The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x

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The dendritic cell specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni

egg antigens and recognizes the glycan antigen Lewis-x

Irma van Die1‡, Sandra J. van Vliet1, A. Kwame Nyame2, Richard D. Cummings2, Christine

M.C. Bank1, Ben Appelmelk3, Teunis B.H. Geijtenbeek1,

and Yvette van Kooyk1

Departments of 1Molecular Cell Biology and 3Medical Microbiology, VU University Medical

Center, van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands and 2Department of

Biochemistry and Molecular Biology, the Oklahoma Center for Medical Glycobiology,

University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

‡ To whom correspondence should be addressed:

Glycoimmunology GroupDepartment of Molecular Cell Biology,VU University Medical Center,van der Boechorststraat 7,1081 BT Amsterdam, the Netherlands.Tel: +31 20 4448157;Fax: +31 20 4448144;email: [email protected]

Copyright (c) 2003 Oxford University Press

Glycobiology Advance Access published February 20, 2003 by guest on June 6, 2013

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Running title: Recognition of Schistosome egg antigen by DC-SIGN

Keywords: adhesion, glycosylation, schistosomiasis, dendritic cells, carbohydrate-interaction

by guest on June 6, 2013http://glycob.oxfordjournals.org/

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Summary

Schistosoma mansoni soluble egg antigens (SEA) are crucially involved in modulating the

host immune response to infection by S. mansoni. We report here that human dendritic cells

bind SEA through the C-type lectin DC-SIGN. Monoclonal antibodies against the

carbohydrate antigens Galβ1-4(Fucα1-3)GlcNAc (Lex) and GalNAcβ1-4(Fucα1-3)GlcNAc

(LDNF) inhibit binding of DC-SIGN to SEA, suggesting that these glycan antigens may be

crucially involved in binding. In a solid-phase adhesion assay DC-SIGN-Fc binds polyvalent

neoglycoconjugates that contain Lex antigen, whereas no binding was observed to Galβ1-

4GlcNAc, and binding to neoglycoconjugates containing only α-fucose or oligosaccharides

with a terminal α1-2-linked fucose is low. These data indicate that binding of DC-SIGN to

Lex antigen is fucose-dependent, and that adjacent monosaccharides and/or the anomeric

linkage of the fucose are important for binding activity.

Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-

type N-glycans. Site-directed mutagenesis within the carbohydrate recognition domain

(CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to

HIV gp120, Lex and SEA. By contrast, mutation of amino acid Val351 abrogates binding to

SEA and Lex, but not to HIV gp120. These data suggest that DC-SIGN recognizes these

ligands through different, but overlapping regions within its CRD.

Our data imply that DC-SIGN is not only a pathogen receptor for HIV gp120, but may also

function in pathogen recognition by interaction with the carbohydrate antigens Lex and

possibly LDNF, that are found on important human pathogens such as Schistosomes and the

bacterium Helicobacter pylori.


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Introduction

Schistosomiasis is a major tropical parasitic disease, caused by trematode worms of the

genus Schistosoma. Pathology results from the host immune response to Schistosome eggs

and the granulomatous reaction evoked by the soluble egg antigens (SEA) that are released

through ultramicroscopic pores in the egg shell (Ross et al., 2002). A major focus of the host

immune response to schistosomes are glycoconjugates that abundantly occur on different

stages of the parasite (Cummings and Nyame, 1996; Cummings and Nyame, 1999). In

particular fucosylated schistosomal glycoconjugates are prominently involved in the host

humoral and cellular immune responses to infection. A strong humoral response has been

found against the glycan epitopes GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) and GalNAcβ1-

4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF) in both infected animals and humans (Nyame et al.,

2000; Van Remoortere et al., 2001; Eberl et al., 2002). Sera of infected hosts also contain

antibodies against Galβ1-4(Fucα1-3)GlcNAc (Lex, CD15), a glycan epitope shared by

humans and schistosomes (Nyame et al., 1998; Van Remoortere et al., 2001; Eberl et al.,

2002). These Lex antibodies may induce autoimmune reactions, as was shown by their ability

to mediate complement-dependent cytolysis of myeloid cells and granulocytes (Nyame et al.,

1996; Nyame et al., 1997; Van Dam et al., 1996). Lex as well as many other glycan epitopes

occur on several stages of the parasite, i.e. cercariae, eggs, schistosomula or adult worms

(Srivatsan et al., 1992; Cummings and Nyame, 1996; Van Remoortere et al., 2000).

Importantly, Lex containing glycoconjugates trigger cellular immune responses. They induce

proliferation of B-cells from infected animals, which secrete IL-10 and PGE2 (Velupillai and

Harn, 1994), and induce the production of IL-10 by peripheral blood mononuclear cells from

schistosome-infected individuals (Velupillai et al., 2000). In a murine schistosome model,

Lex is an effective adjuvant for induction of a Th2 response (Okano et al., 2001), and it has

been demonstrated that sensitization with Lex results in an increased cellular response


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towards SEA-coupled beads implanted in the liver and to the formation of granulomas

(Jacobs et al., 1999).

It is not clear yet how schistosome glycans trigger host immune responses, and which

cellular receptors are involved. Obviously, the immune responsiveness of T-lymphocytes to

antigens and their effector functions in response to inflammation require cell-antigen contact

and cell-cell adhesion. Antigen-presenting cells such as dendritic cells (DC) are expected to

play an important role in recognition of the schistosome antigens. DC are central in directing

Th1-Th2 responses and molecular patterns on the pathogen that are recognized by DC that

capture pathogen determinants, are crucial for biasing the Th immune response (Jankovic et

al., 2002). In mouse models, DC pulsed with SEA potently stimulate Th2 responses both in

vivo and in vitro while failing to undergo a conventional maturation process (MacDonald et

al., 2001). Therefore we hypothesized that receptors on DC, possibly C-type lectins that

recognize glycans, modulate DC functions upon interaction with S. mansoni SEA.

