Fucosyltransferases in Schistosoma mansoni development2

Glycobiology vol. 11 no. 3 pp. 249–259, 2001

Fucosyltransferases in Schistosoma mansoni development2

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

Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fucα1,3GlcNAc, Fucα1,2Fuc, Fucα1,6GlcNAc, and perhaps Fucα1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophoreassisted 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 (Galβ1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by α2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4Glc). A low level of fucosyltransferase that transfers fucose to nosialylated type-1 oligosaccharides (Galβ1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucosetype 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 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, glycosylphosphatidyl-

1To

whom correspondence should be addressed to our colleague and friend Dr. Mette Strand, deceased October 1997.

2Dedicated

© 2001 Oxford University Press

inositol anchors, and glycolipids (Cummings and Nyame, 1996, 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, 1996, 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., 1996; Le Marer et al., 1997). 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, 1985; Weiss et al., 1986, 1987; Levery et al., 1992). Schistosomal fucosylated structures contain α1,3 fucose linkages and other types, including Fucα1,2Fuc, Fucα1,6GlcNAc, and an unusual internal linkage, Fucα1,4GlcNAc (Levery et al., 1992). S. mansoni fucosyltransferase activities have been investigated in extracts of adult worms with the use of a small number of acceptor substrates (LNnT, LNT, NeuAcα2,3Galβ1,4GlcNAc, and NeuAcα2,6Galβ1,4GlcNAc; see Table I for acceptor substrate abbreviations) (DeBose-Boyd et al., 1996). 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 α3- and α2-fucosyltransferases were the main activities identified. One of the products formed by these T. ocellata transferases was determined to be Fucα1,2Fucα1,3GalNAcβ1,4GlcNAc (Hokke et al., 1998). 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., 1998). 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., 2000). 249

E.T.A. Marques et al.

Table I. List of oligosaccharides Number

Name

Structure

Supplier

01

LNT

Galβ1,3GlcNAcβ1,3Galβ1,4Glc

V-Labs

02

LNnT

Galβ1,4GlcNAcβ1,3Galβ1,4Glc

OGS

03

LNFII

Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc

V-Labs

04

LNFIII

Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4Glc

OGS

05

LNDI

Fucα1,2 Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc

OGS

06

LST a

NeuAcα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glc

OGS

07

LSTc


OGS

08

Lex

Galβ1,4(Fucα1,3)GlcNAc

OGS

09

SLex

NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc

OGS

10

Lea

Galβ1,3(Fucα1,4)GlcNAc

OGS

11

SLea

NeuAcα2,3Galβ1,3(Fucα1,4)GlcNAc

OGS

12

3′F-Lac

Galβ1,4(Fucα1,3)Glc

OGS

13

SF-Lac

NeuAcα2,3Galβ1,4(Fucα1,3)Glc

OGS

14

DFH

[Galβ1,4(Fucα1,3)GlcNAcβ1,6][Galβ1,4(Fucα1,3)GlcNAcβ1,3]Galβ1,4Glc

Glycotech

15

SLNnT


See text

16

Chitobiose

GlcNAcβ1,4GlcNAc

Calbiochem

17

Chitotriose

GlcNAcβ1,4GlcNAcβ1,4GlcNAc

Calbiochem

18

Chitotetraose

GlcNAcβ1,4GlcNAcβ1,4GlcNAcβ1,4GlcNAc

Calbiochem

19

Blood group A

GalNAcα1,3(Fucα1,2)Gal

Calbiochem

20

2′F-Lac

Fucα1,2Galβ1,4Glc

Calbiochem

21

Core I

GalNAcβ1,4GlcNAcβ1,4GlcNAcβ1,4GlcNAc

See text

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 welldefined 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 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, NeuAcα2-3Galβ1-4GlcNAc, is available, it cannot be used as an ANTS-labeled acceptor substrate because the sugar ring of the GlcNAc at the reducing 250

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, GalNAcβ1,4GlcNAcβ1,4GlcNAcβ1,4GlcNAc, was based on the carbohydrate structure of the schistosome neutral egg glycolipids identified by Khoo et al. (1997). 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 β1,4-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 fluorophoreassisted carbohydrate electrophoreses (FACE). Previous enzymatic syntheses using this procedure have resulted exclusively in β-linked terminal GalNAc (Palcic and Hindsgaul, 1991; Do et al., 1995). 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

Schistosomal fucosyltransferases

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. 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. 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

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.

