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Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes
Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Gal[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lex and sLex expression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S.haematobium and S. japonicum contain the Lex antigen, but no sialyl Lex antigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lex antigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica, the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus, and the free-living nematode Caenorhabditis elegans. Neither Lex nor sialyl-Lex is detectable in these other helminths. Furthermore, none of the helminths, including schistosomes,express Lea, Leb, Ley, or the H-type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin, which binds Fuc[alpha]1->3GlcNAc, and Wisteria floribunda agglutinin,which binds GalNAc[beta]1->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lex antigen, whereas many different helminths may express [alpha]1,3-fucosylated glycans and the LDN motif.
Key words: Lewis x antigen/Schistosoma mansoni/Schistosoma haematobium/Schistosoma japonicum/Haemonchus contortus/Dirofilaria immitis/Fasciola hepatica/Caenorhabditis elegans
Introduction
Adult S.mansoni express the Lewis x (Lex; Gal[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1->R), but not the sialyl Lewis x (sLex; NeuAc[alpha]2->3Gal[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1->R), antigen on both membrane and secreted glycoproteins (Ko et al., 1990; Srivatsan et al., 1992a; Van Dam et al., 1994; Cummings and Nyame, 1996). Glycans containing the Lex antigen constitute a major target for host immune response during infection. Cytolytic IgM and IgG antibodies reactive to Lex are generated in both S.mansoni infected humans and animals (Ko et al., 1990; Nyame et al., 1996, 1997; Van Dam et al., 1997). Expression of Lex is developmentally regulated in S.mansoni, where the antigen is detectable on S.mansoni schistosomula and adults, but it appears to be absent from cercariae (Koster and Strand, 1994).
Adult S.mansoni also synthesize N-glycans containing the lacdiNAc sequence (LDN; GalNAc[beta]1->4GlcNAc-R) and LDN sequences in which GlcNAc residues are [alpha]1,3-fucosylated to generate LDNF (GalNAc[beta]1->4(Fuc[alpha]1->3)GlcNAc-R) (Srivatsan et al., 1992b). LDNF-related structures have also been described in cercarial and egg glycoconjugates of S.mansoni (Khoo et al., 1995, 1997a,b). Interestingly, LDNF-related structures and Fuc[alpha]1->3GlcNAc linkages, but not the Lex antigen, have been identified in microfilaria of the parasitic nematode D.immitis (dog heartworm) (Kang et al., 1993) and in adults of the ruminant nematode H.contortus (Haslam et al., 1996; R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, unpublished observations). Thus, while LDN and LDNF may be common forms of N-glycosylation for both trematodes (flatworms) and nematodes (roundworms), Lex expression may be restricted to members of the trematode family, and possibly only to schistosomes.
Fucosylated glycans have been shown in mammalian systems to be important recognition determinants in cell-cell interactions. In human neutrophils, [alpha]1,3-fucosylated glycans in cell surface glycoproteins are required for selectin-mediated adhesion to the endothelium (McEver and Cummings, 1997). The roles of Lex, LDN, and LNDF glycans in S.mansoni are not known. Furthermore, the distribution of Lex, sLex, LDN, LNDF, and other biologically important fucosylated glycans among schistosomes and other helminths has not been described.
We have investigated the expression of Lex, sLex, other Lewis antigens, LDN, and LNDF in adults of two other major human schistosome species, S.haematobium and S.japonicum, and in a group of selected flatworms and roundworms including D.immitis, H.contortus, the related parasitic flatworm F.hepatica, and the free-living roundworm C.elegans. Our results demonstrate that Lex expression is common among schistosomes, but absent in other helminths. The presence of Lex antigen in schistosomes is associated with anti-Lex responses in infected humans and animals. Conversely, our new results, combined with recent studies on the glycosyltransferase activities in various helminths (R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, unpublished observations), indicate that all helminths can generate LDN and LDNF-related structures. These findings suggest that [alpha]1,3-fucosylation of glycans may be important in developmental processes for all the worms, whereas Lex expression may be specifically involved in schistosomiasis.
