Schistosoma mansoni–infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennaryN-glycan Man3GlcNAc2-R

Alexandra van Remoortere2,3, Christine M.C. Bank2, A. Kwame Nyame4, Richard D. Cummings4, André M. Deelder3 and Irma van Die1,2

2 Department of Molecular Cell Biology, Glycoimmunology Group, Vu University Medical Center, Van Der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands
3 Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, the Netherlands
4 Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Biomedical Research Center, 975 NW 10th Street, Oklahoma City, OK 73104, USA

Received on September 16, 2002; revised on October 15, 2002; accepted on October 15, 2002


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
To reveal the role of cross-reactive carbohydrate determinants in the host immune response in helminth infections and allergenicity, we developed monoclonal antibodies (mAbs) that recognize glycan epitopes present on glycoconjugates from both helminths and plants. An IgM mAb (100-4G11-A) was selected from a panel of anti-glycan mAbs generated from Schistosoma-infected or immunized mice because it recognized both a plant glycoprotein horseradish peroxidase and phospholipase A2 from honeybee venom. On further characterization, it was shown that mAb 100-4G11-A recognizes the truncated biantennary N-glycan Man3GlcNAc2-R. Immunocytochemical analysis and immunoblotting with this mAb demonstrated that Man3GlcNAc2-R structures occur on many glycoproteins of schistosomes and other invertebrates. Remarkably, Man3GlcNAc2-R is also expressed on a restricted number of vertebrate glycoproteins. Our data indicate that this truncated N-glycan is immunogenic in mice during the course of infection. Nevertheless, no elevated antibody levels against this glycan epitope could be detected in sera of individuals infected with Schistosoma mansoni.

Key words: anti-carbohydrate monoclonal antibodies / antigenicity / N-glycans / schistosomes


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Parasites affect millions of people worldwide and cause tremendous suffering and death, especially in developing countries. Glycoconjugates play a crucial role in the pathology of most parasitic helminth infections and are a major focus of the host immune response. Little is known, however, of the molecular basis of these processes, although many helminth carbohydrate structures have been characterized in recent years (see reviews in Cummings and Nyame, 1996Go, 1999Go; Dell et al., 1999Go; Hokke and Deelder, 2001Go). To understand the role of helminth carbohydrate structures in the induction of host immune responses and to evaluate their potential application in diagnostics or vaccine development, it is essential to determine the subcellular localization and stage-specific expression of these structures and establish whether these structures are unique for specific helminths.

Recently, several monoclonal antibodies (mAbs) have been identified that bind to carbohydrate structures of schistosomes. These mAbs recognize carbohydrate structures that may be specifically related to schistosomes and/or other helminths (e.g., GalNAcß1-4[Fuc{alpha}1-2Fuc{alpha}1- 3] GlcNAc [LDN-DF] and Fuc{alpha}1-3GalNAcGlcNAc [F-LDN]) (Kantelhardt et al., 2002Go; Khoo et al., 1995Go, 1997a; Van Remoortere et al., 2000Go)) as well as glycan structures that are found on glycoconjugates of both mammals and helminths (e.g., Galß1-4[Fuc{alpha}1-3]GlcNAc (Lewisx, Lex), GalNAcß1-4GlcNAc [LDN], and GalNAcß1-4[Fuc{alpha}1-3]GlcNAc [LDNF]) (Dell et al., 1999Go; Huang et al., 2001Go; Nyame et al., 1997Go, 1998Go, 2000Go; Van Dam et al., 1996Go; Van den Eijnden et al., 1998Go; Van Remoortere et al., 2000Go; Wuhrer et al., 1999Go). Most of these mAbs were isolated from Schistosoma-infected mice, indicating that the glycan antigens are immunogenic during the course of infection. In addition, humans and primates infected with schistosomes show elevated levels of IgM and/or IgG antibodies against these carbohydrate structures (Van Remoortere et al., 2001Go; Nyame et al., 1998Go), establishing the importance of these glycan epitopes in induction of an anti-glycan humoral immune response.