C-type lectins bind sugars in a Ca2+ dependent manner using conserved carbohydrate

recognition domains (CRDs) (Drickamer, 1999). DC express a variety of C-type lectins, that

specifically recognize glycan antigens (Figdor et al., 2002) and potential C-type lectins that

have been described previously to interact with pathogens include the mannose receptor

(MR)(Blackwell et al., 1985; Ezekowitz et al., 1990) and the C-type lectin DC-SIGN. DC-

SIGN was originally defined as a DC-specific receptor that supports DC-mediated T-cell

clustering by interaction with ICAM-3 (Geijtenbeek et al., 2000c), and migration of DC by

binding to endothelial expressed ICAM-2 (Geijtenbeek et al., 2000b). In addition, DC-SIGN

binds HIV-1 gp120 to facilitate transport of HIV from mucosal sites to draining lymph nodes

to infect T-cells (Geijtenbeek et al., 2000a). Like other C-type lectins on DC, DC-SIGN

captures and internalizes antigen for presentation to T-cells (Engering et al., 2002). However,

binding of HIV-1 to DC-SIGN escapes the antigen presentation route (Geijtenbeek et al.,


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2000a). Elucidation of the crystal structure of DC-SIGN in combination with binding studies

suggests that high-mannose glycans are recognized by DC-SIGN (Mitchell et al., 2001;

Feinberg et al., 2001). However, the glycan structures present on ICAM-2, ICAM-3 or HIV-1

gp120 that regulate the binding of DC-SIGN to these ligands have not yet been identified.

Here, we demonstrate that DC-SIGN interacts with SEA from Schistosoma mansoni

which may have important implications for its function as a receptor for Schistosomes.


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Results

S. mansoni soluble egg antigens (SEA) bind to human immature dendritic cells (DC) through

interaction with the C-type lectin DC-SIGN

Because DC are central in directing Th1-Th2 responses, we searched for a cell-surface

receptor expressed on DC that interacts with S. mansoni SEA. To detect binding of SEA to

human immature DC, a fluorescent-bead adhesion assay was developed. Fluorescent beads

were precoated with monoclonal antibodies (mAbs) to SEA glycan antigens and then used to

capture SEA. The conjugated beads were allowed to interact with DC, similar to the

approach described by (Geijtenbeek et al., 1999). SEA is a mixture of glycoproteins,

containing many immunogenic glycan antigens (Khoo et al., 1997; Cummings and Nyame,

1999). Major glycan antigens present in SEA, and the anti-glycan mAbs that specifically

recognize them, are depicted in Figure 1. To capture SEA on the fluorescent beads, we used

mAbs against the LDN antigen (Figure 1), a glycan epitope that abundantly occurs on many

glycoconjugates within SEA and which is absent on DC (not shown). The binding of SEA-

coated fluorescent beads to DC was comparable to the binding of HIV-1 gp120 to DC

(Figure 2B). To investigate whether the binding is mediated by a C-type lectin, we

investigated whether EDTA, that removes Ca2+ ions that are essential for carbohydrate

binding, or mannan hapten, could inhibit binding of SEA to the DC. The interaction of SEA

to DC was completely blocked by EDTA (not shown), and mannan (Figure 2B), suggesting

that the receptor on DC is a C-type lectin with mannose-specificity. Because the C-type

lectins DC-SIGN and the MR, that both are expressed by immature DC (Figure 2A), are

potential receptors for the recognition of glycan antigens, we investigated whether antibodies

directed against these molecules could inhibit the binding. Interestingly, the binding could be


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partially blocked by the anti-DC-SIGN mAb AZN-D1, that binds to the CRD of DC-SIGN

(Geijtenbeek et al., 2002), but not by the anti-MR mAb clone 19 (Figure 2B), indicating that

DC-SIGN is a pathogen receptor on DC for a subset of Schistosome SEA.

Glycoproteins within SEA of different Schistosome species contain ligands for DC-SIGN

To further analyse the binding properties of DC-SIGN to S. mansoni SEA, we investigated

binding of soluble chimeric DC-SIGN-Fc to SEA. In a solid phase adhesion assay DC-SIGN-

Fc showed efficient binding to wells coated with SEA (Figure 3A), whereas no binding was

observed using another Fc-protein, ICAM3-Fc (not shown). The interaction was completely

inhibited by the anti-DC-SIGN antibody AZN-D1, or EDTA. By contrast, the binding was

not affected by a DC-SIGN-specific antiserum directed against a peptide epitope (CCRD,

Engering et al., 2002) that is located C-terminal of the CRD (not shown). These results

indicate that binding of DC-SIGN to SEA is mediated by the CRD of DC-SIGN. Binding of

DC-SIGN-Fc to SEA is likely to be high avidity, since binding was observed at very low

coating concentrations of SEA (Figure 3B). Because SEA contains many different

glycoproteins, we investigated which glycoproteins within SEA interact with DC-SIGN-Fc.

SEA glycoproteins of S. mansoni, as well as of S. haematobium and S. japonicum, were

separated by SDS-PAGE and analysed by Western blotting with DC-SIGN-Fc, and as a

control with ICAM-3-Fc. Several glycoproteins within SEA of all three Schistosome species

showed specific interaction with soluble DC-SIGN-Fc (Figure 3C). Silver staining of a SDS-

PAGE polyacrylamide gel containing similar amounts of SEA showed that the DC-SIGN

binding proteins represent a subfraction of the Schistosome SEA.


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DC-SIGN binds to Lex containing glycans

Because it has been reported previously that SEA are heavily fucosylated (Khoo et al., 1995;

Khoo et al., 1997) and that DC-SIGN may exhibit binding to fucose (Curtis et al., 1992;

Mitchell et al., 2001), we explored whether the binding of DC-SIGN to SEA is fucose

mediated and can be blocked by mAbs specific for the fucosylated glycans Lex, LDNF or

LDN-DF in a competitive ELISA. The results show that anti-Lex and anti-LDNF mAbs

indeed inhibit the binding between SEA and DC-SIGN, whereas the binding between DC-

SIGN and HIV-1 gp120 was not affected by these antibodies (Fig. 4A). A combination of

anti-Lex and anti-LDNF mAbs strongly blocked the binding of DC-SIGN to SEA (Fig. 4A).

These data indicate that both Lex and LDNF glycans, that resemble each other by containing

a terminal fucose α1-3 linked to a GlcNAc residue, contribute to the binding of soluble DC-

SIGN-Fc to SEA. By contrast, no inhibition was found using the anti-LDN-DF mAbs in

similar concentration, suggesting that DC-SIGN cannot interact with LDN-DF, in which the

α1-3fucose is capped with an α1-2fucose.