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 (Galβ1,3 GlcNAcβ1,3Galβ1,4Glc) formed a product that comigrated with the fucosylated oligosaccharide LNFII (Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc). The most pronounced fucosyltransferase activity was observed in the presence of the acceptor LNnT (Galβ1,4GlcNAcβ1,3Galβ1,4Glc). This acceptor produced at least five different radioactive bands (Figure 3); the major band, designated LNnT3 (Figure 3), comigrated with the oligosaccharide LNFIII (Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4Glc), which is 251


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.

an expected product (DeBose-Boyd et al., 1996). 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 (Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc), 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. 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 (Fucα1,2Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc) and LSTa (NeuAcα2,3Galβ1,3Glc252

NAcβ1,3Galβ1,4Glc) did not form any detectable products. The sialylated oligosaccharide LSTc (NeuAcα2,6Galβ1,4GlcNAcβ1,3Galβ1,4Glc) 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 (Galβ1,4(Fucα1,3)Glc) as substrate yielded a fucosylated radioactive product, whereas the saccharide 2′F-Lac (Fucα1,2Galβ1,4Glc) 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 (NeuAcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc) was the secondbest acceptor substrate in the schistosome extracts and produced an intense radioactive band. In contrast to these


Table II. Summary of the fucosyltransferase activities identified in S. mansoni (activity, pmol/mg/h)

01)

Substratea

Cercaria

Adult

LNT(T)

—*

—

LNT1 02)

Egg 4.45 3.72

LNnT(T)

6.24

7.60

LNnT1

6.24

2.10

LNnT2

—

4.79

101

LNnT3

—

0.25

248

LNnT4

—

0.18

LNnT5

—

0.15

8.45

03)

LNFII(T)

—

0.14

0.62

04)

LNFIII(T)

1.72

0.51

4.01

LNFIII1

0.95

0.36

2.18

LNFIII2

0.27

0.16

1.23

LNDI

—

—

05)

408 7.01

33.2

—

06)

LSTa

—

—

—

07)

LSTc

—

0.16

15.0

08)

Lex

—

—

—

09)

LeA

—

—

—

10)

SLex

—

—

—

11)

SLeA

—

—

—

12)

3′F-Lac

—

—

13)

SF-Lac

—

—

—

14)

DFH

—

—

—

15)

SLNnT

0.39

0.47

52.7

16)

Chito2

ND**

ND

—

17)

Chito3

ND

ND

18)

Chito4

ND

ND

19)

Blood A

ND

ND

—

20)

2′Flac

ND

ND

—

21)

Core IT

—

—

19.3

2.22

1.61 3.18

*—, no detectible activity. ** ND, not determined. aSubscripts refer to electrophoretically distinct products (e.g., see Figure 3). (T) = total for all products.

results, there was no product formed in the control reactions in the absence of schistosome extract (data not shown). Chitotriose (GlcNAcβ1,4GlcNAcβ1,4GlcNAc) and chitotetraose (GlcNAcβ1,4GlcNAcβ1,4GlcNAcβ1,4GlcNAc) were also acceptors, each producing a single characteristic radioactive band. The smaller saccharide chitobiose (GlcNAcβ1,4GlcNAc) was apparently not used as an acceptor substrate by the Schistosoma. The saccharide blood group A (GalNAcα1,3(Fucα1,2)Gal) was not a substrate. Schistosome

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.

core I (GalNAcβ1,4GlcNAcβ1,4GlcNAcβ1,4GlcNAc) 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. 253


Because fucosidase activities in the schistosome extracts may interfere with the interpretation of the fucosyltransferase data, α2- and α3-fucosidase activities were determined (Figure 4). Fucosidase activity was determined by measuring the formation rate of lactose from the oligosaccharides 3′F-Lac (Galβ1,4(Fucα1,3)Glc) (Figure 4C) and 2′F-Lac (Fucα1,2Galβ1,4Glc) (Figure 4B) in the presence of schistosomal extracts. ANTS-labeled lactose migrates differently than do the ANTSlabeled fucosylated oligosaccharides (Figure 4A). Both α2(Figure 4B) α3-fucosidase (Figure 4C) activities were detected. The results are summarized in Table III. In these experiments the egg extracts showed higher α2- and α3fucosidase-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 α3-fucosidase activity was 2.5- and 10-fold greater than α2-fucosidase activity in the cercarial and adult worm extracts, respectively. In contrast, the α3-fucosidase activity in the egg extract was only one third of the α2-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.

Table III. Fucosidase activity in S. mansoni extracts incubated with 2′F-Lac or 3′F-Lac (pmol/mg/h) Substrate

Cercaria

2′F-Lac

33 (SD 19)

Adult 16 (SD 8)

981 (SD 107)

Egg

3′F-Lac

83 (SD 12)

166 (SD 84)

361 (SD 30)

SD, standard deviation.

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

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.

