Fucosyltransferases in Schistosoma mansoni development

E.T.A. Marques Jr.1, Y. Ichikawa, M. Strand, J.T.  August, G.W. Hart3 and R.L. Schnaar

Department of Pharmacology and Molecular Sciences and 3Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA

Received on August 14, 2000; revised on November 3, 2000; accepted on November 3, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fuc{alpha}1,3GlcNAc, Fuc{alpha}1,2Fuc, Fuc{alpha}1,6GlcNAc, and perhaps Fuc{alpha}1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophore-assisted carbohydrate electrophoresis with detection of radioactive fucose incorporation from GDP-[14C]-fucose into structurally defined acceptors. The total fucosyltransferase-specific activity in egg extracts was 50-fold higher than that in the other life stages tested (cercaria and adult worms). A fucosyltransferase was detected that transferred fucose to type-2 oligosaccharides (Galß1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by {alpha}2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galß1,4(Fuc{alpha}1,3)GlcNAcß1,3Galß1,4Glc). A low level of fucosyltransferase that transfers fucose to no-sialylated type-1 oligosaccharides (Galß1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucose-type iminosugars inhibitors were tested on schistosome fucosyltransferases. A new fucose-type 1-N-iminosugar was four- to sixfold more potent as an inhibitor of schistosome fucosyltransferases in vitro than was deoxyfuconojirimycin. In vivo, this novel 1-iminosugar blocked the expression of a fucosylated epitope (mAb 128C3/3 antigen) that is associated with the pathogenesis of schistosomiasis.

Key words: FACE/fucose/glycosyltransferase/iminosugar/Schistosome/trematode


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Shistosoma infection is one of the most prevalent tropical diseases in the world. The parasite trematode is a complex multicellular organism that synthesizes a diverse variety of O-linked and N-linked glycoproteins, glycosylphosphatidylinositol anchors, and glycolipids (Cummings and Nyame, 1996Go, 1999). Schistosome glycoconjugates differ from typical mammalian glycoconjugates by their relatively high amount of fucose. In fact, in the schistosome glycocalyx, fucose represents more then 50% of the total sugar. Fucosylated glycoconjugates are thought to be directly involved in many aspects of the parasite’s life cycle, as well as in the immune response to and pathogenesis associated with Schistosoma infection (Cummings and Nyame, 1996Go, 1999). The presence of large quantities of fucose on unique complex carbohydrates of the parasite and their roles in important biological functions make the fucosyltransferases suitable therapeutic targets.

As the parasites mature, they follow a specific path within the mammalian host to complete their life cycle. Fucosylation may regulate their trafficking during development, as is the case for certain mammalian saccharide structures on blood cells (Maly et al., 1996Go; Le Marer et al., 1997Go). Fucosylated glycoconjugates are also important in the host immune response to the parasite. Some of the schistosome eggs are trapped in the host tissues, and the immune response to the egg fucose–containing epitopes results in the formation of granulomas. Among the most potent inducers of granuloma formation are fucose-containing glycoconjugates unique to schistosomes and recognized by mAb 128/C3 (Weiss and Strand, 1985Go; Weiss et al., 1986Go, 1987; Levery et al., 1992Go).

Schistosomal fucosylated structures contain {alpha}1,3 fucose linkages and other types, including Fuc{alpha}1,2Fuc, Fuc{alpha}1,6GlcNAc, and an unusual internal linkage, Fuc{alpha}1,4GlcNAc (Levery et al., 1992Go). S. mansoni fucosyltransferase activities have been investigated in extracts of adult worms with the use of a small number of acceptor substrates (LNnT, LNT, NeuAc{alpha}2,3Galß1,4GlcNAc, and NeuAc{alpha}2,6Galß1,4GlcNAc; see Table I for acceptor substrate abbreviations) (DeBose-Boyd et al., 1996Go). These extracts contained a fucosyltransferase that preferentially fucosylated the acceptor LNnT, forming the Lewis x (LeX) determinant, and also showed an activity capable of synthesizing sialyl Lewis x (sLeX). The fucosyltransferase activity in these crude extracts had an apparent KM of 300 µM for GDP-Fuc and a Vmax of 0.23 nmol/mg/h. Fucosyltransferase activities have also been studied in the avian species of Schistosoma, Trichobilharzia ocellata, where {alpha}3- and {alpha}2-fucosyltransferases were the main activities identified. One of the products formed by these T. ocellata transferases was determined to be Fuc{alpha}1,2Fuc{alpha}1,3GalNAcß1,4GlcNAc (Hokke et al., 1998Go). A fucosyltransferase encoded by S. mansoni with a high percent of sequence identity and enzymatic activity comparable to human and mouse fucosyltransferase VII has been cloned (Marques et al., 1998Go). More recently, another S. mansoni cDNA sequence with similarities to mammalian fucosyltransferases was isolated, but its enzymatic activity has not yet been determined (Trottein et al., 2000Go).


