glycans, whereas xylosylation is the major core modification in 0–3-h RPs. These types of modifications are not found in mammalian glycoproteins but are common in plants and invertebrates (48, 49). They induce a glycan-specific Th2-type cellular immune response to schistosomes in mice (50). The {alpha}1->3 core fucose in honeybee venom phospholipase A2 is a major allergenic determinant that induces substantial IgE responses in allergic individuals and in helminth infections (49–53). The core xylose residue is also largely responsible for IgE binding and allergenicity of plant- and insect-derived glycoproteins (48). The majority of 0–3-h RP O-glycans are based on the schistosome-specific core Galß1->3(Galß1->6)GalNAc, whereas ESPs possess minor amounts of this core and higher levels of mammalian-like mucin-type core 1 and 2 structures. From a glycobiological perspective a significant finding is the presence of a novel O-glycan core where a Gal residue is attached to the protein backbone. The biological significance of this new form of protein O-glycosylation remains to be elucidated, but it implicates the presence of a novel polypeptide galactose transferase enzyme in schistosomes.

It is evident from our results that secretions contain stage-specific glycan epitopes that may play a role in the interplay with the immediate host environment for successful parasitism. There are several reasons why the egg may release potentially immunogenic glycoproteins. Eggs are deposited by females in the blood vessels of the gut wall in a completely undeveloped state, and the miracidium larva takes 5–7 days to develop before the egg travels to the gut lumen for excretion in the feces (3). This process requires an intact host immune system because egg excretion via the feces is greatly reduced in immunocompromised mice and humans with human immunodeficiency virus infection (54, 55). From this it can be inferred that the potent immunogenicity of the egg secretions is an integral part of their escape mechanism. Furthermore because antibody responses are overwhelmingly directed to glycan epitopes in ESPs (6), a proportion of the glycan structures we have defined must be the key epitopes. The mechanism of egg passage through the gut tissues thus represents an unusual example of a pathogen recruiting host immune responses for its own purposes. One further possibility, by analogy with glycosaminoglycans that serve to sequester chemokines at the surface of mammalian endothelial cells, is that the extensive glycosylation of the ESPs also has a sequestering function (56). The mature egg releases ESPs to assist its passage through the gut tissues. This process would be made more efficient if the secretions were concentrated in its vicinity instead of diffusing away; the glycans could provide such a mechanism by binding to host tissue constituents or by assembling the ESPs into macromolecular complexes. Another property of the glycosylation of ESPs may be to confer resistance to digestion by the two proteases known to be present in the egg secretions (3). A likely explanation for the strong immunogenicity of the cercarial glycans is that they provide a smoke screen to disguise more vulnerable peptide epitopes from immune attack. A more elaborate version of this hypothesis is that the cercarial epitopes are a "matador's cloak." The schistosomulum in transit from epidermis to blood vessel represents a moving target, and it is easy to envisage how glycans released from the acetabular glands into the skin in the initial stages of penetration might decoy leukocytes away from the parasite during the brief period when it is acquiring its immune disguise (57).

Further glycoproteomics studies are being undertaken to determine the glycans expressed by the individual glycoproteins and their site-specific occupancy. This information will allow issues of glycan clustering and presentation, which have been shown to be of critical importance for the binding of glycans to a whole range of lectins (39, 45, 46), to be investigated.


  FOOTNOTES
 
Received, February 2, 2007, and in revised form, May 1, 2007.

Published, MCP Papers in Press, June 4, 2007, DOI 10.1074/mcp.M700004-MCP200

1 The abbreviations used are: RP, released protein; ESP, egg-secreted protein; PNGase F, peptide-N-glycosidase; PNGase A, peptide-N-glycosidase A; Hex, hexose; HexNAc, N-acetylhexosamine; Xyl, xylose; Fuc, fucose; Man, mannose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylglucosamine; Lex, Lewis X; LacNAc, N-acetyllactosamine; LacdiNAc, N,N'-diacetyllactosediamine; DC, dendritic cell; GC, gas chromatography; IPSE, interleukin-4-inducing principle of S. mansoni eggs; Omega-1, hepatotoxic ribonuclease omega-1; 2DE, two-dimensional electrophoresis; SCP, sperm-coating protein; Sm, S. mansoni; CE, cercarial elastase. Back

2 T. M. Kariuki, O. I. Farah, R. A. Wilson, and P. S. Coulson, unpublished data. Back

* This work was supported in part by the Biotechnology and Biological Sciences Research Council (BBSRC) and the Wellcome Trust and by additional funds from the United Nations Development Programme/World Bank/World Health Organization Special Programme for Research and Training in Topical Diseases.

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. Back

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

A BBSRC professorial research fellow. Back

|| To whom correspondence should be addressed. Tel.: 44-207-594-5222; Fax: 44-207-225-0458; E-mail: s.haslam{at}imperial.ac.uk


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