3Department of Medical Chemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands and 4Department of Parasitology, L4-Q, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands
Received on October 5, 1999; revised on December 10, 1999; accepted on December 18, 1999.
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Abstract |
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Key words: fucose/oligosaccharide/antigenicity/enzymatic synthesis/schistosomiasis
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Introduction |
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The structures of several schistosomal glycoconjugates have been elucidated. For example, monomeric and polymeric Lewisx (Lex) structures have been demonstrated on both membrane-bound and secreted glycoproteins of adult worms (Ko et al., 1990; Srivatsan et al., 1992
; Van Dam et al., 1994
). In addition to Lex structures, the adult worms of Schistosoma mansoni also synthesize N-linked glycans containing GalNAcß14GlcNAc (LacdiNAc, LDN) and GalNAcß14(Fuc
13)GlcNAc (LDN-F) (Srivatsan et al., 1992
), the LacdiNAc analog of the Lex blood group antigen. Multifucosylated structures have been found on O-glycans of the cercarial glycocalyx with the following terminal structure: (Fuc
1
2)±Fuc
1
2Fuc
1
3GalNAcß1
4((Fuc
1
2)±Fuc
1
2Fuc
1
3)-GlcNAcß1
3Gal
1
(Khoo et al., 1995
) and on glycolipids of S. mansoni eggs that consist of a backbone of repeating ß14 linked GlcNAc residues substituted with Fuc
12Fuc
13 side chains (Khoo et al., 1997
).
It has long been known that the humoral immune response in schistosomiasis is mainly directed against glycoconjugates (Nash et al., 1981; Aronstein et al., 1983
; Omer Ali et al., 1988
). More recently, several of these carbohydrates have been identified. In early infections, the most pronounced antibody response (IgM) in humans is directed against the gut-associated circulating cathodic antigen (CCA) (Deelder et al., 1989
). Upon infection with S. mansoni, humans and primates generate cytolytic IgM and IgG antibodies against the Lex structure (Nyame et al., 1996
; Van Dam et al., 1996
). Recent studies of Nyame et al. (1999)
showed that sera of mice infected with S.mansoni show IgM and IgG antibody titers toward LacdiNAc.
The aim of this study was to gain more insight into the expression of fucosylated epitopes such as Lex, LacdiNAc, LDN-F, and GalNAcß14(Fuc12Fuc
13)GlcNAc (LDN-DF) throughout the schistosome life cycle. To this end, we synthesized these fucosylated epitopes and report here that antibodies recognizing these structures are generated by Schistosoma infected mice, indicating that they are immunogenic during infection. MAbs recognizing these structures were used to investigate the localization of these epitopes in various stages of the schistosome by an indirect immunofluorescence assay.
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Results |
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Discussion |
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With the identification of several MAbs, tissue specific oligosaccharides throughout the life-cycle stages in Schistosoma can be detected. In this study, several MAbs were found that recognize the monomeric Lex epitope. Previously, a number of MAbs were identified that bind to CCA, the carbohydrate portion of which consists of repeats of the Lex structure (Van Dam et al., 1994; Deelder et al., 1996
). Of the 15 MAbs against CCA tested, only MAbs 221B3-A and 225G11-A (Deelder et al., 1996
) recognized in addition the monomeric Lex epitope. Both these MAbs bind to the surface of cercariae in contrast to the MAbs that exclusively recognize the monomeric form of Lex. These data strongly suggest that the staining of the surface of cercariae is due to the presence of polymeric Lex. The monomeric Lex epitope is not exposed on the surface of cercariae, but only after their transformation into schistosomula its surface expression is initiated (Koster and Strand, 1994
). MAbs that are specific for monomeric Lex specifically recognize the oral sucker of cercariae, which organ supposedly is involved in attachment of the cercariae. During attachment and penetration of the skin, cercariae produce water insoluble secretions, which can be visualized with fluorescent lectins as "kissing marks." (Linder, 1990
). It was reported that MAb 5045B1, which recognizes the Lex epitope, stimulates cercariae to secrete mucous material at the attachment site to which it subsequently can bind (Koster and Strand, 1994
).
