Abstract
The dendritic cell-specific C-type lectin DC-SIGN functions as a pathogen receptor that recognizes Schistosoma mansoni egg antigens through its major glycan epitope Galbeta1,4(Fucalpha1,3)GlcNAc (Lex). Here we report that L-SIGN, a highly related homologue of DC-SIGN found on liver sinusoidal endothelial cells, binds to S. mansoni egg antigens but not to the Lex epitope. L-SIGN does bind the Lewis antigens Lea, Leb, and Ley, similar as DC-SIGN. A specific mutation in the carbohydrate recognition domain of DC-SIGN (V351G) abrogates binding to all Lewis antigens. In L-SIGN Ser363 is present at the corresponding position of Val351 in DC-SIGN. Replacement of this Ser into Val resulted in a "gain of function" L-SIGN mutant that binds to Lex, and shows increased binding to the other Lewis antigens. These data indicate that Val351 is important for the fucose specificity of DC-SIGN. Molecular modeling and docking of the different Lewis antigens in the carbohydrate recognition domains of L-SIGN, DC-SIGN, and their mutant forms, demonstrate that Val351 in DC-SIGN creates a hydrophobic pocket that strongly interacts with the Fucalpha1,3/4-GlcNAc moiety of the Lewis antigens. The equivalent amino acid residue Ser363 in L-SIGN creates a hydrophilic pocket that prevents interaction with Fucalpha1,3-GlcNAc in Lex but supports interactions with the Fucalpha1,4-GlcNAc moiety in Lea and Leb antigens. These data demonstrate for the first time that DC-SIGN and L-SIGN differ in their carbohydrate binding profiles and will contribute to our understanding of the functional roles of these C-type lectin receptors, both in recognition of pathogen and self-glycan antigens.
Highlights
The dendritic cell-specific C-type lectin dendritic cells (DCs)-SIGN functions as a pathogen receptor that recognizes Schistosoma mansoni egg antigens through its major glycan epitope Gal1,4(Fuc␣1,3)GlcNAc (Lex)
L-SIGN Shows Binding to S. mansoni soluble egg antigens (SEA)—In a previous study [15], we showed that DC-SIGN binds S. mansoni SEA through the recognition of Lex antigens
The LDN-captured SEA beads interacted with K562 cells expressing DC-SIGN; we could not observe any binding to L-SIGN (Fig. 1C)
Summary
Antibodies and Neoglycoconjugates—The following antibodies were used, AZN-D1 (IgG1, anti-DC-SIGN), AZN-D2 (anti-DC-SIGN/anti-LSIGN) [1, 22], and anti-LDN monoclonal antibody SMLDN1.1 [25]. Binding Assay—Stable K562 cells expressing different wild-type and mutant C-type lectins (5 ϫ 104 cells) were incubated in a total volume of 25 l with biotinylated PAA-linked glycoconjugates (5 g/ml) in adhesion buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2, and 0.5% bovine serum albumin) for 30 min at 37 °C. Fluorescent Bead Adhesion Assay—For measuring SEA binding to whole cells, a bead adhesion assay was used as described previously [27]. For the fluorescent beads adhesion assay 50 ϫ 103 cells were resuspended in adhesion buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2, and 0.5% bovine serum albumin). Adhesion was determined using flow cytometry (FACScan) by measuring the percentage of cells that had bound fluorescent beads in the FL-3 channel. Energy minimizations were carried out using the Powell procedure until a gradient deviation of 0.05 kcal1⁄7molϪ11⁄7ÅϪ1 was attained
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