Next we analyzed the potential of soluble DC-SIGN-Fc to bind to neoglycoproteins

containing different glycan moieties in ELISA. The results in Fig. 4B show that DC-SIGN-

Fc strongly interacts with a neoglycoprotein carrying Lex. The binding is fucose-dependent,

since no binding was observed to Galβ1,4GlcNAc (LacNAc). By contrast, binding to

similarly presented LDN-DF is poor. In addition, the binding of DC-SIGN-Fc to different

oligosaccharides linked to polyacrylamide (PAA) was analyzed. Biotinylated polyvalent

PAA-based neoglycoconjugates, carrying a similar density of oligosaccharides, were

captured on Streptavidine-coated ELISA plates and binding with soluble DC-SIGN-Fc was

detected as described above. The results in Figure 4B show that DC-SIGN-Fc binds to Lex-

PAA, whereas no, or low, binding was observed to PAA molecules carrying LacNAc, α-

fucose or Fucα1-2Galβ1-4GlcNAc (H-type2). Similar results were obtained when the


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biotinylated PAA molecules were coated directly to conventional microtiterplates. Our

preliminary data indicate that DC-SIGN also binds LDNF containing neoglycoconjugates

(not shown). These data demonstrate that DC-SIGN shows specific, fucose-dependent

binding to the trisaccharide antigen Lex and that adjacent monosaccharides, anomeric linkage

and/or position of the fucose residue in the oligosaccharide are important for binding activity.

The interaction of SEA with DC-SIGN differs from that of HIV-1 gp120

DC-SIGN recognizes both ligands derived from the pathogens HIV and schistosomes, as

well as ligands from self-molecules. To increase our understanding how DC-SIGN interacts

with these different ligands, we investigated in a DC-binding assay whether SEA inhibits the

interaction of dendritic-cell expressed DC-SIGN to HIV-1 gp120 and to ICAM-3, a T-cell

ligand that has been shown previously to interact with DC-SIGN (Geijtenbeek et al., 2000c).

The results demonstrate that SEA can inhibit the binding of both ICAM-3 and HIV gp120 to

DC (Figure 5A), suggesting that SEA interacts with both the ICAM-3 and HIV gp120 ligand

binding sites of DC-expressed DC-SIGN. To further define the interaction of SEA and Lex

with the CRD within DC-SIGN, we investigated the binding properties of these antigens to

K562 cell transfectants expressing mutant forms of DC-SIGN (Geijtenbeek et al., 2002) (Fig.

5B-D). Both SEA and Lex antigens strongly interact with K562 cells expressing DC-SIGN,

and not to mock-transfected K562 cells (Figure 5B). The C-type lectin domain of DC-SIGN

binds two Ca2+ ions (Geijtenbeek et al., 2000c) and those amino acid residues in close

contact with Ca2+ at site 2 (Glu347, Asn349, Glu354 and Asn365) or with Ca2+ at site 1 (Asp320,

Glu324, Asn350 and Asp355) are essential for ligand binding (Geijtenbeek et al., 2002).

Mutation of either Glu324 to Ala, or Glu347 to Gln in DC-SIGN (Figure 5C) resulted in

complete loss of interaction with both SEA and Lex (Figure 5B and results not shown),

indicating that binding to these antigens is mediated through the primary ligand-binding site


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and is Ca2+ dependent. This is similar to results reported for binding of DC-SIGN to HIV-1

gp120 (Geijtenbeek et al., 2002).

It was recently demonstrated that a specific mutation in the CRD of DC-SIGN (V351G)

allows binding of HIV-1 gp120 (Geijtenbeek et al., 2002), but abrogates binding to ICAM-3.

Our results indicate that the DC-SIGN V351G mutant does not bind to either SEA or Lex,

whereas binding to HIV-1 was still observed, indicating that Val351 is essential for binding to

both SEA and Lex (Figure 5B). These results demonstrate that the SEA binding site on DC-

SIGN may resemble the ICAM-3 binding site and partly overlaps the binding site for HIV-1

gp120.


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Discussion

Interactions between the human blood fluke Schistosoma mansoni and the host immune

system require contacts between parasite antigens and host immune cells. Because

schistosome eggs and the antigens they secrete play a major role in the pathogenesis, we

explored the molecular interactions between S. mansoni soluble egg antigen (SEA) and host

immune cells. In this study we show that S. mansoni SEA binds to human dendritic cells

(DC) and that DC-SIGN, a recently identified DC-specific C-type lectin, is primarily

involved in this interaction through binding glycan antigens carrying the α1-3-fucosylated

structures Lex and/or LDNF.

In schistosomiasis, SEA glycoconjugates are a major focus of the immune

responses generated in infected hosts. Among the SEA glycans, the Lex epitope is of

particular interest, as it has been shown to be involved in both humoral and cellular immune

responses, and is crucially involved in granuloma formation. Despite the accumulating data

about the importance of glycans in modulating the host immune response, not much is known

about the host receptors that interact with SEA or other schistosome glycans. Schistosome

eggs have been demonstrated to interact with soluble L-selectin but the egg glycan ligands

involved were not identified (Elridi et al., 1996). In addition, it has been shown that E-

selectin interacts with low affinity with the LDNF antigen on human protein C, which is also

present on schistosome glycans, but direct interaction between schistosome antigens and E-

selectin has not been demonstrated (Grinnell et al., 1994).

Dendritic cells are the first immune cells that encounter invading pathogens and they express

a variety of C-type lectins that can interact with carbohydrates on pathogen-derived antigens

(Figdor et al., 2002). Our data show that DC interact with SEA, and that a major part of this

binding activity could be blocked by antibodies against the DC-specific antigen receptor DC-


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SIGN. This suggest that DC-SIGN is a receptor for SEA on DC, although it cannot be

excluded that other DC receptors contribute to the binding of SEA. Because binding of DC-

SIGN-Fc to SEA is inhibited by antibodies recognizing Lex and LDNF carbohydrate

antigens, but not by antibodies against the difucosylated LDN-DF antigen, we propose that

specific presentation and anomeric linkage of the fucose residue in an oligosaccharide, such

as in Lex and LDNF, is important for binding. This is supported by our findings that DC-

SIGN specifically binds to polyvalent neoglycoconjugates carrying Lex, whereas binding to

similar polyvalent neoglycoconjugates carrying the monosaccharide αfucose, or

oligosaccharides with a terminal α1-2-linked fucose was poor. The glycan epitopes Lex and

LDNF are strongly related, as both contain a fucose that is α1-3-linked to GlcNAc, and both

epitopes contain a non-reducing sugar β1-4-linked to GlcNAc, which is galactose in Lex and

N-acetylgalactosamine in LDNF. Their occurrence differs though, since as far as known

expression of Lex in invertebrates is unique for schistosomes and the nematode Dictyocaulus

viviparus, whereas LDNF is a common glycan epitope in many helminths (Nyame et al.,

1998; Haslam et al., 2000). Future studies that involve isolation and characterization of the

SEA glycoproteins that bind DC-SIGN, will be required to establish the identity and

structural properties of the SEA ligands in more detail.