Results
Lex antigen expression in S.haematobium and S.japonicum and lack of expression in other helminths
To determine whether Lex and/or sLex is expressed by other schistosome species and helminths in general, extracts of S.haematobium and S.japonicum, F.hepatica, D.immitis, H.contortus, and C.elegans were analyzed by ELISA using mAb CD-15 to Lex and mAb CSLEX-1 to sLex. As positive controls, extracts of COS7 cells expressing human FTIII (COS7/FTIII), which express both Lex and sLex determinants (Kukowska-Latallo et al., 1990), and extracts of S.mansoni, which express Lex, but not sLex, were also analyzed. Extracts of wild-type COS7 cells, which lack Lex and sLex determinants, were analyzed as negative controls (Nyame et al., 1996; R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, unpublished observations). Lex was detected in extracts of both S.haematobium and S.japonicum, but not in extracts of F.hepatica, H.contortus, D.immitis, or C.elegans (Figure
Figure 1. ELISA of Lex and sialyl Lex antigen expression. Microtiter wells were coated with 10 mg/ml solution of total detergent extracts of the indicated adult trematodes and nematodes and probed for the presence Lex and sialyl Lex antigens by ELISA using mAbs specific for Lex (A) or sialyl Lex (B) as described in Materials and methods. Extracts of COS7 cells were analyzed as negative controls, while extracts of S.mansoni and COS7/FTIII were analyzed as positive controls. Each assay was performed in triplicate, and the results represent averages of the three determinations.
In a complementary approach, Western blot analyses for expression of Lex were also performed using total glycoproteins from these organisms. Extracts of S.mansoni, COS7/FTIV cells and wild type COS7 cells were analyzed as controls. Lex expression was observed in a number of high molecular weight glycoproteins in S.haematobium and S.japonicum (Figure
Figure 2. Western blot analysis for glycoproteins bearing Lex. Extracts of the indicated trematodes and nematodes (25 µg) were separated by SDS-PAGE under reducing conditions and the presence of glycoproteins bearing Lex were identified by immunoblotting using anti-CD-15 mAb and ECL detection system as described in Materials and methods. Extracts of COS7 cells were used as negative controls, while extracts of S.mansoni and COSFTIV were positive controls.
Absence of other Lewis antigens on schistosome species and other helminths
We next examined whether human schistosomes and the other helminths synthesize glycans containing other Lewis blood group antigens. Extracts of helminths were analyzed by ELISA using mAbs to Lea, Leb, Ley, and blood group H determinants. Human milk derived bile salt-activated lipase (BAL), which contains many of the human blood group antigens (Wang et al., 1995; Landberg et al., 1997), and extracts of wild-type COS7 cells were analyzed as positive and negative controls, respectively. The extracts of the helminths and COS7 cells do not express Lea, Leb, or Ley, whereas all three mAbs reacted with BAL (Figure
Figure 3. Absence of Lea, Leb, and Ley from trematodes and nematodes. Microtiter wells were coated with extracts from the indicated helminths and analyzed for Lea, Leb, and Ley antigens by ELISA using mAb specific for the three glycans. Human bile-activated lipase (BAL) was analyzed as a positive control, while extracts of COS7 cells were analyzed as negative controls. The ELISAs were performed in triplicate and the results represent averages of the three determinations.
Presence of anti-Lex Abs in rodents infected with S.haematobium and S.japonicum
We previously observed that sera from animals infected with S.mansoni contained IgM and IgG responses to Lex-containing glycans (Nyame et al., 1997). We tested whether a similar response exists in animals infected with S.haematobium and S.japonicum. For this analysis a neoglycoprotein prepared from BSA was used. BSA is the carrier in preparations of neoglycoproteins because it is devoid of carbohydrate (Stowell and Lee, 1980). LNFPIII-BSA, which contains the Lex antigen, was used as the target. The neoglycoprotein was coated onto microtiter plates and incubated with serially diluted sera from infected or uninfected mice and hamsters. The results demonstrate that the sera from mice infected with S.japonicum and hamsters infected with S.haematobium, but not sera from uninfected animals, contain IgM and IgG reactive with LNFPIII-BSA (Figure
Figure 4. Analysis or sera from rodents for antibodies to Lex antigen. Sera from 10 S.japonicum infected mice and 10 S.haematobium infected hamsters were each diluted serially and analyzed for IgM and IgG antibodies to Lex by ELISA using LNFPIII-BSA as the target antigen and techniques described in Materials and methods. Sera from 10 uninfected mice and 10 uninfected hamsters were similarly analyzed as controls. Each dilution was analyzed in triplicate and the results represent averages of the three determinations from all 10 animals in each group of infected and uninfected rodents. Open circles represent sera from infected rodents, and solid circles represent sera from uninfected rodents.