In schistosomes and other helminths, some N-glycans contain an {alpha}1->3-fucose ({alpha}3-Fuc) linked to the proximal core GlcNAc and/or a ß1->2-xylose (ß2-Xyl) linked to the core ß-mannose (Khoo et al., 1997b, 2001Go; Van Die et al., 1999Go). These N-glycans not only are found on helminth glycoproteins but are typical for glycoproteins derived from plants, insects, and mollusc, but are not found in mammalian glycoproteins (D'Andrea et al., 1988Go; Kubelka et al., 1993Go; Lerouge et al., 1998Go; Van Die et al., 1999Go; Van Kuik et al., 1985Go, 1986Go). Cross-reactive carbohydrate antigens observed in immunoassays of helminth, plant, arthropod, and mollusc extracts have been thought to be due to the presence of these unusual N-glycan core structures. Both core {alpha}3-Fuc and/or ß2-Xyl residues are epitopes for the binding of IgE antibodies to plant allergens, horseradish peroxidase (HRP), honeybee venom phospholipase A2 (PLA2), and helminth glycoproteins (Tretter et al., 1993Go; Van Die et al., 1999Go; Van Ree et al., 2000Go). This is of particular interest as a Th2 type immune response, and high serum IgE levels are typical for helminth-infected hosts as well as for individuals suffering from atopic diseases (Bell, 1996Go).

To unravel the role of these cross-reactive glycoconjugates in the host immune response in helminth infections and allergenicity, we set out to identify and characterize mAbs that bind to carbohydrate epitopes shared by helminths and plants. Here we report the isolation and characterization of mAb 100-4G11-A that recognizes a truncated high-mannose-type N-glycan, Man3GlcNAc2-R, which is expressed on many glycoproteins of invertebrate organisms, as well as on a limited amount of mammalian glycoproteins.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Isolation of an mAb that binds HRP and PLA2
To isolate anti-glycan mAbs that show cross-reactivity between helminths and allergenic plant glycoproteins, a panel of 188 anti-carbohydrate mAbs derived from fusions with spleen cells of mice infected with cercariae of Schistosoma spp. or immunized with hatching fluid from Schistosoma eggs (Van Remoortere et al., 2000Go), was screened for reactivity with HRP. Six mAbs recognized HRP, and not human transferrin (Tr) or BSA (Table I). To investigate whether the mAbs showed cross-reactivity with other glycoproteins, they were tested in a subsequent screening for binding to several glycoproteins with defined glycan structures. A structural overview of the major N-linked glycans of the glycoproteins tested is depicted in Figure 1. Of the six HRP-positive mAbs, five also recognized PLA2, which resembles HRP because it contains truncated N-glycans modified with a core {alpha}3-Fuc epitope, but in contrast to HRP, PLA2 carries no ß2-Xyl epitope (Table I). None of the six mAbs showed binding to a neoglycoprotein carrying a biantennary N-glycan modified with a ß2-xylose (data not shown). These data demonstrate the occurrence of mAbs derived from Schistosoma-infected or immunized mice that cross-react with both HRP and PLA2, indicating that these mAbs recognize a shared carbohydrate epitope on these glycoproteins.


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Table I. mAbs isolated from S. mansoni-infected or immunized micethat react with HRP

 


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Fig. 1. Overview of carbohydrate structures used in this study. (AD) Structures of the major N-linked glycans from the glycoproteins HRP, PLA2, human transferrin, and RNAse B (Man5GlcNAc2); (E, F) carbohydrate structures coupled to BSA: as/agGP-F2 (E) and core-{alpha}1,3-fucosylated as/agGP-F2 (F); (G) recombinant proteinGST-SfManI, produced in insect cells; and (H) Man3GlcNAc2, the proposed minimal epitope of 100-4G11-A.