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Table I. List of oligosaccharides
 
Here we used a novel fucosyltransferase assay to study the acceptor substrate specificities of schistosome fucosyltransferases and their variations during different stages of development. This assay is based on the labeling of saccharides at the reducing end with a negatively charged fluorophore, 8-aminonaphthalene-1,3,6 trisulfonic acid (ANTS) as described by Jackson (1990, 1996). The ANTS-labeled oligosaccharides were used as acceptor substrates for fucosyltransferases, with radiolabeled GDP-Fuc as donor. The reaction mixtures were separated by high-resolution polyacrylamide gel electrophoresis (PAGE) and radioactive fucosylated products detected. This assay allowed us to detect femtomole quantities of product formed by the parasite enzymes using several well-defined oligosaccarides as acceptors. Finally, we tested the inhibitory effects of novel fucose-type iminosugars on parasite fucosyltransferases in vitro and in vivo. Some of these compounds inhibit the expression of fucosylated epitopes in living parasites.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
New substrate oligosaccharides: SLNnT and schistosome core I
Two oligosaccharides, SLNnT and schistosome core I, were enzymatically synthesized for this study. Although the trisaccharide 3'-sialyl-N-acetyllactosamine, NeuAc{alpha}2-3Galß1-4GlcNAc, is available, it cannot be used as an ANTS-labeled acceptor substrate because the sugar ring of the GlcNAc at the reducing end, where the fucosylation occurs, is opened during ANTS derivatization, leading to loss of its natural conformation and loss of acceptor activity for schistosomal enzymes (data not shown). The loss of the sugar ring conformation of the saccharide at the reducing end by ANTS-labeling reduces the utility of labeled disaccharides as substrates for fucosyltransferase assays. The longer oligosaccharide SLNnT, which has two more saccharide residues at the reducing end, is an acceptor.

The schistosome core I structure, GalNAcß1,4GlcNAcß1,4GlcNAcß1,4GlcNAc, was based on the carbohydrate structure of the schistosome neutral egg glycolipids identified by Khoo et al. (1997)Go. It was predicted that the oligosaccharide backbone of that structure would be the natural substrate for the schistosomal fucosyltransferases. This oligosaccharide was biosynthetically made using ß1,4-galactosyltransferase, which mediates the transfer of GalNAc to the nonreducing end of chitotriose to yield schistosome core I. The addition of GalNAc to chitotriose was determined using fluorophore-assisted carbohydrate electrophoreses (FACE). Previous enzymatic syntheses using this procedure have resulted exclusively in ß-linked terminal GalNAc (Palcic and Hindsgaul, 1991Go; Do et al., 1995Go).

FACE fucosyltransferase assay
The use of FACE to separate the substrates and products of the fucosyltransferase assay allowed quantitative analyses of reaction products. Furthermore, multiple radiolabeled products in a single reaction could be detected and quantified. The FACE fucosyltransferase assay was first optimized using commercial enzymes and compared to standard methods (data not shown). To quantify radioactivity in the products we used a radioactive scale that was exposed simultaneously with the phosphorimager plate. The procedure demonstrated a linear correlation between pixel density and d.p.m. The lowest concentration of radioactivity, which yielded pixel density above background, was 2.5 d.p.m. We set the pixel density of the 5 d.p.m. standard as the limit of detection for all subsequent experiments.

A limitation of the current assay is that product characterization is not definitive. However, transfer of radiolabeled fucose to a well-defined acceptor, combined with product electrophoretic mobility compared to known standards, allowed tentative assignments to be made in many cases.

When schistosome extracts were incubated with GDP-[14C]-fucose and the ANTS-labeled acceptor oligosaccharide, LNnT, a linear correlation was observed between the amount of product formed and the amount of protein extract from schistosome that was used in the reaction mixture. As expected, the quantity of product formed was found to increase with time (Figure 1A and B). The multiple products formed using this acceptor are more fully described in Fucosyltransferase and fucosidase activities are regulated during S.mansoni development.



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Fig. 1. Fucosyltransferase activities in schistosome egg extracts. (A) Correlation between amount of schistosome egg protein extract used in the fucosyltransferase assay using LNnT as acceptor substrate and the amount (pixel density) of the product formed. Below the graph are shown (i) a fluorescence image of the FACE gel showing the ANTS-labeled substrate, and (ii) the autoradiograph of the radioactive products that were quantified as pixel density. (B) The effect of time on the formation of product in the fucosyltransferase reaction. The graph shows the pixel density of the bands corresponding to the products formed as a function of time. The corresponding fluorescent and autoradiographic images are presented below the graph. In these reactions 16 µg of schistosome egg extract were used.

 
Fucosyltransferase and fucosidase activities are regulated during S. mansoni development
The fucosyltransferase and fucosidase activities in adult, cercaria and egg extracts were determined for a series of acceptor substrates. Substantial differences in the activities of the enzymes among the three life stages were apparent.