Figure 5. Specificity of anti-Lex antibody responses in infected rodents. Pooled sera from S.japonicum infected mice and S.haematobium infected hamsters were serially diluted and analyzed for either IgM or IgG antibody reactivity toward LNFPIII-BSA ([bull]), LNFPII-BSA ([cir]), sialyl Lex-BSA ([squ]), and LNnT-BSA (o) by ELISA, as described in Materials and methods. Each ELISA was done in triplicate, and the results represent averages of the three determinations.
Presence of anti-Lex Abs in humans infected with S.haematobium and S.japonicum
We previously reported that sera from humans infected with S.mansoni contained antibodies to the Lex antigen (Nyame et al., 1996). To determine whether this autoimmune response also exists in humans infected with the other schistosome species, pooled sera from uninfected humans or humans infected with either S.haematobium or S.japonicum were incubated with either wild-type COS7 cells, which lack Lex antigens, or COS7/FTIV cells, which express Lex, but not sLex, antigens.The cells were analyzed for bound Abs by incubation with goat anti-human IgM-FITC conjugate followed by FACS analysis on a flow cytometer. Pooled sera from S.mansoni infected humans was also analyzed as a positive control. Sera from humans infected with all the three schistosome species stained COS/FTIV cells, while staining of control COS7 cells was weak (Figure
Figure 6. Presence of anti-Lex antibodies in sera of S.japonicum and S.haematobium infected humans. Pooled sera from S.japonicum and S.haematobium infected humans were diluted 1:20 and incubated with COSFTIV or COS7 cells as described in Materials and methods. Bound antibodies were detected by flow cytometry following an incubation with goat anti-mouse IgM-FITC. Pooled sera from uninfected humans and S.mansoni infected humans were analyzed as negative and positive controls, respectively.
Evidence for expression of LDN and [alpha]1,3-fucosylated glycans in glycoproteins from all helminths analyzed
Both S.mansoni and D. immitis synthesize glycoconjugates containing the LDN motif GalNAc[beta]1->4GlcNAc and the GlcNAc residue in many of the LDN residues are [alpha]1,3-fucosylated to generate LDNF (Srivatsan et al., 1992; Kang et al., 1993). In recent studies we found that both H.contortus and C.elegans contain an [alpha]1,3-fucosyltransferase ([alpha]1,3FT) activity capable of transferring Fuc to GlcNAc residues in either lactosamine Gal[beta]1->4GlcNAc or LDN acceptors (R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, unpublished observations). In addition, we detected the [beta]1,4-N-acetylgalactosaminyltransferase ([beta]1,4GalNAcT) capable of synthesizing LDN in extracts of S.mansoni (Srivatsan et al., 1994), H.contortus and C.elegans. However, we did not detect [beta]1,4-galactosyltransferase ([beta]1,4GalT) in extracts of these latter two helminths (R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, unpublished observations). The results suggest that helminths may generally be able to synthesize LDN-type structures and LDNF, but only schistosomes may be unable to synthesize the Lex antigen, because schistosomes contain a [beta]1,4GalT (Rivera-Marrero and Cummings, 1990).