 
mAb 100-4G11-A binds Man3GlcNAc2-R
Because mAb 100-4G11-A (hereafter designated as 100-4G11-A) showed the strongest binding to both HRP and PLA2, this mAb was selected for further characterization. To identify which glycoform of PLA2 was recognized by 100-4G11-A, PLA2 glycoforms were separated by affinity chromatography with Conacavalin A (ConA), a lectin that specifically recognizes biantennary complex-type, hybrid-type, and high-mannose-type N-glycans, but does not recognize either tri-/tetraantennary complex-type N- glycans or O-glycans (Figure 2A). Four pools were made from the individual fractions as indicated in Figure 2A and analyzed by western blotting for binding with 100-4G11-A. 100-4G11-A recognized only glycoproteins in fraction 4, which consists of PLA2 proteins that bound to the ConA column (Figure 2B). Because a core {alpha}3-Fuc epitope is present on both PLA2 and HRP, we tested whether this core fucose is required for recognition by 100-4G11-A by analyzing the fractions for reactivity with affinity purified polyclonal anti-serum specific for core {alpha}3-Fuc residues (Faye et al., 1993Go). The anti-core {alpha}3-Fuc antibodies recognized fractions corresponding with both ConA-binding and nonbinding fractions. These data demonstrate that 100-4G11-A is not specific for the core {alpha}3-Fuc epitope but only recognizes PLA2 glycoform(s) that bind to ConA.




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Fig. 2. Analysis of PLA2 glycoforms by ConA affinity chromatography. (A) Honeybee PLA2 glycoforms were separated by FPLC affinity chromatography with the lectin ConA. Elution of the bound glycoforms of PLA2 was performed with an increasing gradient from 0 to 33 mM methyl {alpha}-D-mannopyranoside in PBS in 40 min as indicated. (B) Western blot of pooled fractions (as indicated in A) with 100-4G11-A. Approximately 2.5 µg of each of the four fractions and PLA2 were analyzed by western blot using affinity-purified anti-serum specific for core {alpha}3-Fuc and mAb 100-4G11-A. As secondary antibody, AP-conjugated goat anti-rabbit or mouse antibody, was used, respectively.

 
In addition, several monosaccharides were tested for their ability to inhibit the binding between 100-4G11-A and PLA2 in enzyme-linked immunosorbent assay (ELISA) (Figure 3). Methyl {alpha}-D-mannopyranoside completely blocked the binding of the mAb at a concentration of 50 mM, whereas D-mannose was also effective as an inhibitor. In contrast, D-GlcNAc, L-fucose, or D-xylose did not interfere with the binding even at high concentrations. These results suggest that mannose is an essential constituent of the epitope that is recognized by 100-4G11-A.



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Fig. 3. Inhibition of binding of 100-4G11-A to PLA2 by monosaccharides. The ability of several monosaccharides to inhibit the binding between 100-4G11-A and PLA2 was determined by ELISA. Plates were coated with PLA2. mAb 100-4G11-A was preincubated with monosaccharides in concentrations as indicated; binding of 100-4G11-A was measured using AP-labeled goat-anti mouse IgM.

 
To characterize the carbohydrate specificity of 100- 4G11-A in more detail, the recognition profile of this mAb was analyzed by ELISA using several (neo)glycoproteins that contain glycan epitopes resembling those found on PLA2 (for structural details, see Figure 1). 100-4G11-A did not bind to RNAse B, containing high mannose-type N-glycans with the composition Man5–9-GlcNAc2. No significant binding was observed to glutathione-S- transferase (GST)-SfManI, a soluble GST fusion protein produced in insect cells that carries a monoantennary N-glycan containing a single terminal Galß1,4GlcNAc (LacNAc) unit on the {alpha}1,3-linked mannose of the N-glycan core structure. These data indicate that neither high mannose of the size composition Man5–9-GlcNAc2- nor hybrid-type N-glycans are recognized by 100-4G11-A. Similarly, 100-4G11-A did not recognize either neoglycoproteins carrying the biantennary glycopeptides as/agGP-F2 or as/agGP-F2 modified with a core-{alpha}1,3Fuc or neoglycoproteins containing the oligosaccharides LDN or LDNF (not shown).