Several MAbs were isolated that exclusively recognized the LDN-DF epitope. This structure could be detected on miracidia, egg shells, and whole cercariae and in the excretory system of adult worms including the flame cells. It should be mentioned that one of the MAbs that recognize the LDN-DF epitope (MAb 1145B1-A) was defined in previous studies as a MAb that recognizes repeating carbohydrate epitopes of S.mansoni soluble egg and hatching fluid antigens (Nourel Din et al., 1994). Furthermore this antibody has been reported to show a strong immunoreactivity with schistosomulum surface and with germinal cells in miracidia (Bogers et al., 1994
, 1995). A very similar fluorescence pattern was observed with MAb 128C3/3 (Koster and Strand, 1994
) which recognizes fucose-containing carbohydrate epitopes present on both glycolipids and glycoproteins (Weiss and Strand, 1985
; Weiss et al., 1986
; Dalton and Strand, 1987
). However, while the binding of MAb 128C3/3 could be inhibited with monomeric L-fucose (Levery et al., 1992
), such an inhibition could not be shown for MAb 1145B1-A (H.A.M.Nibbeling, personal communication). Therefore, it is unlikely that MAb 1145B1-A recognizes exactly the same epitope as MAb 128C3/3. Yet another MAb (485D2/7) has been described that reacts with a part of the antigens reactive with MAb 128C3/3 and appears to be S.mansoni specific (Weiss and Strand, 1985
). In this respect MAb 485D2/7 differs from MAb 128C3/3, which also is reactive with other schistosome species. It thus seems that the different MAbs each recognize unique epitope sites in fucosylated structures. It would be of interest to know whether the underlying sugars also form part of these epitopes, for instance by comparing the reactivity of LDN-DF with that of Fuc
12Fuc
13GalNAc (Kamath and Hindsgaul, 1996
).
Of the MAbs tested three reacted with both the LDN-F and LDN-DF structures. Apparently these MAbs recognize a common epitope in LDN-F and LDN-DF. They reacted not only with the excretory system, egg shells, and miracidia, but also with the parenchyma. Comparison of the differences in reactivity of the anti-LDN-DF MAbs and the anti-LDN-F/LDN-DF MAbs (Table II) leads to the conclusion that the LDN-F epitope is probably expressed in the parenchyma and in the head of cercariae. By contrast, whole cercariae were not recognized by these MAbs. It is possible that the third fucose of the multifucosylated side-chain, present on O-glycans of the cercarial glycocalyx (Khoo et al., 1995), blocks the recognition by anti-LDN-F/LDN-DF MAbs. Although LDN-F is present on complex-type N-glycans (Nyame et al., 1989
; Srivatsan et al., 1992
), no MAb was found that exclusively reacted with the LDN-F epitope. One explanation may be that the LDN-F structure as such is not presented to the immune system, because it is only present inside the parasite. Presentation of LDN-DF might in some instances lead to the generation of antibodies that only recognize the LDN-F part of this structure and hence show reactivity with both LDN-F and LDN-DF.
The advantage of using SPR analysis for screening MAbs against neoglycoproteins is that it gives an indication of the affinity of the MAbs for the neoglycoproteins in terms of association rate and affinity constants. Subsequently, the most promising MAbs can be selected and used for improving the immunodiagnosis of schistosomal infection. Indeed, initial studies have shown that SPR technology can be used to analyze the human antibody response against neoglycoproteins in schistosomiasis (A.van Remoortere, G.J.van Dam, D.H.van den Eijnden, I.van Die, and A.M.Deelder, unpublished observations).
In conclusion, the use of different neoglycoproteins can lead to the identification of MAbs that recognize antigens of S.mansoni and may lead to a better understanding of the expression and function of glycans in schistosomes. The identification of the different carbohydrate structures in the life cycle stages of S.mansoni provides more insight into the development of the parasite and into the immune response during infection.
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Materials and methods |
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Parasites
S.mansoni (Puerto Rico strain) adult worms were collected by perfusion of the hepatic portal system of golden hamsters at seven weeks after infection with 1500 cercariae. Cercariae of T.ocellata (Hokke et al., 1998) and S. mansoni were obtained as described previously (Van Dam et al., 1996
).
Monoclonal antibodies
Production of MAbs used in this study has previously been described (Van Dam et al., 1993; Deelder et al., 1996
; Nibbeling et al., 1998
). From a few thousand hybridomas produced over the years in the department of Parasitology (LUMC), a panel of 188 MAbs was selected for putative probable reactivity with carbohydrate epitopes on basis of their reactivity in IFA on male adult worms fixed with Rossmans fixative and on worms or eggs present in frozen sections of infected hamster livers and/or the recognition of repetitive epitopes in immunoelectrophoresis. Isotypes of the MAbs were generally determined in IFA or Dot Immuno Binding Assay using anti-mouse isotype-specific fluorescein isothiocyanate or peroxidase conjugates (Van Dam et al., 1993
).