An example of the fucosyltransferase data is shown in Figure 2, and a summary of the activities of each acceptor in extracts of cercariae, adult worms, and eggs is shown in Table II. For all life stages tested, the oligosaccharide LNnT was the best acceptor. Surprisingly, regardless of the acceptor, the fucosyltransferase-specific activities of the egg extracts were higher than that of the adult worms or cercarial extracts, with the activity for any specific acceptor varying up to 100-fold from one life stage to another. For example, the use of SLNnT as substrate yielded a specific activity of 52.7 pmol/mg/h for the egg extract, 13-fold the activity observed with the use of the LNFIII for this life stage, while the specific activity for the SLNnT was only 0.5 pmol/mg/h in an extract of adult worms and is equivalent to the activity for LNFIII in the same extract. In contrast, fucosyltransferase activity for some other acceptors varied by only two- to fourfold from one life stage to another. For example, the specific activity for the acceptor LNFIII was 4 pmol/mg/h for the egg extract, 1.8 pmol/mg/h for the cercarial extract, and 0.5 pmol/mg/h for the adult worm extract. Some acceptor oligosaccharides, such as 3'F-Lac, only showed activity in egg. The differences of fucosyltransferase activities in the life stages indicates the presence of more than one enzyme in the extracts and also suggests that specific enzymes and fucosylated products may exert their main functions during particular life stages.



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Fig. 2. Representative results of fucosyltransferase assays of schistosome egg extracts using a variety of defined acceptors. Fucosyltransferase assays used 16 µg of egg extract incubated for 6 h with the indicated acceptors as described in the text. (Right Panel) Fluorescent images (left) and autoradiographs (right) of the same gels. The ANTS-labeled monosaccharide fucose is included as an electrophoretic marker. Control "–" is the reaction mixture in the absence of schistosome extract or acceptor substrate; the radioactive band indicates the donor substrate, GDP-Fuc. Control "-+" contains the schistosome extract but no acceptor. (Left Panel) The same acceptors used in the fucosyltransferase assays were incubated under the same conditions but without schistosome extract. The radioactive standards used to convert to d.p.m. are also shown. The structures of the oligosaccharides are shown in Table I.

 

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Table II. Summary of the fucosyltransferase activities identified in S. mansoni (activity, pmol/mg/h)
 
Control reactions (Figure 2), which did not contain schistosomal extract or acceptor substrate, had no fluorescent band and radioactivity only near the bottom of the lane, representing donor nucleotide sugar GDP-Fuc. In a second control reaction (Figure 2), which contained the schistosomal extract but did not contain an acceptor substrate, there is a reduction of radiolabel comigrating with GDP-Fuc, as compared to absence of schistosome extract, although no detectable products were formed. The reaction containing the acceptor LNT (Galß1,3 GlcNAcß1,3Galß1,4Glc) formed a product that comigrated with the fucosylated oligosaccharide LNFII (Galß1,3(Fuc{alpha}1,4)GlcNAcß1,3Galß1,4Glc). The most pronounced fucosyltransferase activity was observed in the presence of the acceptor LNnT (Galß1,4GlcNAcß1,3Galß1,4Glc). This acceptor produced at least five different radioactive bands (Figure 3); the major band, designated LNnT3 (Figure 3), comigrated with the oligosaccharide LNFIII (Galß1,4(Fuc{alpha}1,3)GlcNAcß1,3Galß1,4Glc), which is an expected product (DeBose-Boyd et al., 1996Go). Two bands, LNnT1 and LNnT2, migrated below this marker and may represent the combined actions of fucosyltransferases and glycosidases. The LNnT4 product comigrated with the oligosaccharide LNDI (Fuc{alpha}1,2Galß1,3(Fuc{alpha}1,4)GlcNAcß1,3Galß1,4Glc), suggesting the presence of more than one fucose molecule in the product. LNnT5 presented a migration pattern suggestive of the presence of a third fucosylation.



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Fig. 3. Multiple products are formed when LNnT is incubated with schistosome egg extract and GDP-Fuc. Phosphorimager image of the fucosyltransferase assay using LNnT as acceptor substrate and 16 µg of egg extract as described in the text. On the left side are indicated the positions of various fucosylated products as referred to in Table II, and on the right side are the positions of standard oligosaccharides.

 
The reaction using the LNFII as the acceptor saccharide yielded two radioactive products. The lower one comigrated with LNFII and the upper one with LNDI. Use of LNFIII as the acceptor also resulted in the formation of two products, one comigrated with LNFIII and the other with LNDI. The oligosaccharides LNDI (Fuc{alpha}1,2Galß1,3(Fuc{alpha}1,4)Glc-NAcß1,3Galß1,4Glc) and LSTa (NeuAc{alpha}2,3Galß1,3GlcNAcß1,3Galß1,4Glc) did not form any detectable products. The sialylated oligosaccharide LSTc (NeuAc{alpha}2,6Galß1,4GlcNAcß1,3Galß1,4Glc) was a relatively good substrate, and a radioactive product was formed that migrated close to LNDI. No detectable radioactive product was found for reactions involving the oligosaccharides Lea, Lex, sLea, or sLex. It is interesting that the use of the oligosaccharide 3'F-Lac (Galß1,4(Fuc{alpha}1,3)Glc) as substrate yielded a fucosylated radioactive product, whereas the saccharide 2'F-Lac (Fuc{alpha}1,2Galß1,4Glc) did not (lactose also failed to act as an acceptor; this may be due to linearization of the Glc residue by ANTS derivatization). The oligosaccharide SLNnT (NeuAc{alpha}2,3Galß1,4GlcNAcß1,3Galß1,4Glc) was the second-best acceptor substrate in the schistosome extracts and produced an intense radioactive band. In contrast to these results, there was no product formed in the control reactions in the absence of schistosome extract (data not shown).