The results suggested the possibility that 100-4G11-A might recognize a truncated high-mannose-type N-glycan smaller than Man5–9-GlcNAc2. To address this, we treated both as/agGP-F2-BSA and as/agGP-F2-{alpha}1,3Fuc-BSA with N-acetylglucosaminidase to generate the core N-glycan Man3-GlcNAc2-R. Likewise, GST-SfManI was treated with both ß-galactosidase and N-acetylglucosaminidase to generate Man3-GlcNAc2-R. The resulting Man3-GlcNAc2-R structures on as/agGP-F2-BSA, as/agGP-F2-{alpha}1,3Fuc-BSA, and GST-SfManI were strongly bound by 100-4G11-A (Figure 4). These results demonstrate that 100-4G11-A recognizes the truncated high-mannose-type N-glycan Man3-GlcNAc2-R. Our data strongly suggest that the antibody requires both a free terminal {alpha}1,3-linked and {alpha}1,6-linked mannose in the trimannosyl core structure Man{alpha}1-3(Man{alpha}1-6)Manß- to recognize its ligand. 100-4G11-A does not recognize either Man5GlcNAc2-R, the major N-glycan on RNAse B that contains a free {alpha}1,3-mannose, or the monoantennary N-glycan on GST-SfManI that has a free {alpha}1,6-mannose. The presence of a core {alpha}1,3-Fuc linked to the inner GlcNAc does not affect the binding (Figure 4).



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Fig. 4. mAb 100-4G11-A binds to Man3GlcNAc2-R. The binding of 100-4G11-A to different glycoproteins and neoglycoproteins that contain glycan structures that occur on PLA2 was determined by ELISA. Some antigens were pretreated with glycosidases, as indicated by H (N-acetylglucosaminidase) or G (ß4-galactosidase). mAb 100-4G11-A only binds to antigens that were pretreated with the glycosidases, resulting in the structure Man3GlcNAc2-R.

 
Antibody 100-4G11-A binds to many glycoproteins of schistosomes and other helminths
To determine the occurrence and localization of Man3-GlcNAc2-R epitopes in Schistosoma and other helminth species, the reactivity of 100-4G11-A to protein-linked glycans of different life-cycle stages of S. mansoni and S. haematobium was determined by western blotting. All life-cycle stages of both Schistosoma species contain glycoproteins that react strongly with this mAb (Figure 5), whereas no binding was observed to (glyco)lipids extracted from S. mansoni eggs and adult worms (data not shown). Immunofluorescence analysis (IFA) of different schistosome life-cycle stages showed that 100-4G11-A predominantly recognizes the tegument and cells in the parenchyma of the adult worm, spots in the eggs, and the oral sucker of cercaria (Figure 6). Several extracts derived from other helminths were analyzed for the presence of glycoproteins binding to 100-4G11-A. Most of the extracts contained several glycoproteins that were recognized by this mAb (Figure 7A). Interestingly, some proteins of the excretory/secretory antigen of Haemonchus contortus showed strong binding, whereas no reactivity was observed in the adult stage (Figure 7A, lane 7–8). Western blot of adult Caenorhabditis elegans extracts showed little if any staining with 100-4G11-A, whereas staining was observed toward extracts from the eggs and larval stages (Figure 7A, lanes 1–5). Our data demonstrate that the truncated N-glycan recognized by 100-4G11-A is present in all helminths investigated and that its expression in some of these helminths is stage-specific.



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Fig. 5. Western blot analysis of glycoproteins of various life-cycle stages of Schistosoma. Proteins within extracts of Schistosoma were separated by SDS–PAGE, and the presence of glycoproteins carrying Man3GlcNAc2-R was identified by immunoblotting with 100-4G11-A. Extracts werederived from adults (A), eggs (E), and cercariae (C) from S. mansoni(lanes 1–3) and from S. haematobium (lanes 4–6).

 


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Fig. 6. Immunofluorescence patterns of schistosomes with 100-4G11-A. Cercaria (left) and a frozen section of a liver of S. mansoni–infected hamster (right) were incubated with 100-4G11-A, followed by staining with a fluorescein isothiocyanate conjugate of a rabbit anti-mouse Ig antibody. Fluorescence (illustrated as white spots in the photographs) was observed on the tegument (T), in structures in the parenchyma (P) of the adult worm, in structures in the egg (S), and on the oral sucker of the cercaria (O).