The binding of DC-SIGN to the Lex antigen is remarkable, since previous studies

showed that DC-SIGN interacts with mannose containing structures, especially when

presented in multivalent form (Mitchell et al., 2001; Feinberg et al., 2001). Sofar, the glycan

moieties on HIV gp120 and the self-glycoproteins ICAM-2 and ICAM-3 that are recognized

by DC-SIGN have not been characterized. ICAM-3 contains a small amount of N-linked high

mannose-type oligosaccharides (Funatsu et al., 2001) that may be essential for DC-SIGN

binding (Geijtenbeek et al., 2002). HIV gp120 contains many high-mannose type glycans

(Geyer et al., 1988) which may account for its high affinity binding to DC-SIGN. Since our


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data show that the carbohydrate recognition profile of DC-SIGN is broader than originally

thought, future studies are required to establish which carbohydrate structures are recognized

by DC-SIGN on the different ligands.

The primary binding site of DC-SIGN is situated in the C-type lectin domain and the

Ca2+ at site 2 and the amino acid residues Glu347, Asn349, Glu354 and Asn365 form the core of

this site (Feinberg et al., 2001; Geijtenbeek et al., 2002). Here, we demonstrate that SEA and

Lex specifically bind to this site, similar to the other DC-SIGN ligands ICAM-3 and HIV-1

gp120. The ligand binding site of DC-SIGN forms a hydrophobic surface resembling a

shallow trough, with the amino acid residue Val351 forming the edge of the binding pocket.

This Val351 is essential for the interaction of DC-SIGN with ICAM-3, whereas HIV-1 gp120

still binds a mutant DC-SIGN in which Val351 is converted to Gly (Geijtenbeek et al., 2002).

We here showed that binding of both SEA and Lex to DC-SIGN requires Val351,

demonstrating that their binding sites on DC-SIGN differ from that of HIV-1 gp120. By

binding to different carbohydrate ligands, DC-SIGN may trigger distinct effector functions in

DC, allowing them to induce pathogen-specific immune responses. Such a differential

binding capacity may also allow DC-SIGN to discriminate between the self-antigens (i.e.

ICAM-2 and ICAM-3) and pathogens, interactions that require the induction of different DC

effector functions.

The binding of DC-SIGN to SEA and Lex and/or LDNF containing glycoconjugates

suggests that DC-SIGN may function in the host immune response to schistosomes through

recognition of these glycan determinants. The occurrence of Lex and LDNF carbohydrate

antigens on different schistosome stages and species has been described (Srivatsan et al.,

1992; Van Dam et al., 1994; Nyame et al., 2000; van Remoortere et al., 2000; Wuhrer et al.,

2000; Khoo et al., 2001; Nyame et al., 2002). Thus, the recognition of these glycan antigens

by DC-SIGN may allow an early and continuous interaction with DC which may result in


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antigen-presentation and explain the specific anti-glycan humoral immune responses found in

schistosome infected hosts. It may also be possible that interaction between DC-SIGN and

Lex early in infection results in priming the host for vigourous granuloma formation when

challenged with egg antigens, as was proposed by (Jacobs et al., 1999). In addition, SEA

induce a predominant Th2 type immune response in murine as well as human

schistosomiasis, and a role for Lex antigens in this process has been proposed (Pearce et al.,

1991; Grzych et al., 1991; Velupillai and Harn, 1994; Araujo et al., 1996; Okano et al., 2001;

Terrazas et al., 2001). Our data may imply that DC-SIGN is involved in this process by

interaction with Lex, an hypothesis that is currently under study.


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Materials and Methods

Materials

Crude S. mansoni SEA extract was kindly provided by Dr. F. Lewis (Biomedical Research

Institute, Rockville, Maryland). The crude extract was centrifuged for 90 min at 100,000 x g

at 4ºC and sterilized by passing through a 0.2 µm filter. Unless otherwise indicated, binding

studies were performed with S. mansoni SEA. The following antibodies were used: anti-MR

(Clone 19, BD Pharmingen, NJ), CD11b (bear-1), CD11c (SHCL3), anti-DC-SIGN (AZN-

D1) (Geijtenbeek et al., 2000c), and the PE-conjugated antibodies CD80, CD83, CD86 and

HLA-DR (BD Pharmingen, NJ). Several anti-glycan monoclonal antibodies (mAbs) were

used: the IgG anti-LDN-DF mAb 114-5B1-A (van Remoortere et al., 2000), the IgM anti-

LDN mAb SMLDN1.1 (Nyame et al., 1999), the IgG anti-LDNF mAb SMFG4.1 and the IgG

anti-Lex Mab F8A1.1. The description and development of the latter two antibodies

SMFG4.1 and F8A1.1 will be described elsewhere (AK Nyame and RD Cummings,

manuscript in preparation). The neoglycoprotein Lex-HSA (containing approx. 20 mol

oligosaccharide/mol HSA) was from Isosep AB, Tullinge, Sweden. The neoglycoprotein

LDN-DF-BSA, containing on average 12 mol oligosaccharide/mol BSA, was previously

synthesized enzymatically as described (Van Remoortere et al., 2000). The polyvalent

neoglycoconjugates Lex-PAA, α-L-Fucose-PAA, Fucα1-2Galβ1-4GlcNAc (H-type2) and

LacNAc-PAA, were from Syntesome, Munich, Germany (saccharide 20% mol., biotin 5%

mol.). Coating of the neoglycoconjugates was established in all experiments with appropriate

lectins and glycan-specific antibodies.


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Cells

Immature DC were cultured from monocytes in the presence of IL4 and GM-CSF (500 and

800 U/ml, respectively; Schering-Plough, Brussels, Belgium) (Sallusto and Lanzavecchia,

1994). At day 7 the phenotype of the cultured DC was confirmed by flowcytometric analysis.

The DC express high levels of MHC class I and II, CD11b, CD11c and ICAM-1, moderate

levels of LFA-1 and CD80, and low levels of CD14. K562 transfectants expressing wild-type

DC-SIGN or mutant DC-SIGN (Geijtenbeek et al., 2002) were generated by transfection of

K562 cells with 10 µg pRc/CMV-DC-SIGN or mutant DC-SIGN plasmid by electroporation

as described previously (Geijtenbeek et al., 2000a).