Chitotriose (GlcNAcß1,4GlcNAcß1,4GlcNAc) and chitotetraose (GlcNAcß1,4GlcNAcß1,4GlcNAcß1,4GlcNAc) were also acceptors, each producing a single characteristic radioactive band. The smaller saccharide chitobiose (GlcNAcß1,4GlcNAc) was apparently not used as an acceptor substrate by the Schistosoma. The saccharide blood group A (GalNAc{alpha}1,3(Fuc{alpha}1,2)Gal) was not a substrate. Schistosome core I (GalNAcß1,4GlcNAcß1,4GlcNAcß1,4GlcNAc) was the third-best acceptor identified for the schistosome egg extracts (Table II), and it formed more than one radioactive product, possibly including the putative epitope of the mAb 128C3/3, although more experiments are required to test this hypothesis. No products were detected when schistosome core I was tested using adult or cercaria extracts.

For some substrates, especially those that are both sialylated and fucosylated, such as sLex, sLea, and SF-Lac, there were several visible fluorescent bands that had more rapid electrophoretic mobility than that of the starting oligosaccharide and were absent from the control reactions (Figure 2; fluorescence). These bands may be degradation products catalyzed by glycosidases present in the schistosome extract. However, neither these sugars nor their degradation products yielded radioactive fucosylated products. Several other fluorescent bands with slower migration were also visible in the reactions with several of the substrates. In most cases these do not appear to be fucosylation products, since they do not appear as corresponding fucosylated bands on the autoradiograph; therefore, they may be products of other reactions. For example, desialylation, reduces the total negative charge of an oligosaccharide reducing its migration.

Because fucosidase activities in the schistosome extracts may interfere with the interpretation of the fucosyltransferase data, {alpha}2- and {alpha}3-fucosidase activities were determined (Figure 4). Fucosidase activity was determined by measuring the formation rate of lactose from the oligosaccharides 3'F-Lac (Galß1,4(Fuc{alpha}1,3)Glc) (Figure 4C) and 2'F-Lac (Fuc{alpha}1,2Gal-ß1,4Glc) (Figure 4B) in the presence of schistosomal extracts. ANTS-labeled lactose migrates differently than do the ANTS-labeled fucosylated oligosaccharides (Figure 4A). Both {alpha}2- (Figure 4B) {alpha}3-fucosidase (Figure 4C) activities were detected. The results are summarized in Table III. In these experiments the egg extracts showed higher {alpha}2- and {alpha}3-fucosidase-specific activities than the other life stages did. This result indicates that the higher fucosyltransferase activity detected in egg extract is not due to lower fucosidase activity in this extract. It is noteworthy that {alpha}3-fucosidase activity was 2.5- and 10-fold greater than {alpha}2-fucosidase activity in the cercarial and adult worm extracts, respectively. In contrast, the {alpha}3-fucosidase activity in the egg extract was only one third of the {alpha}2-fucosidase activity. These high fucosidase activities at substrate concentrations of 0.5 mM may interfere with the determination of the fucosyltransferase activity by degrading the products, resulting in underestimation of the fucosyltransferase activities. However, the fucosylated substrates, LNFII, LNFIII, and LNDI did not display degradation products, nor did the sialylated substrates LSTa or LSTc.



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Fig. 4. Fucosidase assay. (A) Representative fluorescent image of the results of a fucosidase assay using schistosome egg extracts. The oligosaccharide substrate is indicated above each lane. The ANTS-labeled lactose standard is shown to indicate the electrophoretic migration of the fucosidase product. (B) Fucosidase assay using 2'F-Lac as the substrate. (C) Fucosidase assay using 3'F-Lac as the substrate. The life stage of the extract used in each reaction is indicated above each lane. The substrate and product of the reactions are indicated on the right.

 

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Table III. Fucosidase activity in S. mansoni extracts incubated with 2'F-Lac or 3'F-Lac (pmol/mg/h)
 
Iminosugars inhibit S. mansoni fucosyltransferases in vitro
Egg fucosyltransferase activity was determined using LNnT as the acceptor in the presence and absence of various iminosugars (Figure 5) at concentrations up to 17.5 mM (Figure 6A). The ß-type iminosugars A and B reduced the formation of products by as much as 85% at 17.5 mM. Iminosugar A had an IC50 of 15.1 ± 4.8 mM (mean ± SE; p = 0.014 compared to no inhibitor) and Ki of 11.6 ± 2.8 mM, whereas iminosugar B had an IC50 of 10.4 ± 2.8 mM (p = 0.0004) and Ki of 7.8 ± 1.1 mM. The {alpha}-type iminosugar C did not show inhibition; it appeared to have an inhibitory effect at lower concentrations (up to 5 mM) and to increase the amount of product formed at higher concentrations (17.5 mM). Iminosugar D showed an IC50 > 17.5 mM and a high Ki ~74 mM [similar to the values reported by Qiao et al. (1996)Go], which was not significantly different from no inhibitor. Similarly, fucose showed no inhibitory effect. The inhibitory effect of the iminosugars A, B, and C at 5 mM was also tested for other acceptor molecules (Figure 6B), SLNnT, and LNFIII. Whereas the inhibitory potencies of the iminosugars for the fucosylation of LNnT and its sialylated form, SLNnT, were comparable, they were more effective in inhibiting the reaction using LNFIII as acceptor, with nearly complete inhibition at 5 mM iminosugar B (Figure 6B). In addition, a significant inhibition of the egg {alpha}-fucosidase activity was observed for iminosugars A and B at 5 mM, although these iminosugars are much less potent inhibitors of {alpha}-fucosidase than the iminosugars C and D (data not shown).