 


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Fig. 7. Binding of mAb 100-4G11-A to glycoproteins within extracts of different tissues or cell lines of invertebrates and mammals. Reactivity of100-4G11-A with extracts of different helminths (A) and extracts of different tissues or cell lines of invertebrates (B) and mammals (C). Proteins within the extracts were separated by SDS–PAGE and transferred to nitrocellulose by western blotting. The blots were incubated with 100-4G11-A, followed by incubation with goat anti-mouse IgM AP or PO conjugates. (A) Samples tested were various stages of the free living nematode C. elegans (egg [E], larval stages L1, L2/3, L4/young adult [L4], and adult worm [A]), the parasitic nematode Haemonchus contortus (excretory secretory antigens [ES] and adultworms [A]), and extracts derived from the adult stage from the parasitic nematodes Toxocara canis (Tc), Trichinella spiralis (Ts), Dirofilaria immitis (Di), the trematode Fasciola hepatica (Fh), and the cestode Hymenolepos diminuta (Hd). (B) The insect glycoprotein PLA2 and glycoproteins of the albumen gland of Lymnea stagnalis. (C) Extracts of veal kidney, veal brain, rat macrophage cell line (NR 8383), human liver, and human cerebellum.

 
Antibody 100-4G11-A recognizes many invertebrate glycoproteins and a restricted number of glycoproteins in mammals
Several extracts of different tissues or cell lines from vertebrates and invertebrates were analyzed for the presence of glycoproteins recognized by 100-4G11-A. The mAb showed a strong reactivity with a range of glycoproteins of the albumen gland and prostate gland of the snail Lymnaea stagnalis (i.e., Figure 7B, lane 2), the intermediate host for the avian schistosome Trichobilharzia ocellata. Remarkably, several glycoproteins in tissues of vertebrate species showed specific binding with the mAb, such as glycoproteins in human liver and the rat macrophage cell line NR 8383 (Figure 7C). Because 100-4G11-A recognizes glycoproteins in brain tissues, we were interested in whether myelin contains Man3GlcNAc2 N-glycans. Analysis of myelin by FACScan demonstrated that this mAb indeed bound to mouse and rat myelin (Figure 8). In contrast, the mAb does not recognize glycoproteins in the white matter of the brain, in astrocytes, or in the cerebral endothelial cell line HBZCZ, nor glycoproteins in veal kidney and heart, or in human placenta (data not shown). In conclusion, the truncated N-glycan recognized by 100-4G11-A is abundantly expressed on glycoproteins of the snail L. stagnalis and is found on insect glycoproteins (i.e., honeybee PLA2). Remarkably, this carbohydrate epitope is also found on a restricted number of glycoproteins in some vertebrate tissues.



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Fig. 8. Binding of 100-4G11-A to myelin. FACScan analysis of mouse myelin (continuous line), myelin with R{alpha}M-PE (bold continuous line) or with 100-4G11-A followed by incubation with R{alpha}M-PE (broken line). Similar results were obtained with rat myelin (not shown).

 
No significant antibody levels are found against Man3GlcNAc2-R in individuals infected with schistosomes
Because Man3GlcNAc2-R is a glycan that is expressed on all stages of schistosomes, allowing an early and continuous contact with the host immune system, we investigated whether sera of individuals infected with S. mansoni contain antibodies against this epitope by ELISA. Sera of individuals infected with S. mansoni contained both significant levels of IgM and IgG antibodies against honeybee PLA2 as compared to the antibody levels in sera of uninfected individuals. However, these antibodies did not recognize the truncated N-glycan Man3GlcNAc2-R because no increase in antibody levels (IgG or IgM) could be detected in N-acetylglucosaminidase treated as/agGP-F2 (Figure 9). In addition, no IgE antibodies could be detected that reacted with Man3GlcNAc2-R, whereas an IgE response was observed against PLA2 in several patients (data not shown).



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Fig. 9. Levels of IgG and IgM recognizing the neoglycoprotein Man3GlcNAc2-BSA and PLA2 in sera of individuals, infected (I) with S. mansoni, and uninfected (C). The binding of antibodies to PLA2 and Man3GlcNAc2-BSA was measured by ELISA. Glycoproteins were coated at a concentration of 5 µg/ml.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this study we searched for mAbs recognizing glycan epitopes that are shared by antigens of schistosomes and allergenic plants, such as the immunogenic core {alpha}1,3-Fuc or ß1,2-Xyl epitopes that are epitopes for binding of cross-reactive IgE antibodies to plant allergens and helminth glycoproteins (Tretter et al., 1993Go; Van Die et al., 1999Go; Van Ree et al., 2000Go). To select mAbs specific for such cross-reactive glycan epitopes, a panel of anti-carbohydrate mAbs isolated from Schistosoma-infected or immunized mice was screened with HRP. Out of six mAbs that recognized HRP, five also showed reactivity with honeybee PLA2, implying that the epitope is a glycan structure present on both glycoproteins. Our results showed that 100-4G11-A recognizes truncated N-glycans that contain a trimannosyl unit Man3GlcNAc2-R with both a free {alpha}1,3- and {alpha}1,6-linked mannose. The presence of a core {alpha}1,3-Fuc residue on the inner core GlcNAc does not affect the binding.