Enzymatic synthesis of oligosaccharides
An outline of the enzymatic synthesis of LacdiNAc, LDN-F, LDN-DF and Lex is given in Figure 1 linked to the carboxymethyl octyl group (R). Five micromoles of GlcNAc-R was incubated with 6 µmol of UDP-[3H]GalNAc (0.09 Ci/mol) and partially purified UDP-GalNAc:GlcNAcß ß14-N-acetylgalactosaminyltransferase (ß4-GalNAcT) from the albumen gland of Lymnea stagnalis (Mulder et al., 1995
) in an incubation mixture containing 100 mM sodium cacodylate pH 6.5, 20 mM MnCl2, 100 mM NaCl, 600 mM GlcNAc, 4 mM ATP, and 0.5% (v/v) Triton X-100 for 18 h at 37°C. Three micromoles of the produced LacdiNAc-R was fucosylated with 4 µmol of GDP-Fuc and partially purified GDP-Fuc:[Galß1
4]GlcNAcß
1
3-fucosyltransferase (
3-FucT) (1.4 mU) from human milk (DeVries et al., 1993
) in an incubation mixture containing 100 mM sodium cacodylate pH 7.0, 100 mM NaCl, 20 mM MnCl2, 4 mM ATP and 0.5% (v/v) Triton X-100, for 48 h at 37°C (Bergwerff et al., 1993
). Two µmol of LDN-F was further fucosylated with partially purified GDP-Fuc:Fuc
1
2-fucosyltransferase (
2-FucT) from T.ocellata (Hokke et al., 1998
) under the same conditions as described previously, for 72 h at 24°C. During the incubations the progress of the reactions was monitored by HPAEC with pulsed amperometric detection on a CarboPac PA-1 pellicular anion-exchange column (0.9 x 2.5 cm, Dionex) as described previously (Joziasse et al., 1993
). Under very similar conditions GlcNAc-R was incubated with UDP-[3H]Gal (0.09 Ci/mol), UDP-Gal:GlcNAcß ß1
4-galactosyltransferase (ß4-GalT) from bovine milk (Sigma), GDP-Fuc and
3-FucT to synthesize Lex-R (Bergwerff et al., 1993
).
The products of all incubations were isolated on Sep-Pac C-18 cartridges (1 g) and eluted with methanol (Palcic et al., 1988). The eluate was dissolved in 4 ml 50 mM ammonium acetate pH 5.2 and applied to a column (1.6 x 200 cm) of Bio-Gel P-4 (200400 mesh) equilibrated and eluted at a flow of 16 ml/h with 50 mM ammonium acetate, pH 5.2 at 25°C. Fractions of 2 ml were collected and monitored for 3H-radioactivity. Fractions containing the products were pooled and lyophilized. The structure of each product was verified by 1H-NMR spectroscopy as described previously (Hokke et al., 1998
).
Construction of neoglycoproteins
During the enzymatic synthesis, hydrolysis of the carboxymethyl group occurred. To assure total conversion of this group to a carboxylic acid function, the synthesized oligosaccharides were treated with 0.1 M NaOH for 10 min at 80°C. Each of the oligosaccharides LacdiNAc, LDN-F, Lex, and LDN-DF was coupled to BSA via the carboxyl group of the spacer according to a slightly modified procedure of Andersson et al. (1993). Briefly, to a solution of 75 µl dioxane:water (2:1) which contained 1 µmol of oligosaccharide and 1 µmol of triethylamine, 2.4 µmol of N, N, N ',N ''-tetramethyl-(succinimido)uronium tetrafluoroborate (TSTU) was added. The reaction mixture was rotated head over head for 30 min at room temperature. It was subsequently added to 450 µl of a solution of BSA (2 mg/ml) in 0.1 M sodium borate pH 8.5 and incubated head over head for 4 h at room temperature. By addition of hydroxylamine to a concentration of 25 mM, unreacted succinimido esters were inactivated. Each mixture was dissolved in 4 ml 50 mM ammonium acetate pH 5.2 and applied to a Bio-Gel P-4 column (1.6 x 200 cm, 200400 mesh) as described above. Two milliliter fractions were collected and monitored for radioactivity. The void volume peak containing the neoglycoprotein, was collected and lyophilized. The degree of coupling of the oligosaccharides to BSA was estimated by matrix assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry (LDI-1700-XP instrument) using the center of the distribution of the singly-charged molecular ion. Unreacted oligosaccharides were collected and lyophilized for re-use. The obtained neoglycoproteins (LacdiNAc-BSA, LDN-F-BSA, LDN-DF-BSA and Lex-BSA) were used to characterize the MAbs in ELISA and SPR analysis.