Fluorescent bead adhesion assay

To demonstrate binding of SEA to whole cells, a fluorescent bead adhesion assay was used

as described (Geijtenbeek et al., 1999). Streptavidin was covalently coupled to the

TransFluorSpheres (488/645 nm, 1.0 µm; Molecular Probes, Eugene, OR) as described

previously (Geijtenbeek et al., 1999) and the streptavidin-coated beads were incubated with

biotinylated F(ab’)2 fragment of goat anti-mouse IgG (6 µg/ml; Jackson Immunoresearch),

followed by an overnight incubation at 4°C with anti-LDN mAb. The beads were washed and

incubated with 1 µg/ml SEA overnight at 4°C. Essentially, 50x103 cells were pre-incubated

in adhesion buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2,

0.5% BSA) with or without blocking mAbs (20 µg/ml) or mannan (50 µg/ml) for 10 minutes

at room temperature. Ligand-coated fluorescent beads (20 beads/cell) were added to the cells

and the suspension was incubated for 45 minutes at 37°C. Cells were washed and adhesion


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was determined using flow cytometry (FACScan, Becton Dickinson, Oxnard, CA), by

measuring the percentage of cells that had bound fluorescent beads. HIV-1 gp120 fluorescent

beads were prepared as described previously (Geijtenbeek et al., 2000a).

Solid phase DC-SIGN-Fc adhesion assay (ELISA) and inhibition by anti-glycan-Mabs

DC-SIGN-Fc consists of the extracellular portion of DC-SIGN (amino acid residues 64-404)

fused at the C-terminus to a human IgG1-Fc fragment into the Sig-pIgG1-Fc vector

(Geijtenbeek et al., 2002). DC-SIGN-Fc was produced in Chinese Hamster Ovary K1 cells by

co-transfection of DC-SIGN-Sig-pIgG1 Fc (20 µg) and pEE14 (5 µg) vector. DC-SIGN-Fc

concentrations in the supernatant were determined by an anti-IgG1 Fc ELISA. Recombinant

human ICAM3-Fc (Fawcett et al., 1992) was produced in CHO cells. The solid phase

adhesion assay was performed by coating neoglycoproteins (1 µg/ml) or SEA (0,03 - 4

µg/ml) in ELISA plates overnight at 4°C, followed by blocking with 1% BSA in TSM (20

mM Tris-HCl pH 7.4 containing 150 mM NaCl, 2 mM CaCl2 and 2 mM MgCl2) for 30 min.

at room temperature (RT). Alternatively, biotinylated PAA-based neoglycoconjugates in

TSM (1 µg/ml) were incubated for 1 hour in preblocked streptavidine-coated ELISA-plates

(Pierce), or coated directly in conventional ELISA plates similarly to the neoglycoproteins.

After washing, soluble DC-SIGN-Fc (0.5 - 1 µg/ml in TSM buffer) or when indicated

ICAM3-Fc (1 µg/ml in TSM buffer) as a negative control, was added and the adhesion was

performed for 60 min. at room temperature. Unbound Fc-protein was washed away and

binding was determined by an anti-IgG1 Fc ELISA using a peroxidase conjugate of goat anti-

human IgG1-Fc. DC-SIGN-specificity was determined in the presence of either 20 µg/ml

anti-DC-SIGN mAb (AZN-D1), or 5 mM EDTA. When indicated, anti-glycan mAbs were

used as competitive inhibitors. In those experiments SEA was coated at 0,5 µg/ml. After


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blocking, coated SEA were preincubated with antiglycan mAbs (at concentration 0,1 mg/ml

of total mAb) for 30 min at RT before adding DC-SIGN-Fc.

SDS-PAGE and Western blotting

SEA (2 µg/lane) were separated by SDS-PAGE under reducing conditions on a 15 %

polyacrylamide gel, using the Mini-Protean II system (BioRad) and blotted onto a

nitrocellulose membrane. The membrane was blocked in a solution of 5 % BSA in TSM for 2

h, followed by incubation with DC-SIGN-Fc or ICAM3-Fc (both 1 µg/ml in TSM buffer

containing 1% BSA) for 1 h. After washing (in TSM containing 0,1% Tween) and incubation

with alkalic phosphatase conjugated goat anti-human IgG, bound antibodies were detected

using x-phosphate/5-bromo-4-chloro-3-inodyl-phosphate (BCIP) (Boehringer Mannheim)

and 4-nitrobluetetrazoliumchloride (NBT) (Boehringer Mannheim).

Acknowledgements

We thank Wietske Schiphorst for technical assistence in part of the experiments. This work

was supported by an NIH grant (RO1 AI 47214) to R.D.C.


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Abbreviations

The abbreviations used are: LN, LacNAc, Galß1,4GlcNAc; Lewis x, Lex, Galβ1-4(Fucα1-

3)GlcNAc; LDN, LacdiNAc, GalNAcβ1-4GlcNAc; LDNF, GalNAcβ1-4(Fucα1-3)GlcNAc;

LDN-DF, GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc; DC, dendritic cells; DC-SIGN, dendritic

cell specific ICAM-3 grabbing non-integrin; MR, mannose receptor


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References

Araujo,M.I., Dejesus,A.R., Bacellar,O., Sabin,E., Pearce,E., and Carvalho,E.M. (1996).

Evidence of a T helper type 2 activation in human schistosomiasis. Eur. J. Immunol. 26,

1399-1403.

Blackwell,J.M., Ezekowitz,R.A.B., Roberts,M.B., Channon,J.Y., Sim,R.B., and Gordon,S.

(1985). Macrophage complement and lectin-like receptors bind Leishmania in absence of

serum. J. Exp. Med. 162, 324.

Cummings,R.D. and Nyame,A.K. (1996). Glycobiology of schistosomiasis. FASEB J. 10,

838-848.

Cummings,R.D. and Nyame,A.K. (1999). Schistosome glycoconjugates. Biochim. Biophys.

Acta 1455, 363-374.

Curtis,B.M., Scharnowske,S., and Watson,A.J. (1992). Sequence and expression of a

membrane-associated C-type lectin that exhibits CD4-independent binding of human

immunodeficiency virus envelope glycoprotein gp120. Proc. Natl. Acad. Sci. USA 89, 8356-

8360.