Relatively large amounts of the truncated N-glycan recognized by 100-4G11-A have been found previously on type II variant surface glycoproteins of the parasite protozoan Trypanosoma brucei (Zamze et al., 1991Go), and on immunodominant antigens of the parasite helminth Trichinella spiralis (Reason et al., 1994Go). Here we show that other helminths from different orders of the phyla platyhelminths and nematodes also synthesize glycoproteins containing this truncated N-glycan. In some helminths, such as H. contortus and the free-living nematode C. elegans, expression of this glycan epitope is developmentally regulated. In invertebrates, the truncated N-glycan structure is apparently occurring abundantly. Recently, the presence of these structures has been demonstrated on recombinant neuropsin, synthesized in insect cells (Takahashi et al., 1999Go). 100-4G11-A may therefore be a useful tool in identifying the glycosylation on recombinant glycoproteins produced in insect cells. As far as we know, however, this is the first report that shows the presence of these truncated structures in vertebrates, and this observation poses questions about the role or significance of such N-glycans that may be formed by degradation of complex-type N-glycans.

Despite the isolation of mAbs from infected mice against the Man3GlcNAc2 glycan structure, we could not detect significantly higher levels of antibodies against this epitope in sera of individuals infected with S. mansoni in comparison with sera of controls. The Man3GlcNAc2-R epitope is found on human endogenous glycoproteins, which may explain why only low antibody levels against this glycan epitope are found in infected individuals. Similarly, antibody levels against the endogenous monomeric Lex carbohydrate epitope are low in individuals infected with schistosomes, whereas mAbs recognizing this structure have been isolated, as reported previously (Nyame et al., 1998Go; Van Remoortere et al., 2001Go). Despite the low antibody levels against Lex, however, LNFPIII, which contains Lex, is a potent inducer of a Th2 response and acts as an adjuvant (Okano et al., 2001), indicating that glycan structures may have indirect effects on the host humoral immune system.

Unexpectedly, none of the 188 mAbs tested recognized neoglycoproteins carrying N-glycans with a core ß1,2-Xyl or a core {alpha}1,3-Fuc. Because these core modifications have been demonstrated on both adult worms and the eggs of schistosome glycoproteins (Khoo et al., 1997b, 2001Go; Van Die et al., 1999Go) and do not occur on mammalian glycoproteins, they are expected to be immunogenic in mice. The reason for our failure to detect hydridomas secreting such mAbs is not known.

Our data indicate that the generation of antibodies against glycan antigens differs in mouse and human infections. The murine model has been widely used to dissect the immune responses in S. mansoni infection. Recently it has become clear that immunological data obtained from schistosome-infected mice and humans may differ profoundly, one of the differences being that a Th2 response in infected mice is associated with pathology, whereas in infected humans the same type of response is thought to be beneficial for the host (Boros, 1994Go; Cheever et al., 1997Go; Fallon, 2000Go). Because glycoconjugates play an important role in the pathogenesis and host immune responses in schistosomiasis, such differences may be partially due to differences in endogenous glycosylation between mice and humans, contributing to differences in the discrimination between self and non-self antigens upon infection.