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Fig. 5. Iminosugars used in this study. (A) General classification; (B) specific structures.

 


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Fig. 6. Effect of iminosugars on S. mansoni egg fucosyltransferase activities. (A) The indicated concentrations of the iminosugars A (filled circles; —), B (open circles; ...), C (closed triangles; ---), and D (closed squares; –·) were tested on formation of product by the egg fucosyltransferases using LNnT as acceptor. The effect of fucose (open triangles; ··–) is presented for comparison. The lines represent the curve fitting of the data based on the model V = Km / 1 + (I / KI) when [S]<<Km. This equation was applied to calculate the Ki of the iminosugars. (B) The effect of 5 mM of the iminosugars A (black bars), B (dark gray bars), and C (light gray bars) on egg fucosyltransferase activities using acceptors LNFIII, SLNnT, and LNnT. Open bars represent the activity in absence of the iminosugar.

 
A novel fucose-related iminosugar inhibits the expression of a pathogenic fucosylated epitope by adult worms in culture
Adult worms were cultured in the presence of iminosugars, and the effects of the inhibitors on protein synthesis and the expression of fucosylated epitopes were investigated. The level of expression of fucosylated epitopes was determined by radioimmunoprecipitation of fucosylated glycoproteins. Total incorporation of [35S]methionine was only modestly affected (<30%) by concentrations of iminosugars up to 500 µM (data not shown). Radiolabeled worm extracts were immunoprecipitated with mAb 128C3/3, which recognizes a highly expressed fucosylated epitope (Weiss and Strand, 1985Go); and with mAb 103A5, an isotype-matched control mAb, that does not react with schistosomal proteins (Figure 7). The amount of [35S]methionine-labeled glycoprotein expressing the 128C3/3 epitope that was immunoprecipitated by mAb 128C3/3 was reduced to background (the level seen for mAb 103A5) when the worms were incubated in presence of >=5 µM of iminosugar A.



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Fig. 7. Inhibition of biosynthesis of a fucosylated glycan by iminosugar A. The amount of radiolabeled protein immunoprecipitated by mAbs 128C3/3 (squares), and the mAb 103A5 (circles) (an isotype control antibody used to define the background radioactivity), after incubation of worms in the presence of the indicated concentration of iminosugar.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A major finding of this study was that much higher total fucosyltransferase activity was found in egg extracts compared to cercarial or worm extracts. The presence of higher fucosyltransferase activities in the egg extracts may reflect the importance of the fucosylated structures in mediating adhesion and trafficking (Lejoly-Boisseau et al., 1999Go), induction of Th2 (Okano et al., 1999Go; Velupillai et al., 2000Go), and granulomatous response by the egg (Weiss et al., 1987Go). The relative enzymatic activities for different acceptor substrates changed at each of the developmental stages. The fucosyltransferase activity that uses sialylated acceptor SLNnT in the egg extract was 100 times higher than that in the adult worm or cercarial extracts. For other substrates the difference was not as large. For example, the activity in the egg extract was only twice that in cercariae and four times higher than that in adult worms when using LNFIII as substrate. Although the use of crude extracts in this study limits interpretations, differences in the relative levels of enzymatic activities indicate that fucosyltransferases are differentially regulated during development. The data suggest the presence of more than one enzyme in schistosome extracts, allowing the expression of distinct fucosylated structures at specific life stages. As an example, the expression of LeX has been shown to be developmentally regulated in schistosomes (Köster and Strand, 1994Go).

In general, the schistosome fucosyltransferases prefer type-2 uncharged sugars, such as LNnT, as acceptors and may preferentially form {alpha}1,3 linkages (Figure 2; LNnT and Figure 3). The type-2 sialylated oligosaccharides, SLNnT and LSTc, also showed significant activity as acceptor substrates when incubated with egg extracts. Fucosylation of these oligosaccharides may form the sLex structure. The formation of sLex by schistosome fucosyltransferases extracts (DeBose-Boyd et al., 1996Go) and a fucosyltransferase-type VII encoded by the S. mansoni able to express the sLex epitope in recombinant cells (Marques et al., 1998Go) have been reported previously. In addition, L-selectin, a high-affinity lectin for sLex, has been found bound to egg miracidia (El Ridi et al., 1996Go).

Schistosomal extracts could also fucosylate LNFIII, forming a product that comigrated with LNDI, which is a hexasaccharide containing two fucoses. The LNFIII-fucosylated product may contain Fuc{alpha}1,2Fuc in that the LNFIII oligosaccharide is similar to the acceptor substrate used by Hokke and collaborators (Hokke et al., 1998Go) to identify the Fuc{alpha}1,2Fuc fucosyltransferase. An alternative possibility is that the fucosylated structure formed is Fuc{alpha}1,2Gal, but this structure seems less likely because the other acceptor substrates that terminate in Galß1,4GlcNAc did not serve as substrates.