Drickamer,K. (1999). C-type lectin-like domains. Curr. Opinion Struct. Biol. 9, 585-590.

Eberl,M., Langermans,J.A.M., Vervenne,R.A., Nyame,K.A., Cummings,R.D., Thomas,A.W.,

Coulson,P.S., and Wilson,R.A. (2002). Antibodies to glycans dominate the host response to

schistosome larvae and eggs: is their role protective or subversive? J. Infect. Dis. 183, 1238-

1247.


Dow

nloaded from


22

22

Elridi,R., Velupillai,P., and Harn,D.A. (1996). Regulation of schistosome egg granuloma

formation - host-soluble L-selectin enters tissue-trapped eggs and binds to carbohydrate

antigens on surface membranes of miracidia. Infect & Immun 64, 4700-4705.

Engering,A., Geijtenbeek,T.B.H., van Vliet,S.J., Wijers,M., van Liempt,E., Demaurex,N.,

Lanzavecchia,A., Fransen,J., Figdor,C.G., Piguet,V., and van Kooyk,Y. (2002). The dendritic

cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells. J

Immunol 168, 2118-2126.

Ezekowitz,R.A.B., Sastry,K., Bailly,P., and Warner,A. (1990). Molecular characterization of

the human macrophage mannose receptor: demonstration of multiple carbohydrate

recognition-like domains and phagocytosis of yeasts in Cos-1 cells. J. Exp. Med. 172, 1785-

1794.

Fawcett J., Holness C.L., Needham L.A., Turley H., Gatter K.C., Mason D.Y., Simmons D.L.

(1992). Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on

resting leukocytes. Nature 360, 481-484.

Feinberg,H., Mitchell,D.A., Drickamer,K., and Weis,W.I. (2001). Structural basis for

selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 294, 2163-

2166.

Figdor,C.G., van Kooyk,Y., and Adema,G.J. (2002). C-type lectin receptors on dendritic cells

and langerhans cells. Nature Rev. Immunol. 2, 77-84.

Funatsu,O., Sato,T., Kotovuori,P., Gahmberg,C.G., Ikekita,M., and Furukawa,K. (2001).

Structural study of N-linked oligosaccharides of human intercellular adhesion molecule-3

(CD50). Eur. J. Biochem. 268, 1020-1029.


Dow

nloaded from


23

23

Geijtenbeek,T.B.H., Kwon,D.S., Torensma,R., van Vliet,S.J., van Duijnhoven,G.C.F.,

Middel,J., Cornelissen,I.L.M.H.A., Nottet,H.S.L.M., KewalRamani,V.N., Littman,D.R.,

Figdor,C.G., and van Kooyk,Y. (2000a). DC-SIGN, a dendritic cell-specific HIV-1-binding

protein that enhances trans-infection of T-cells. Cell 100, 587-597.

Geijtenbeek,T.B.H., Krooshoop,D.J.E.B., Bleijs,D.A., van Vliet,S.J., van Duijnhoven,G.C.F.,

Grabovsky,V., Alon,R., Figdor,C.G., and van Kooyk,Y. (2000b). DC-SIGN-ICAM-2

interaction mediates dendritic cell trafficking. Nature Immunology 1, 353-357.

Geijtenbeek,T.B.H., Torensma,R., van Vliet,S.J., van Duijnhoven,G.C.F., Adema,G.J., van

Kooyk,Y., and Figdor,C.G. (2000c). Identification of DC-SIGN, a novel dendritic cell-

specific ICAM-3 receptor that supports primary immune responses. Cell 100, 575-585.

Geijtenbeek,T.B.H., van Duijnhoven,G.C.F., van Vliet,S.J., Krieger,E., Vriend,G.,

Figdor,C.G., and van Kooyk,Y. (2002). Identification of different binding sites in the

dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1. J.

Biol. Chem. 277, 11314-11320.

Geijtenbeek,T.B.H., van Kooyk,Y., van Vliet,S.J., Renes,M.H., Raymakers,R.A.P., and

Figdor,C.G. (1999). High frequency of adhesion defects in B-Lineage acute lymphoblastic

leukemia. Blood 94, 754-764.

Geyer,H., Holschbach,C., Hunsmann,G., and Schneider,J. (1988). Carbohydrates of human

immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein

120. J. Biol. Chem. 263, 11760-11767.

Grinnell,B.W., Hermann,R.B., and Yan,S.B. (1994). Human protein C inhibits selectin-

mediated cell adhesion: role of unique fucosylated oligosaccharides. Glycobiology 4, 221-

225.


Dow

nloaded from


24

24

Grzych,J.M., Pearce,E., Cheever,A., Caulada,Z.A., Caspar,P., Heiny,S., Lewis,F., and

Sher,A. (1991). Egg deposition is the major stimulus for the production of Th2 cytokines in

murine schistosomiasis mansoni. J Immunol 146, 1322-1327.

Haslam,S.M., Coles,G.C., Morris,H.R., and Dell,A. (2000). Structural characterization of the

N-glycans of Dictyocaulus viviparus: discovery of the Lewisx structure in a nematode.

Glycobiology 10, 223-229.

Jacobs,W., Deelder,A.M., and Van Marck,E. (1999). Schistosomal granuloma modulation. II.

Specific immunogenic carbohydrates can modulate schistosome-egg-antigen-induced hepatic

granuloma formation. Parasitol. Res. 85, 14-18.

Jankovic,D., Liu,Z., and Gause,W.C. (2002). Th1-and Th2-cell commitment during

infectious disease: asymmetry in divergent pathways. Trends in Immunol. 22, 450-457.

Khoo,K.-H., Chatterjee,D., Caulfield,J.P., Morris,H.R., and Dell,A. (1997). Structural

mapping of the glycans from the egg glycoproteins of Schistosoma mansoni and Schistosoma

japonicum - identification of novel core structures and terminal sequences. Glycobiology 7,

663-677.

Khoo,K.-H., Sarda,S., Xu,X., Caulfield,J.P., McNeil,M.R., Homans,S.W., Morris,H.R., and

Dell,A. (1995). A unique multifucosylated -3GalNAc β1-4GlcNAcβ1-3Galα1- motif

constitutes the repeating unit of the complex O-glycans derived from the cercarial glycocalyx

of Schistosoma mansoni. J. Biol. Chem. 270, 17114-17123.

Khoo,K.H., Huang,H.H., and Lee,K.M. (2001). Characteristic structural features of

schistosome cercarial N-glycans: expression of Lewis X and core xylosylation. Glycobiology

11, 149-163.