To further investigate this possibility, we utilized two plant lectins that bind to glycans containing the LDN and LDNF motifs. Lotus tetragonolobus agglutinin(LTA)binds to Fuc[alpha]1->3GlcNAc linkages in either Lex or LDNF (Yan et al., 1996) and Wisteria floribunda agglutinin (WFA) binds to GalNAc[beta]1->4GlcNAc (lacdiNAc or LDN) linkages (Torres et al., 1988; Srivatsan et al., 1992b). Total extracts from the different helminths were separated by SDS/PAGE, transblotted, and analyzed by lectin binding analysis using biotinylated LTA and WFA followed by streptavidin-peroxidase. Glycoproteins from all the helminths bound both LTA and WFA and the binding was largely inhibited by the appropriate haptenic sugars, Fuc and GalNAc, respectively (Figure
Figure 7. Analysis of helminths for reactivity with WFA and LTA. Detergent extracts of each parasite was preabsorbed on streptavidin-Sepharose and 3 µg of each indicated helminth was separated by SDS-PAGE under reducing conditions and blotted onto nitrocellulose. The membranes were incubated with 1 µg/ml solution of biotinylated WFA or LTA in the absence or presence of GalNAc or Fuc, which are hapten inhibitors of WFA and LTA, respectively. Reactive bands were visualized by treatments with streptavidin-peroxidase and the ECL detection system.
Discussion
Our results demonstrate that adult S.haematobium and S.japonicum synthesize high molecular weight glycoproteins containing Lex glycans. Expression of the Lex antigen is highly specific, since none of the three human schistosomes express the sLex, Lea, Leb, Ley, or blood group H-type 1 antigens. In addition, the results indicate that animals and humans infected with S.haematobium and S.japonicum exhibit specific immune responses to the Lex antigen, as is observed for infections with S.mansoni (Ko et al., 1990; Nyame et al., 1996, 1997; Van Dam et al., 1996).
Although parasitic helminths represent a sizable fraction of all animal parasites, very little is known about the overall structures of their glycoconjugates, despite extensive evidence that glycans from parasitic helminths are immunogenic. Most of the emphasis to date has been on schistosomes, where they have been found to contain polylactosamine sequences and Lex-antigens on complex-type N- and O-glycans (Srivatsan et al., 1992a; Van Dam et al., 1994), LDN- and LDNF-containing structures on complex-type N-glycans (Nyame et al., 1989; Srivatsan et al., 1992b), high mannose-type N-glycans (Nyame et al., 1988a) and glycans containing an unusual trisaccharide repeating structure that is fucosylated (Levery et al., 1992; Khoo et al., 1995, 1997a). Interestingly, some of the 1,3-fucosyl residues in S.mansoni egg glycolipids are 1,2-fucosylated to generate Fuc1->2Fuc1-> 3GlcNAc structures, but these difucosylated structures are lacking in S.japonicum (Khoo et al., 1997a). In addition, unusual core structures containing xylose and [alpha]3- and [alpha]1-6-linked fucosyl residue were described in S.mansoni and S.japonicum and the relative amounts of these modifications differ between the two species (Khoo et al., 1997b).
The synthesis of these structures in S.mansoni and other schistosomes is consistent with their known complement of glycosyltransferases. Schistosomes contain a [beta]1,4GalNAcT (Neeleman et al., 1994; Srivatsan et al., 1994), an [alpha]1,3FT (DeBose-Boyd et al., 1996) and a [beta]1,4GalT (Rivera-Marrero and Cummings, 1990). The [beta]1,4GalNAcT is required for LDN biosynthesis, whereas the [beta]1,4GalT is required for N-acetyllactosamine biosynthesis, which is the precursor for synthesis of the Lex antigen. However, the expression of a functional [beta]1,4GalT may be the limiting factor that precludes expression of Lex in most helminths. For example, extracts of adult H.contortus contain both [alpha]1,3FT and [beta]1,4GalNAcT activity, but [beta]1,4GalT activity is not detectable (R.A.DeBose-Boyd, A.K.Nyame, D.P.Jasmer, and R.D.Cummings, in preparation). The expression of the [beta]1,4GalT activity in schistosomes may be developmentally regulated, since little galactose is incorporated into glycoconjugates by transformed schistosomula, whereas galactose incorporation is abundant in adult schistosomes (Nyame et al., 1988b). Thus, developmentally regulated expression of the [beta]1,4GalT, rather than the [alpha]1,3FT, may be responsible for the observed developmentally regulated expression of the Lex antigen in schistosomes (Koster and Strand, 1994). The observation that all the helminths tested in our study contain glycoproteins recognized by WFA and LTA suggests that all helminths synthesize LDN, rather than N-acetyllactosaminyl-based structures and these contain [alpha]1,3-fucosylated GlcNAc residues. However, detailed carbohydrate structural analyses are required for glycoconjugates from all these helminths before this can be definitively established.