3'F-Lac, which terminates in Galß1,4Glc, was used as an acceptor by the schistosomal fucosyltransferases, and the SF-Lac, 2'F-Lac and Lex were not. These results suggest that there is a fucosyltransferase that can form Fuc{alpha}1,2Galß1,4(Fuc{alpha}1,3)Glc but may not form Fuc{alpha}1,2Galß1,4(Fuc{alpha}1,3)GlcNAc. It is interesting that a lectin has been identified in the intermediate host snail that binds to Fuc{alpha}1,2Galß1,4Glc, and this saccharide is known to be highly expressed in the egg miracidia. This interaction between the snail lectin and the miracidial sugar Fuc{alpha}1,2Galß1,4Glc may play a role in mediating the infection of the intermediate host (Mansour et al., 1995Go; Mansour, 1996Go). Alternatively, the product formed using 3'F-Lac as acceptor may contain a Fuc{alpha}1,2Fuc linkage.

Chitotriose, chitotetraose, and schistosome core I were also substrates for the schistosomal fucosyltransferases. These oligosaccharides resemble the repetitive units of the putative epitope of mAb 128C3/3. Chitotetraose and schistosome core I differ only in their nonreducing terminal saccharide (GlcNAc vs. GalNac). This difference was apparently significant, because schistosome core I was six times more efficient as an acceptor than was the chitotetraose. In addition, schistosome core I formed more than one product. It is interesting that the blood type A oligosaccharide, which also has a GalNAc at the nonreducing end, was not a substrate.

The fucose-related ß-type iminosugars A and B were four- to sixfold more potent inhibitors of the schistosome fucosyltransferases in vitro than were the conventional {alpha}-type iminosugars C and D. The iminosugars homofuconojirimycin and deoxyfuconojirimycin have been shown to be fucosyltransferase inhibitors with IC50 values of ~70 mM (Qiao et al., 1996Go). That study also demonstrated a synergistic effect between the inhibitors and GDP, with the potency increasing by 20- to 80-fold (IC50s ~1–4 mM) in the presence of 30 µM GDP. This synergism is relevant to the use of iminosugars as inhibitors in vivo, because eukaryotic cells typically have an intracellular concentration of 15–50 µM GDP. In in vivo experiments, iminosugar A blocked the expression of the epitope recognized by mAb 128C3/3 at a concentration of 5 µM, whereas its IC50 in vitro was 15 mM, suggesting that the synergism may be occurring in live schistosomes. The inhibitory effects of iminosugars on the expression of fucosylated structures may also be the result of effects on other fucose-metabolizing enzymes, such as fucosidases or those involved in the biosynthesis of GDP-Fuc. Although the compounds were not toxic to the adult worms in vitro, they may affect the parasite life cycle in vivo.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Parasites
Adult S. mansoni (Puerto Rico strain) were harvested from mice 46 days after infection with approximately 200 cercariae by perfusion of the descending aorta, and extensively washed as described previously (Hawn and Strand, 1994Go). Eggs were obtained from infected mouse liver tissue according to standard methods (Lewis et al., 1977Go). The eggs produced by this procedure had less then 1% visible debris by microscopy. Shed cercariae were obtained from the intermediate snail host, Biomphalaria glabrata (Lewis and Colley, 1977Go). No visible debris was observed in the cercarial preparations.

Preparation of S. mansoni extracts
The schistosome homogenates were prepared according to DeBose-Boyd et al. (1996)Go with minor modifications. Adult worms, cercariae, and eggs of S. mansoni were washed in cacodylate buffer (50 mM, pH 7.0) and sonicated for 10 s at 50% of the maximum output in a cell disrupter (Branson Sonic Power Co.). The homogenates were made to 1% in Triton X-100 and incubated on ice for 30 min, then centrifuged for 10 min at 15,000 r.p.m. at 4°C. The resulting supernatants were divided in aliquots and stored at –80°C. The protein concentration of each extract was determined by the BCA protein assay (Pierce, Rockford, IL) according to manufacturer’s protocol.

Materials
ANTS was obtained from Molecular Probes (Eugene, OR) as its disodium salt. Oligosaccharides were obtained from Oxford Glyco Systems (Oxford, UK), V-Labs (Convington, LA), Glycotech (Rockville, MD), or Calbiochem (La Jolla, CA), as presented in Table I. The electrophoretic reagents were obtained from Bio-Rad (Hercules, CA). Guanosine diphospho-[U14C]fucose, (GDP-fucose), ammonium salt (310 mCi/mmol) was obtained from Amersham Life Science (Piscataway, NJ). The imaging plates BAS-TR2040 were purchased from Fuji Photo Film (Tokyo). All other reagents were reagent grade.