Dow

nloaded from


25

25

MacDonald,A.S., Straw,A.D., Bauman,B., and Pearce,E.J. (2001). CD8- dendritic cell

activation status plays an integral role in influencing Th2 response development. J Immunol

167, 1982-1988.

Mitchell,D.A., Fadden,A.J., and Drickamer,K. (2001). A novel mechanism of carbohydrate

recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and

binding to multivalent ligands. J. Biol. Chem. 276, 28939-28945.

Nyame,A.K., Debose-Boyd,R., Long,T.D., Tsang,V.C.W., and Cummings,R.D. (1998).

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. Glycobiology 8, 615-624.

Nyame,A.K., Leppanen,A.M., Debose-Boyd,R., and Cummings,R.D. (1999). Mice infected

with Schistosoma mansoni generate antibodies to LacdiNAc (GalNAcβ1,4GlcNAc)

determinants. Glycobiology 9, 1029-1035.

Nyame,A.K., Pilcher,J.B., Tsang,V.C.W., and Cummings,R.D. (1996). Schistosoma mansoni

infection in humans and primates induces cytolytic antibodies to surface Lex determinants on

myeloid cells. Exp. Parasitol. 82, 191-200.

Nyame,A.K., Pilcher,J.B., Tsang,V.C.W., and Cummings,R.D. (1997). Rodents infected with

Schistosoma mansoni produce cytolytic IgG and IgM antibodies to the Lewis x antigen.


Nyame,A.K., Yoshino,T.P., and Cummings,R.D. (2002). Differential expression of LDN,

LDNF and Lewis x glycan antigens in intramolluscan stages of Schistosoma mansoni. J.

Parasitol. In Press.


Dow

nloaded from


26

26

Nyame,K.A., Leppanen,A., Bogitsh,B.J., and Cummings,R.D. (2000). Antibody responses to

the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-infected mice and

expression of the glycan among schistosomes. Exp. Parasitol. 96, 202-212.

Okano,M., Satoskar,A.R., Nishizaki,K., and Harn,D.A., Jr. (2001). Lacto-N-fucopentaose III

found on Schistosoma mansoni egg antigens functions as adjuvant for proteins by inducing

Th2-type response. J Immunol 167, 442-450.

Pearce,E.J., Caspar,P., Grzych,J.M., Lewis,F.A., and Sher,A. (1991). Downregulation of Th1

cytokine production accompanies induction of Th2 responses by a parasitic helminth,

Schistosoma mansoni. J. Exp. Med. 173, 159-166.

Ross,A.G.P., Bartley,P.B., Sleigh,A.C., Olds,G.R., Li,Y., Williams,G.M., and McManus,D.P.

(2002). Schistosomiasis. N Engl J Med 346, 1212-1220.

Sallusto,F. and Lanzavecchia,A. (1994). Efficient presentation of soluble antigen by cultured

human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor

plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp.Med.179, 1109-

1118.

Srivatsan,J., Smith,D.F., and Cummings,R.D. (1992). The human blood fluke Schistosoma

mansoni synthesizes glycoproteins containing the Lewis X antigen. J. Biol. Chem. 267,

20196-20203.

Terrazas,L.I., Walsh,K.L., Piskorska,D., McGuire,E., and Harn,D.A., Jr. (2001). The

Schistosome oligosaccharide lacto-N-neotetraose expands Gr1+ cells that secrete anti-

inflammatory cytokines and inhibit proliferation of naive CD4+ cells: A potential mechanism

for immune polarization in helminth infections. J Immunol 167, 5294-5303.


Dow

nloaded from


27

27

Van Dam,G.J., Bergwerff,A.A., Thomas-Oates,J.E., Rotmans,J.P., Kamerling,J.P.,

Vliegenthart,J.F.G., and Deelder,A.M. (1994). The immunologically reactive O-linked

polysaccharide chains derived from circulating cathodic antigen isolated from the human

blood fluke Schistosoma mansoni have Lewis x as repeating unit. Eur. J. Biochem. 225, 467-

482.

Van Remoortere,A., Hokke,C.H., Van Dam,G.J., Van Die,I., Deelder,A.M., and Van den

Eijnden,D.H. (2000). Various stages of Schistosoma express Lewisx, LacdiNAc, GalNAcβ1-

4(Fucα1−3)GlcNAc, and GalNAcβ1-4(Fucα1−2Fucα1-3)GlcNAc carbohydrate epitopes:

detection with monoclonal antibodies that are characterized by enzymatically synthesized

neoglycoproteins. Glycobiology 10, 601-609.

Van Remoortere,A., Van Dam,G.J., Hokke,C.H., Van den Eijnden,D.H., Van Die,I., and

Deelder,A.M. (2001). Profiles of immunoglobulin M (IgM) and IgG antibodies against

defined carbohydrate epitopes in sera of Schistosoma-infected individuals determined by

surface plasmon resonance. Infect. Immun. 69, 2396-2401.

Van Dam,G.J., Claas,F.H.J., Yazdanbakhsh,M., Kruize,Y.C.M., Van Keulen,A.C.I.,

Ferreira,S.T.M.F., Rotmans,J.P., and Deelder,A.M. (1996). Schistosoma mansoni excretory

circulating cathodic antigen shares lewis-x epitopes with a human granulocyte surface

antigen and evokes host antibodies mediating complement-dependent lysis of granulocytes.

Blood 88, 4246-4251.

Velupillai,P., Dos Reis,E.A., Dos Reis,M.G., and Harn,D.A. (2000). Lewisx-containing

oligosaccharide attenuates schistosome egg antigen-induced immune depression in human

schistosomiasis. Human Immunology 61, 225-232.


Dow

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28

28

Velupillai,P. and Harn,D.A. (1994). Oligosaccharide-specific induction of interleukin 10

production by B220+ cells from schistosome-infected mice: a mechanism for regulation of

CD4+ T-cell subsets. Proc. Natl. Acad. Sci. USA 91, 18-22.

Wuhrer,M., Dennis,R.D., Doenhoff,M.J., Lochnit,G., and Geyer,R. (2000). Schistosoma

mansoni cercarial glycolipids are dominated by Lewis X and pseudo-Lewis Y structures.