Enzyme-linked immunosorbent assay
Flat-bottom 96-well polystyrene microtitration plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with 50 µl of LacdiNAc-BSA, LDN-DF-BSA, or Lex-BSA (1 µg/ml in 0.035 M phosphate-buffered saline (PBS), pH 7.8) or LDN-F-BSA (5 µg/ml), for 15 min at 37 °C while shaking. After each incubation step, the plates were washed with 20-fold diluted PBS. Non-specific binding sites were blocked with 100 µl of 0.3% BSA in PBS. Subsequently the wells were incubated with 50 µl of hybridoma cell supernatant containing 0.3% Tween 20 (Sigma, St. Louis, MO). For detection of bound antibodies, peroxidase conjugates of anti-mouse immunoglobulin were used. After color development using 3,3',5,5'-tetramethylbenzidine as a substrate, the absorbance was measured at 630 nm with an automated microplate reader EL311 (Biotek Instruments, USA).
Surface plasmon resonance spectroscopy
All experiments were performed at 25°C and all injections were carried out automatically by a BIAcore 3000 instrument with a computer interface for system control, data acquisition and data analysis (Biacore AB, Uppsala, Sweden). All buffers were filtered (0.2 µm) and degassed before use. The neoglycoproteins were immobilized at a flow rate of 5 µl/min in 10 mM sodium acetate (pH 4.0) onto a carboxylmethylated dextran CM5 sensor chip by covalent amine coupling according to the instructions of the manufacturer. In short, 35 µl of a mixture containing 100 mM N-hydroxysuccinimide and 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was injected to activate the sensor chip. Initially, the neoglycoproteins (such as BSA, LacdiNAc-BSA, Lex-BSA, LDN-DF-BSA) to be immobilized were injected at a concentration of 20 µg/ml in 10 mM sodium acetate pH 4.0 until an increase of ~4000 response units (RU) was observed. This chip was used to study the binding of MAbs that recognize the LacdiNAc-, the Lex-, or the LDN-DF-epitope. For the determination of MAbs that recognize the LDN-F epitope, a new chip was prepared. LDN-F was immobilized to a level of 7000 RU, while for Lex-BSA, LDN-DF-BSA, or BSA an immobilization level of 2500 RU was accomplished to compensate for differences in carbohydrate density on BSA. Unreacted N-hydroxysuccinimide esters were subsequently deactivated by 35 µl 1 M ethanolamine. All analyses were performed at a flow rate of 5 µl/min using HEPES-buffered saline (HBS) as an eluent. Ten microliters of the MAb supernatants was injected, and after each sample the sensor chip was regenerated with 10 µl of 100 mM HCl. Analysis of the data was performed using the BIA evaluation 3.0 software. After subtraction of the blank value (BSA), the binding pattern of each MAb was interpreted as "binding" or "no binding."
IFA
The IFA was carried out on 5 µm-thick frozen liver sections of S.mansoni infected hamsters, on sections of adult worms fixed with Rossmans fixative (Nash et al., 1977; Nash, 1978
). and on whole cercariae fixed according to the method described by Habeeb (Habeeb, 1987
). Slides were incubated with MAb culture supernatant in a humid atmosphere at 37°C for 60 min, then washed and incubated for 45 min with a FITC conjugate of rabbit anti-mouse immunoglobulin antibody (Nordic, Immunological Laboratories, Tilburg, the Netherlands), diluted 1/50 in PBS containing 0.1 mg/ml Evans blue in a humid atmosphere at 37°C. The slides were observed with a Leica DM-RB fluorescence microscope with the appropriate filter combination for FITC fluorescence. The fluorescence was interpreted visually as positive or negative (Table II). As a negative control fresh culture medium was used.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 Present address: Department of Parasitology, L4-Q, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands
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References |
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