Oligosaccharide synthesis
Synthesis of Sialyl{alpha}2,3-LNnT. The biosynthesis of oligosaccharide NeuAc{alpha}2-3Galß1-4GlcNAcß1-3Galß1-4Glc, sialyl{alpha}2,3-LNnT (SLNnT) was performed with rat {alpha}2,3-(N)-sialyltransferase (Calbiochem). The enzyme (10 mU) was incubated with 800 nmoles of LNnT and 4 µmoles of CMP-ß-D-sialic acid (Calbiochem), in 50 mM MOPS, pH 7.4, with 0.2% Triton CF-54 and 0.5 mg/ml of ultrapure BSA (Boehringer Mannheim, Indianapolis, IN), a total volume of 160 µl for 6 h at 37°C. The reaction product was passed through an anion exchange column QAE Sephadex® A-50 (Pharmacia), and the sialylated sugars were eluted with 1 M ammonium formate, then lyophilized and resuspended in distilled water. A separate reaction mixture was also prepared using 0.1 mU of enzyme, 5 nmoles of LNnT, and 0.34 nmoles of CMP-ß-D-[U14C]sialic acid (300 mCi/mmol) (Amersham Life Science) in 10 µl and incubated under the same conditions. The sialylated oligosaccharide product was characterized by treatment with 1 mU of Newcastle disease virus neuraminidase (Boehringer Mannheim) for 1 h according to the manufacturer’s protocol. The purified products of the reactions were analyzed and the presence of sialic acid confirmed using FACE (data not shown).

Synthesis of schistosome core I. The biosynthesis of the oligosaccharide GalNAcß1,4GlcNAcß1,4GlcNAcß1,4GlcNAc, schistosome core I, was performed through adaptations of the method described by Palcic and Hindsgaul (1991)Go. The reaction mixture was prepared as follows: 4.77 µmoles of N, N', N''-triacetylchitotriose (Calbiochem) was incubated with 15.3 µmoles of UDP-{alpha}-D-N-acetylgalactosamine (Calbiochem) and 6 U of ß1,4-galactosyltransferase from bovine milk, which has been demonstrated to form GalNAcß1,4GlcNAc linkages (Calbiochem) in 100 mM sodium cacodylate buffer, pH 7.5, and 0.5 mM MnCl2 at 37°C in a volume of 1.9 ml for 24 h, after which an additional 15.3 µmoles of UDP-{alpha}-D-N-acetylgalactosamine and 3 U of ß1,4-galactosyltransferase were added for final volume of 2.4 ml and the reaction was incubated for an additional 24 h. Finally, 7 U of alkaline phosphatase from calf intestine (Boehringer Mannheim) was added to the reaction, and the MnCl2 was adjusted to 5 mM and incubated for an other 48 h. The reaction rate was determined by removing a sample of the mixture every 24 h and submitting it to FACE analysis. The reaction was continued until it reached 95% completion. The ß1,4-galactosyltransferase reaction product presented a migration profile distinct from that of the starting material. The migration profile of the oligosaccharide product, schistosome core I, was consistent with the migration profile of chitotetraose (GlcNAcß1,4GlcNAcß1,4 GlcNAcß1,4 GlcNAc), which has identical mass and charge (data not shown).

Fluorescent labeling
All oligosaccharides used for the fucosyltransferase assay were prelabeled with ANTS as follows (Jackson, 1990Go, 1996): 100 nmoles of each oligosaccharide in water was placed in individual tubes and lyophilized in a centrifugal vacuum evaporator. To each dry sample was added 5 µl of 0.2 M ANTS in acetic acid/water (3:17, v/v) and 5 µl of 1.0 M NaCNBH3 solution in DMSO. The contents were mixed and centrifuged, incubated at 37°C for 15 h, and then dried under vacuum at 45°C for 4 h. Finally, the ANTS-labeled oligosaccharides were resuspended in water at 10 nmoles/µl and stored at –80°C until use. The fluorescent intensities of the labeled oligosaccharides were compared to a standard ANTS labeled glucose ladder (Bio-Rad), revealing that each oligosaccharide was >95% converted to its ANTS derivative (data not shown).

FACE fucosyltransferase assay
Fucosyltransferase reactions were performed in a 10-µl reaction volume containing 100 mM cacodylate buffer (pH 7.0), 5 mM ATP, 10 mM MnCl2, 50 µg extract protein from cercariae or adult worms or 16 µg protein from egg homogenates, 0.5 mM of ANTS-labeled acceptor (unless otherwise indicated), and 16.6 µM (~115,000 c.p.m.) GDP-[14C]-Fuc (310 mCi/mmol). The reaction mixture was incubated at 37°C for times optimized for each reaction. At the end of the incubation the reaction was stopped by the addition of the electrophoresis loading buffer and the mixture was stored at –20°C prior to FACE analysis.

FACE analysis
The carbohydrate gel electrophoretic procedure was adapted from the protocol described by Jackson (1990, 1996). The fucosyltransferase reaction mixtures were subjected to PAGE using a Power PAC 3000 power supply (Bio-Rad) and a Mini-protean® II Cell. The electrophoretic buffer used was based on the Tris/HCl/glycine discontinuous system of Laemmli, without SDS. The polyacrylamide gels consisted of 32% (w/v) acrylamide and 2.0% bis-N,N'-methylenebisacrylamide as a cross-linker. The polymerization was initiated by addition of 8 µl of 5% ammonium persulfate solution and 4 µl N, N, N', N'-tetramethylethylenediamine per 5 ml gel solution. The samples were electrophoresed at 20 mA constant current at approximately 0°C. At the end of the electrophoresis fluorographic images were captured using a Kodak digital camera DC120 Zoom, with UV transillumination or by using the FACE Imager (Glyko, Novato, CA). The gels were then dried in a gel drier at 80°C for 1 h and exposed to an imaging plate (BAS-TR2040 phosphorimager plate; Fuji) for 3 days or to film (Biomax MR; Kodak, Rochester, NY) to quantify radioactivity.