Characterization of the structure of parasite glycans by conventional analytical methods such as mass spectrometry and nuclear magnetic resonance (NMR), and analysis of their functions is hampered by the small amounts of parasite material that are usually available. Anti-glycan mAbs with a well-defined specificity, such as 100-4G11-A, will be very valuable research tools to provide us with more insight in the expression, distribution, and function of glycan epitopes in the different life-stages of schistosomes and other parasites and, importantly, also in defining interactions between parasite glycans and the host immune system.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials and parasites
HRP, honeybee venom PLA2, human transferrin, methyl {alpha}-D-mannopyranoside, D-mannose, L-fucose, D-xylose, N-acetylglucosamine (GlcNAc), and jackbean ß-galactosidase were purchased from Sigma (St. Louis, MO). All parasitic helminths were obtained as described previously (Van Die et al., 1999Go). Affinity-purified polyclonal antiserum specific for core {alpha}1-3-fucose was provided by Dr. Veronique Gomord (Faye et al., 1993Go). GST-SfManI is a soluble, GST-tagged version of an Sf9 class I mannosidase (Kawar et al., 2000Go) produced in the insect cell line Sf+f4GalT (Hollister et al., 1998Go) and contains a monoantennary ({alpha}1,3-mannose elongated) N-glycan, terminating in galactose (provided by Dr. Jason Hollister, University of Wyoming).

mAbs
Production of mAbs used in this study has been described previously (Van Dam et al., 1993Go). From a few thousand hybridomas produced over the years in the Department of Parasitology (LUMC), a panel of 188 mAbs was selected for screening based on putative reactivity with carbohydrate epitopes (Van Remoortere et al., 2000Go).

Construction of neoglycoproteins
Asialo/agalacto glycopeptides from human fibrinogen (as/agGP-F2) were prepared from asialo-fibrinogen by extensive pronase digestion as described previously (Nemansky and Van den Eijnden, 1993Go) followed by enzymatic degalactosylation with jackbean ß-galactosidase. Core {alpha}3-fucosylated as/agGP-F2 (as/agGP-F2-{alpha}1,3Fuc) was synthesized as described in Van Tetering et al. (1999)Go. All products were structurally characterized by 1H-NMR spectrometry as described (Van Tetering et al., 1999Go). The glycopeptides as/agGP-F2 and as/agGP-F2-{alpha}1,3Fuc (75 nmol each) were activated with ninhydrin and coupled to bovine serum albumin (BSA) by reductive amination essentially as described in Mencke et al. (1987)Go. Subsequently, the resulting neoglycoproteins were separated from unincorporated BSA by ConA lectin affinity chromatography. Matrix-assisted laser desorption and ionization time-of-flight analysis showed the presence of about two N-glycans per molecule BSA. The oligosaccharides LDN and LDN-F were enzymatically synthesized and coupled to BSA as described previously (Van Remoortere et al., 2000Go).

ELISA
Flat-bottom 96-well polystyrene microtitration plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with 50 µl antigen (5 µg/ml in 0.035 M phosphate buffered saline [PBS], pH 7.8) for 1 h at 37°C. After each incubation step, the plates were washed with 20-fold diluted PBS. Nonspecific binding sites were blocked with 100 µl 0.3% BSA in PBS. Sera and secondary antibodies were diluted in PBS containing 0.1% BSA and 0.3% Tween 20. For detection of bound antibodies, alkaline phosphatase (AP)- or peroxidase (PO) conjugates of anti-mouse or anti-rabbit immunoglobulin were used. As substrate for AP, 3 mM 4-nitrophenyl phosphate (Boehringer Mannheim, Mannheim, Germany) in 0.1 M diethanolamine containing 0.5 mM MgCl2, pH 9.6, were used; the absorbance was read after 30 min and overnight at a wavelength of 405 nm. 3,3,5,5'-Tetramethylbenzidine and H2O2 in 0.1 M acetate, pH 5.5, was used as a substrate for PO, the reaction was terminated with H2SO4, and the absorbance was measured at 450 nm.

In the inhibition study 100-4G11-A was preincubated with {alpha}-D-mannopyranoside, D-mannose, L-fucose, D-xylose, and D-GlcNAc in a final concentration range of 2.5 to 500 mM for 1 h at 37°C before use.

When indicated, neoglycoproteins were treated after coating with N-acetylglucosaminidase (12 mU/ml) in 50 mM sodium citrate, pH 5.0, at 37°C overnight.

To identify the presence of human antibodies binding to PLA2 and N-acetylglucosaminidase-treated as/agGP-F2-BSA, human sera were diluted 1/200 for IgM and IgG and 1/10 for IgE and biotin-conjugated goat anti-human IgG (1/10,000 dilution; Vector, Burlingame, CA), goat anti-human IgE (1/1000; Vector), or goat anti-human IgM (1/5000, Vector) were used. Bound antibodies were detected after incubation with streptavidin-AP (1/4000) as described.