Very little is known about glycoconjugate structures in helminths other than schistosomes. The only detailed structural analysis reported for C.elegans glycoconjugates was concerned with neutral glycosphingolipids, where they were shown to contain unusual core structures with Glc, Man and GlcNAc (Gerdt et al., 1997). It has been reported that N-glycans in H. contortus contain unusual fucosylated core structures with both Fuc[alpha]1->3GlcNAc and Fuc[alpha]1->6GlcNAc linkages (Haslam et al., 1996). These unusual structures may be related to the observation that H. contortus produce several gut-derived galactose-containing antigens (H-gal-GP; Redmond et al., 1997) and membrane glycoproteins that contain highly antigenic glycans (Jasmer and McGuire, 1991; Jasmer et al., 1993; Andrews et al., 1995). D.immitis microfilaria are known to synthesize complex-type N-glycans containing the LDNF motif (Kang et al., 1993), but nothing is known about the glycan structures in adult organisms. Finally, there are no reports on the structural definition of glycoconjugates in F.hepatica.
In previous studies, it was observed that S.mansoni glycans lack sialic acid residues (Nyame et al., 1987; Robertson and Cain, 1985). Consistent with this observation, neither S.mansoni nor the other schistosome species, express sLex determinants (this study and Srivatsan et al., 1992). The current findingssuggests that sialic acid may be lacking in schistosome glycans in general.
The functions of Lex glycans and other [alpha]1,3-fucosylated glycans in schistosomes and the other helminths are not known. In mammalian systems, fucosylated glycans, and in particular sLex, are involved in leukocyte-selectin interactions in the inflammatory response (McEver and Cummings, 1997; Varki, 1997). sLex is a critical component of the glycoprotein PSGL-1 on neutrophils recognized by P- and E-selectins. Lex and its derivatives occur on many human tissues and cells, in addition to leukocytes, but their functions in these other tissues are not known (Macher and Beckstead, 1990; Ohta et al., 1993; Satoh and Kim, 1994; McEver and Cummings, 1997).
The possibility that [alpha]1,3-fucosylated glycans in helminths may interact directly with selectins has been explored recently. Schistosome eggs, which express a variety of fucosylated glycans, interact with L-selectin, but whether binding involves Lex or a related fucose-containing glycan, is not yet known (El Ridi et al., 1996). In addition, oligosaccharides containing the LDNF structure bind with low affinity to E-selectin (Grennel et al., 1994), but whether the LDNF structures in helminthic glycoconjugates bind to E-selectin has not been investigated. It is also possible that Lex of schistosomes and the LDNF-containing glycans in all helminths interact with an unknown carbohydrate-binding protein(s) during parasite invasion or development in infected humans. In addition to possible roles in adhesion, fucose-containing glycans may alter the host immune response. There is evidence that Lex glycans may alter cellular immunity in infected hosts, by facilitating a shift from Th1 to Th2 response (Velupillai and Harn, 1994; Palanivel et al., 1996). Whether these phenomena occur in infections with S.haematobium and S.japonicum is not yet known.