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Figure Legends

Fig. 1: Structures of S. mansoni SEA carbohydrate antigens implicated in immune

responses, and monoclonal antibodies (mAbs) recognizing these antigens

Fig. 2: Interaction of SEA with immature human DC. (A) Immature human DCs were

cultured from monocytes in the presence of GM-CSF and IL-4. DCs express high levels of

DC-SIGN, in addition to expressing MR, CD83, CD86, CD80 and HLA-DR as determined

by FACScan analysis. (B) DC-SIGN, expressed by immature DC, shows a similar binding to

SEA as to HIV-1 gp120, a previously defined ligand of DC-SIGN. The adhesion to both SEA

and HIV-1 gp120 was determined using the fluorescent bead adhesion assay. Mannan and

anti-DC-SIGN mAb AZN-D1, but not anti-MR mAb clone 19, block adhesion of SEA to the

DC. The experiment was performed twice in triplicate with DC derived from a different

batch of monocytes. The results shown the mean values of a triplicate assay from one

representative experiment. The standard deviation was <5% in all assays.

Fig. 3: Binding of DC-SIGN to Schistosome SEA. (A) DC-SIGN binds to S. mansoni SEA,

as was determined by an ELISA based assay using soluble DC-SIGN-Fc. Antigens were

coated at a concentration of 1 µg/ml, and binding of DC-SIGN-Fc was measured using

peroxidase conjugated anti-human IgG. No binding to SEA was detected using ICAM3-Fc

(not shown), or using DC-SIGN-Fc in the presence of the anti-DC-SIGN blocking antibody

AZN-D1 (20µg/ml), or EDTA (5 mM). (B) Binding of soluble DC-SIGN-Fc to S. mansoni

SEA, coated to wells in different concentrations, was measured by an anti-IgG-Fc ELISA,

similar as in A. (C) DC-SIGN-Fc binds SEA of different Schistosome species (Sm, S.

mansoni; Sj, S. japonicum; Sh, S. haematobium). SEA (approx. 2 µg each lane) were


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separated by SDS-PAGE on a 15% polyacrylamide gel. The left panel shows a silverstained

gel. Panels 2 and 3 show Western blots incubated with soluble DC-SIGN-Fc and ICAM3-Fc,

respectively.

Fig. 4: DC-SIGN binds to αααα1-3-fucosylated glycans (A) Competitive inhibition of the

binding of soluble DC-SIGN-Fc to SEA and HIV-1 gp120 by anti-glycan mAbs in ELISA.

Coated antigens were preincubated with antiglycan mAbs before adding DC-SIGN-Fc.

Binding of DC-SIGN-Fc was measured by an anti-IgG-Fc ELISA. The experiments were

performed twice in duplicate. The results shown represent the mean percentage of binding ±

SD. (B) DC-SIGN-Fc binds to neoglycoconjugates carrying Lex, but poorly to other

neoglycoconjugates in ELISA. Neoglycoproteins (1 µg/ml) were coated directly to the

microtiter plates in coating buffer, and biotinylated PAA neoglycoconjugates (50 ng/well)

were captured on streptavidine coated microtiter plates. Coated neoglycoconjugates were

incubated with soluble DC-SIGN-Fc. The experiments were performed three times in

duplicate, with essentially similar results. The Figure shows the mean values ± SD of one

representative experiment.

Fig. 5: Binding of SEA and Lex to mutant forms of DC-SIGN (A) SEA blocks binding of

DC-SIGN, expressed by human immature DC, to ICAM-3 and HIV-1-gp120. The adhesion

of both ICAM-3 and HIV-1 gp120 to DC was determined using the fluorescent bead

adhesion assay. Inhibitors were present at a concentration of 20 µg/ml. The experiment was

performed twice in triplicate (S.D. <5%), with DC derived from different batches of

monocytes. The results of both experiments were essentially similar, and the mean values of

one experiment are shown. (B) Binding of SEA and Lex-PAA coated beads to K562

transfectants expressing wild-type DC-SIGN or the E324A, E347Q and V351G DC-SIGN


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mutants, was measured using the fluorescent bead adhesion assay. The experiment was

performed twice in triplicate (S.D. <5%), with different cell cultures. The results show the

mean values of one representative experiment. (C) Amino acid sequence of part of the CRD

of DC-SIGN (AAK20997). The position of the E324A, E347Q and V351G mutations are

indicated by an arrow. (D) K562 transfectants and different mutant forms of DC-SIGN

express similar levels of DC-SIGN as determined by FACScan analysis using AZN-D2 mAb.


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R Lewis x

LDN SMLDN1.1

GlcNAc GalNAcGal Fucose

β4

β4

α3

LDNFβ4

α3

LDN-DF 114-5B1-Aβ4

α3

α2

R

R

R

Carbohydrate Antigens in S. mansoni SEACarbohydrate epitope Abbreviation Anti-glycan mAb

Fig. 1

F8A1.1

SMFG4.1


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0

20

40

60

80

No inhibitor+ anti-DC-SIGN mAb+ anti-MR mab + Mannan

% D

C a

dhes

ion

Expression (log fluorescence)

DC-SIGN

MR

CD80

CD83

CD86

HLA-DR

Cou

nts

A B

SEAHIV-1 gp120

Fig. 2


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Silverstain - DC-SIGN-Fc - ICAM3-Fc

Sm Sj Sh - Sm Sj Sh - Sm Sj Sh

kD

93

48

35

28

0,2

0,4

0,6

0,8

1,0

1,2

1,4

SEA

no inhibitor

+ anti-DC-SIGN MAb+ EDTA

HIV-1 gp120

DC

-SIG

N-F

c bi

ndin

g(A

bsor

banc

e)

Figure 3

A

0

0.4

0.8

1.2

1.6

2

4.00 1.00 0.25 0.06

DC

-SIG

N-F

c bi

ndin

g(A

bsor

banc

e)

B

C

coated SEA (ug /ml)


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50 100

anti-DC-SIGNanti-Lex + anti-LDNF

anti-LDNFanti-Le x

no inhibitor

anti-DC-SIGNanti-Lex + anti-LDNF

anti-LDN-DFanti-LDNFanti-LDNanti-Le x

no inhibitor

7525% binding of DC-SIGN-Fc to coated antigen

Com

petit

ive i

nhib

itors

HIV gp120

SEA

0 0.4 0.8 1.2 1.6

LacNAc -PAA

αFuc -PAAH-type2-PAA

Lex-PAA

BSA

LDN-DF-BSA

LacNAc-HSA

Lex-HSA

Binding of DC-SIGN-Fc to coated antigen(Absorbance)

Figure 4

A

B


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Documents

The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x