Quantification of [14C] fucose
The dried gels were exposed to the imaging plates simultaneously with an autoradiographic [14C] micro-scale (Amersham Life Science), ranging from 0.045 to 45 pCi (approx. 0.1–100 d.p.m.). The imaging plates were analyzed using a BAS 1000 phosphorimager (Fuji). The pixel density of the standards was determined using MacBAS 2.5 software (Fuji) and plotted against d.p.m., and a correlation curve was determined by linear regression using StatView 4.5 (Abacus Concepts, Berkeley, CA). The correlation curve was then used to the convert the pixel densities of the fucosyltransferase product bands to d.p.m.

Fucosidase assay
Fucosidase assays were performed in 10 µl of 100 mM cacodylate buffer, pH 5.0, containing 5 nmoles of the ANTS-labeled substrate oligosaccharide 3'F-Lac (Galß1,4(Fuc{alpha}1,3)Glc-ANTS) or 2'F-Lac (Fuc{alpha}1,2Galß1,4Glc-ANTS), which form lactose (Galß1,4Glc-ANTS) after cleavage of the attached fucose. The reaction mixtures were incubated at 37°C in the presence of 50 µg protein from cercariae or adult worms or 16 µg protein from egg homogenates. The reaction was stopped by addition of 2.5 µl 5x gel electrophoresis sample buffer, and the reaction products were analyzed by FACE. The migration pattern of the ANTS-labeled lactose product of the fucosidase reactions on FACE was distinct from that of the substrates 3'F-Lac and 2'F-Lac. The rate of product formation was determined in triplicate by quantifying the fluorescence of each of the bands corresponding to the substrates and products and comparing these to standards, using Glyko FACE Analytical Software (Glyko).

Iminosugars
A new class of iminosugars, 1-N-iminosugars, was designed as described previously (Ichikawa et al., 1998Go). These 1-N-iminosugars contain a nitrogen atom in the anomeric position, whereas the conventional deoxynojirimycin-type iminosugars have a nitrogen atom in place of the ring oxygen of the monosaccharide (Figure 5A) (Ichikawa et al., 1998Go). The 1-N-iminosugars are more potent inhibitors of ß-glycosidases than the deoxynojirimicyn-type iminosugars, which are potent inhibitors of {alpha}-glycosidases (Ichikawa et al., 1998Go). The iminosugars used in this study were designated iminosugar A, iminosugar B, and iminosugar C (Figure 5B). Iminosugar D (deoxyfuconojirimycin) was purchased from Calbiochem.

Radioisotopic labeling of adult worms with [35S] methionine
Adult worms were cultured in vitro for 48 h in the presence or absence of iminosugar A at concentrations of 5–500 µM in DMEM with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES buffer (pH 7.5), 0.375% sodium bicarbonate, 0.2 U/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. The worms (three males and three females per well) were cultured in 24-well plates, 0.5 ml medium, at 37°C under a 5% CO2 atmosphere using duplicate wells for each condition. The medium and the iminosugars were replaced every 24 h. After 48 h the worms were metabolically labeled with [35S]methionine (600 Ci/mmol, 0.6 mCi/ml, Amersham) for 18 h in methionine-free DMEM plus the above supplements. At the end of the labeling period the worms were washed with DMEM and solubilized.

Radioimmunopreciptation
Tegumental membranes of [35S]methionine-labeled worms were solubilized with 0.2% Triton X-100, 10 mM CaCl2, 50 mM Tris–HCl (pH 7.8), and protease inhibitor cocktail (Complete EDTA-free, Boehringer Mannheim) for 30 min on ice. An aliquot of each labeled extract was then precipitated with trichloroacetic acid and the total radioactivity measured in triplicate. The labeled extracts were immunoprecipitated by standard protocols (Norden and Strand, 1985Go) with mAb 128C3/3 [which recognizes a highly fucosyltated structure described by Levery et al. (1992)Go] and mAb 103A3 (used as a negative control).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. Yang Lu for valuable comments on this manuscript. We also thank Dr. Brian Bradley and Glyko, Inc., for supplying the imaging system and associated technical expertise. Dr. Y. Ichikawa is grateful for support from NIH (GM 52324). Dr. E.T.A. Marques Jr. is supported by grants from CAPES (Brazilian Foundation for Advanced Education) number 1219/94-2, NIH R37AI19217, and WHO TDR960158.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ANTS, 8-aminonaphthalene-1,3,6 trisulfonic acid; FACE, fluorophore-assisted carbohydrate electrophoresis; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; Th2, T cell helper response type 2, associated with humoral immunological responses.


    Footnotes
 
1 To whom correspondence should be addressed 2Dedicated to our colleague and friend Dr. Mette Strand, deceased October 1997. Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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