On the basis of the generation of cytolytic autoantibodies to Lex glycans in infected humans and primates,it has been predicted that an autoimmune disorder may accompany severe and chronic schistosomiasis in humans infected with S.mansoni (Nyame et al., 1996; Van Dam et al., 1996). The presence of anti-Lex antibodies in S.haematobium and S.japonicum infected humans raises the potential for an autoimmune disorder during infections by all human schistosome species. In addition to their cytolytic activity, antibodies to Lex can have profound effects on human neutrophil function (Skubitz et al., 1985; Skubitz and Snook, 1987). Schistosome-derived Lex antigens may also be involved in antibody-dependent cell-mediated cytotoxicity (Trottein et al., 1997). Further studies are needed to determine if the anti-Lex response plays an important role in the pathology of schistosomiasis. Because of the specificity we have observed in the expression of Lex antigen by schistosomes, the presence of anti-Lex might be useful in diagnosing and distinguishing schistosome infections from other parasitic infections. This possibility should be investigated in the future by examining the dynamics and longevity of anti-Lex production in individuals experiencing acute and chronic schistosomiasis.
Materials and methods
Parasites
Adult S.mansoni and S.haematobium were derived from LVG hamsters infected for 8 weeks and 12 weeks, respectively, with cercariae of the two schistosome species. Adult S.japonicum were obtained from CD1 female mice infected for 8 wk with S.japonicum cercariae. Parasites were recovered from the rodents by portal perfusion. D.immitis adults were obtained from NIH Repository at the University of Georgia. Frozen C.elegans adults were the kind gift of Dr. James B. Rand of Oklahoma Medical Research Foundation. H.contortus adults were derived as described previously (Jasmer and McGuire, 1991). F.hepatica adults were derived from the livers of an infected cow obtained from a local abattoir.
Preparation of parasite extracts
Extracts of adult parasites were prepared in a solubilization buffer of phosphate-buffered saline (PBS, pH 7.4) containing protease inhibitors; EDTA (37 µg/ml), trypsin inhibitor (10 µg/ml), PMSF (1000 µM), aprotinin (2 µg/ml), leupeptin (0.5 µg/ml), pepstatin (0.7 µg/ml), and 3,4-dichloroisocoumarin (200 µM). S.mansoni, S.haematobium, S.japonicum, and F.hepatica were disrupted by direct sonication at 4°C on a Branson Sonifier using three, 30 s bursts. H. contortus, D.immitis, and C.elegans were first disrupted on ice with Biohomogenizer (model M133/1281-0, Biospec Products, Inc., Bartlesville, OK) using three, 30 s pulses. The homogenates were disrupted further by sonication as described above. The homogenates were subsequently adjusted to 1 % Triton X-100 and left on ice for 30 min to allow complete solubilization of proteins. The homogenates were first centrifuged at 3000 r.p.m. for 30 min at 4°C to remove large debris, and supernatant fractions were centrifuged further at 50,000 r.p.m. for 1 h at 4°C. The supernatants were recovered and used directly or aliquots were prepared and stored at -80°C.
Cell culture
COS7/FTIII and COS7/FTIV cells were generated by stably transfecting wild type COS7 cells with pcDNA I plasmids containing a cDNA insert for either human fucosyltransferase III or IV and a neomycin-selectable marker (Kukowska-Latallo et al., 1990; Kumar et al., 1991; Lowe et al., 1991). The COS7/FTIII and COS7/FTIV cells were maintained in Dulbecco's Modified Eagle (DME) media containing 10% fetal bovine serum (FBS), 2 mM glutamine, 100 µU/ml penicillin, 100 µg/ml streptomycin, and 400 µg/ml neomycin (G418). Wild-type COS7 cells were maintained in the same media without G418. All cells cultures were maintained at 37°C in a humidified incubator containing 5% CO2.
Preparation of cell extracts
COS7, COS7/FTIII, and COS7/FTIV cells were harvested at 80% confluence and washed 4× with Hanks' buffered saline solution (HBSS). The cells were suspended in solubilization buffer and sonicated by three, 30 s bursts on a Branson Sonifier at 4°C. The homogenates were centrifuged at 14,000 r.p.m. for 30 min at 4°C. The supernatants were recovered and used directly for assays, or aliquots were prepared and stored at -80°C.