Fractionation of different glycoforms of honeybee PLA2
Honeybee PLA2 (500 µg) was subjected to FPLC ConA-affinity chromatography using a flow rate of 0.5 ml/min with detection at 280 nm. Coupling of 30 mg Con A (Sigma) to a 1 ml HiTrap-column (Amersham Biosciences AB, Uppsala, Sweden) was performed according to the manufacturer's instructions. The column was equilibrated with PBS. Elution of ConA binding PLA2 glycoforms was performed by gradient elution with 0–33 mM methyl {alpha}-D-mannopyranoside in PBS in 40 min, followed by elution with 33 mM methyl {alpha}-D-mannopyranoside in PBS for 20 min.

SDS–PAGE and western blotting
Helminth extracts and control samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 12.5–15% gels using the Mini-Protean II system (Bio-Rad, Veenendaal, the Netherlands). Western blotting and antibody reactions were performed essentially as described previously (Agterberg et al., 1993Go). For detection of 100-4G11-A and polyclonal anti-core {alpha}3-Fuc antibodies, AP or PO conjugates of anti-mouse or anti-rabbit immunoglobulins, respectively, were used. Bound antibodies were detected using x-phosphate/5-bromo- 4-chloro-3-inodyl-phosphate (Boehringer Mannheim) and 4-nitrobluetetrazoliumchloride (Boehringer Mannheim) in the case of AP conjugates or using Enhanced ChemiLuminescence reagent (Amersham) and autoradiography in the case of PO conjugates.

IFA
The IFA was performed on frozen liver sections (5 µm thick) of S. mansoni-infected hamsters and whole cercariae as reported previously (Van Dam et al., 1993Go) using 100-4G11-A and a fluorescein isothiocyanate conjugate of rabbit anti-mouse immunoglobulin antibody (Nordic Immunological Laboratories, Tilburg, the Netherlands). The slides were observed with a Leica DM-RB fluorescence microscope.

Analysis of binding of the mAb 100-4G11-A to myelin
Rat myelin was incubated with 100-4G11-A for 30 min on ice. Subsequently, the myelin was incubated with rabbit anti-mouse-phycoerythrin (R{alpha}M-PE) for 30 min on ice. After each step the myelin was washed with PBS containing 0.1% BSA. As control, myelin was incubated with the second antibody. The binding was analyzed by FACScan (Becton-Dickinson, Franklin Lakes, NJ).


    Acknowledgements
 
We thank Drs. J. Hollister and D. Jarvis for their generous gift of the recombinant protein GST-SfManI, Dr. J.J.A. Hendriks for gift of myelin, and Dr. Y. van de Burght for MALDI-TOF analysis. The technical assistance and advice of Dr. D. Poland, Mr. Rene van Zeyl, Ms. Dieuwke Kornelis, Ms. Wietske Schiphorst, and Ms. Angelique van Tetering is highly appreciated. We are grateful to Dr. C. H. Hokke and Prof. D. H. van den Eijnden for helpful discussion. This study was supported by the Netherlands Foundation for Chemical Research and Life Sciences Foundation with financial aid from the Netherlands Organization for Scientific Research and by a grant (AI 47214) from the National Institutes of Health.

1 To whom correspondence should be addressed; e-mail: im.van_die.medchem{at}med.vu.nl Back


    Abbreviations
 
AP, alkaline phosphatase; BSA, bovine serum albumin; Concavalin A, ConA; ELISA, enzyme-linked immunosorbent assay; F-LDN, Fuc{alpha}1-3GalNAcGlcNAc; GST, glutatione-S-transferase; HRP, horseradish peroxidase; IFA, immunofluorescence analysis; LDN, LacdiNAc, GalNAcß1-4GlcNAc; LDN-DF, GalNAcß1-4(Fuc{alpha}1-2Fuc{alpha}1-3)GlcNAc; LDN-F, GalNAc({alpha}1-3Fuc)GlcNAc; Lex, Lewisx, Galß1-4(Fuc{alpha}1-3)GlcNAc; mAb, monoclonal antibody; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; PLA2, phospholipase A2; PO, peroxidase; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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