254

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)], 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 α-fucosidase activity was observed for iminosugars A and B at 5 mM,


mAb 128C3/3, which recognizes a highly expressed fucosylated epitope (Weiss and Strand, 1985); and with mAb 103A5, an isotype-matched control mAb, that does not react with schistosomal proteins (Figure 7). The amount of [35S]methioninelabeled 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. Discussion

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]3[Gal(NAc)β1->4]GlcNAc sequence. Glycobiology, 8, 393–406. Ichikawa, Y., Igarashi, Y., Ichikawa, M., and Suhara, Y. (1998) 1-N-Iminosugars: potent and selective inhibitors of β-glycosidases. J. Am. Chem. Soc., 120, 3007–3018. Jackson, P. (1990) The use of polyacrylamide-gel electrophoresis for the highresolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device. Biochem. J., 270, 705–713. Jackson, P. (1996) The analysis of fluorophore-labeled carbohydrates by polyacrylamide gel electrophoresis. Mol. Biotechnol., 5, 101–123. Khoo, K.H., Chatterjee, D., Caulfield, J.P., Morris, H.R., and Dell, A. (1997) Structural characterization of glycosphingolipids from the eggs of Schistosoma mansoni and Schistosoma japonicum. Glycobiology, 7, 653–661. Köster, B. and Strand, M. (1994) Schistosoma mansoni: immunolocalization of two different fucose-containing carbohydrate epitopes. Parasitology, 108, 433–446. Le Marer, N., Palcic, M.M., Clarke, J.L., Davies, D., and Skacel, P.O. (1997) Developmental regulation of α 1,3-fucosyltransferase expression in CD34 positive progenitors and maturing myeloid cells isolated from normal human bone marrow. Glycobiology, 7, 357–365. Lejoly-Boisseau, H., Appriou, M., Seigneur, M., Pruvost, A., Tribouley-Duret, J., and Tribouley, J. (1999) Schistosoma mansoni: in vitro adhesion of parasite eggs to the vascular endothelium. Subsequent inhibition by a monoclonal antibody directed to a carbohydrate epitope. Exp. Parasitol., 91, 20–29. Levery, S.B., Weiss, J.B., Salyan, M.E., Roberts, C.E., Hakomori, S., Magnani, J.L., and Strand, M. (1992) Characterization of a series of novel fucose-containing glycosphingolipid immunogens from eggs of Schistosoma mansoni. J. Biol. Chem., 267, 5542–5551. Lewis, F.A. and Colley, D.G. (1977) Modification of the lung recovery assay for schistosomula and correlations with worm burdens in mice infected with Schistosoma mansoni. J. Parasitol., 63, 413–417. Lewis, F.A., Sher, A., and Colley, D.G. (1977) Failure of plasma from human schistosomiasis mansoni patients to protect mice from Schistosoma mansoni cercarial challenge. Am. J. Trop. Med. Hyg., 26, 723–726. Maly, P., Thall, A., Petryniak, B., Rogers, C.E., Smith, P.L., Marks, R.M., Kelly, R.J., Gersten, K.M., Cheng, G., Saunders, T.L., and others. (1996) The α(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell, 86, 643–653. Mansour, M.H. (1996) Purification and characterization of SM 37: a fucosyllactose determinant-bearing glycoprotein probed by a Biomphalaria alexandrina lectin on adult male schistosomes. J. Parasitol., 82, 586–593.


Mansour, M.H., Negm, H.I., Saad, A.H., and Taalab, N.I. (1995) Characterization of Biomphalaria alexandrina-derived lectins recognizing a fucosyllactoserelated determinant on schistosomes. Mol. Biochem. Parasitol., 69, 173–184. Marques, E.T., Jr., Weiss, J.B., and Strand, M. (1998) Molecular characterization of a fucosyltransferase encoded by Schistosoma mansoni. Mol. Biochem. Parasitol., 93, 237–250. Norden, A.P. and Strand, M. (1985) Identification of antigenic Schistosoma mansoni glycoproteins during the course of infection in mice and humans. Am. J. Trop. Med. Hyg., 34, 495–507. Okano, M., Satoskar, A.R., Nishizaki, K., Abe, M., and Harn, D.A., Jr. (1999) Induction of Th2 responses and IgE is largely due to carbohydrates functioning as adjuvants on Schistosoma mansoni egg antigens. J. Immunol., 163, 6712–6717. Palcic, M.M. and Hindsgaul, O. (1991) Flexibility in the donor substrate specificity of β1,4- galactosyltransferase: application in the synthesis of complex carbohydrates. Glycobiology, 1, 205–209. Qiao, L., Murray, B., Shimazaki, M., Schultz, J. and Wong, C. (1996) Synergistic inhibition of human α1,3-fucosyltransferase V. J. Am. Chem. Soc., 118, 7653–7662.

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