Antibodies
Anti-Lex (CD-15) and anti-sLex (CSLEX-1) mAbs were obtained from Becton Dickinson (San Jose, CA). Anti-Lea (BG 5), anti-Leb (BG 6), anti Ley (BG 8) and blood group H (BG4) mAbs were obtained from Signet (Dedham, MA). Goat anti-mouse IgM-peroxidase, goat anti-mouse IgG-peroxidase ([gamma]-chain specific) were obtained from Kirkegaard and Perry (Gaithersburg, MD). Anti-hamster IgG mAb (biotin-conjugated) was purchased from Pharmingen, (San Diego, CA). Goat anti-mouse IgG/IgM-peroxidase conjugate was obtained from Boehringer-Mannheim (Indianapolis, IN).
Human antisera
Sera from S.mansoni infected humans were obtained from infected patients from Puerto Rico. S.haematobium infected sera were obtained from patients in Egypt and Nigeria while S.japonicum infection sera were obtained from patients from China. Each patient was determined to be free from infections with other helminths by stool analysis. Normal human sera were obtained from healthy individuals with no history of international travel.
Rodent antisera
Sera from S.haematobium infected rodents were derived by cardiac puncture from LVG Golden Syrian hamsters infected percutaneously with [sim]360 S.haematobium cercariae for 12 weeks. Sera from S.japonicum infected rodents were similarly obtained from Swiss Webster mice infected for 8 weeks with 40 cercariae.
ELISA
ELISA for detection of Lex and other fucose-containing antigens was performed using buffers and procedures described previously (Nyame et al., 1996, 1997). Microtiter plates (96 wells) were coated overnight, at room temperature with 100 µl of 10 µg/ml solution of parasite extracts in coating buffer and subsequently blocked by incubation with 5% BSA in PBS at 37°C for 2 h. The wells were washed and incubated with 100 µl of 10 µg/ml solutions of mAb to Lex, sLex, Lea, Leb, Ley, and blood group H in dilution buffer at room temperature for 1 h. The wells were washed 4× with PBS-Tween (0.05% Tween 20) and incubated at room temperature for 1 h with 100 µl of 1:500 dilution of goat anti-mouse IgG/IgM-peroxidase conjugate. The wells were washed 4× with PBS-Tween, followed by two washes with deionized water and incubated at room temperature for 2 h with 100 ml of ABTS-peroxidase purchased from Kirkegaard and Perry (Gaithersburg, MD). The absorbance of the wells was measured at 405 nm on a microtiter plate reader (Molecular Devices, Sunnydale, CA).
ELISA analysis for detection of circulating antibodies to Lex in the sera of infected animals was carried out essentially as described previously (Nyame et al., 1997). The neoglycoproteins prepared from bovine serum albumin, LNFPIII-BSA, LNnT-BSA, LNFPII-BSA, and sLex-BSA were purchased from V-Labs Inc. (Covington, LA). Microtiter plates were coated with 50 µl of 5 µg/ml LNFPIII-BSA, LNFPII-BSA, LNnT-BSA or sLex-BSA, blocked with BSA, and incubated with 50 µl of serially diluted sera from infected rodents. The wells were subsequently incubated with goat anti-mouse IgM-peroxidase (1:10,000) or goat anti-mouse IgG-peroxidase (1:5000), and reactive wells were detected by incubation with ABTS-peroxidase substrate and absorbance measurements at 405 nm on a microtiter plate reader. For detection of bound hamster IgG, the wells were incubated with mAb to hamster IgG (1 µg/ml) for 1 h before incubation with goat anti-mouse IgG-peroxidase.
Western blot
Approximately, 30 mg of parasite or cell extracts were analyzed by SDS-PAGE on 10% acrylamide gel under reducing conditions and transferred onto nitrocellulose filter. The filter was blocked by incubation with 5% solution of nonfat dry milk in TBS (10 mM Tris, 150 mM NaCl, pH 7.5) for 1 h at room temperature. The filter was washed 4× with TBS-Tween (10 mM Tris, 300 mM NaCl, pH 7.5, 0.05% Tween) and incubated with 10 mg/ml solution of anti-Lex mAb in dilution buffer (10 mM Tris, 300 mM NaCl, pH 7.5, 1% BSA, 0.3% Tween-20) for 1 h at room temperature. The filter was washed once again (4×) and incubated with 1:500 dilution of goat anti-mouse IgM-peroxidase conjugate for 1 h at room temperature. The filter was washed (4×) and bound antibodies were detected by a 30 s incubation with Enhanced ChemiLuminescence (ECL) reagent (Amersham, Arlington Heights, IL) and autoradiography.
Lectin blots
Total detergent extracts of parasites or cells ([sim]3 mg) were preabsorbed on streptavidin-Sepharose to remove endogenous biotin-containing proteins and then fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked by incubation with 3% BSA solution in TBS (10 mM Tris, 150 mM NaCl, pH 7.5), washed 4× in TTBS-2 (10 mM Tris, 0.5 M NaCl, 0.3% Tween-20), and incubated in 1 µg/ml solution of biotinylated WFA or LTA in 10 mM Tris, 150 mM NaCl, 0.05% Tween-20, and 1% BSA, with or without 200 mM GalNAc for the WFA and 500 mM fucose for LTA treatments, respectively. The membranes were washed 4× with TTBS-2 and incubated with 1:5000 dilution of streptavidin-peroxidase (Boehringer-Mannheim, Indianapolis, IN) in 10 mM Tris, 150 mM NaCl, 0.05% Tween-20 and 1% BSA. The membranes were washed with TTBS-2 four times. Incubations were carried out at room temperature for 1 h, and the washes were done for 10 min per each wash. Reactive bands were visualized by the ECL technique.
Flow cytometry
COS7 and COS7/FTIV cells were harvested at 80% confluent density and washed 4× with HBSS. Approximately, 5 × 105 cells were incubated for 30 min at 4°C with 100 µl of pooled human sera diluted 1:20 in HBSS containing 10% NGS (HBSS/NGS). The cells were washed 4× with 1 ml HBSS/NGS and resuspended in 100 µl 1:50 dilution of goat anti-human IgM-FITC conjugate and incubated for 30 min at 4°C. The cells were washed again (4×) with HBSS/NGS and bound antibodies were analyzed analysis on a FACStar flow cytometer (Becton Dickinson, San Jose, CA).
Acknowledgments
This work was supported by NIH Grant AI26725 to R.D.C. Infected mice and hamsters used in this study were provided by a NIAID supply contract (AI#052590). Normal goat serum was kindly donated by Mr. Gary L. Ferrell of the Oklahoma Medical Research Foundation, Oklahoma City, OK. The flow cytometric data was generated with the assistance of Mr. Jim Henthorn and the Flow Cytometry and Cell Sorting Laboratory, University of Oklahoma Health Sciences Center.
Abbreviations
Lex, Lewis x; sLex, sialyl Lewis x; Lea, Lewis a; Leb, Lewis b; Ley, Lewis y; LTA, Lotus tetragonolobus agglutinin; WFA, Wisteria floribunda agglutinin; [beta]1,4GalNAcT, [beta]1,4-N-acetylgalactosaminyltransferase; [alpha]1,3FT, [alpha]1,3-fucosyltransferase; FTIII, [alpha]1,3/4-fucosyltransferase IV; FTIV, [alpha]1,3-fucosyltransferase IV; [beta]1,4GalT, [beta]1,4-galactosyltransferase; BAL, bile-activated lipase; LDN, lacdiNAc (GalNAc[beta]1->4GlcNAc); LDNF, GalNAc[beta]1->4(Fuc[alpha]1->3)GlcNAc; BSA, bovine serum albumin; LNFPIII-BSA, Gal[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1->3Gal[beta]1->4Glc-BSA; LNFPII-BSA, Gal[beta]1->3(Fuc[alpha]1->4)GlcNAc[beta]1->3Gal[beta]1->4Glc-BSA; LNnT-BSA, Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc-BSA; HBSS, Hanks' buffered saline solution; NGS, normal goat serum.
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