Salp15 Binding to DC-SIGN Inhibits Cytokine Expression by Impairing both Nucleosome Remodeling and mRNA Stabilization (original) (raw)
Figure 2
Salp15 Interacts with DCs through the C-Type Lectin DC-SIGN
(A) Salp15 binds to DC-SIGN on DCs. A fluorescent bead adhesion assay was performed using fluorescent beads coated with HIV-1 gp120 (positive control) and Salp15. As negative control beads we used antibody (anti-V5) coated beads. Immature DCs were incubated with the ligands and binding was measured by FACS analysis. Specificity was determined by preincubation of DCs with EDTA or mannan (significance determined by a two-sided one way ANOVA, implementing a one way analysis of variance and a Dunnett multiple comparison test).
(B) Salp15 contains mannose and galactose-specific glycosylations. The glycosylation pattern of Salp15 was analysed by a plant lectin ELISA. For abbreviations see Materials and Methods.
(C) Salp15 binding to DCs is inhibited by carbohydrates that interact with DC-SIGN. Specificity of binding of DCs to Salp15 was further assessed by pretreating DCs with mannan, free carbohydrate structures (Galactose, N-acetylgalactosamine [GalNac], N-acetyl-D-glucosamine [GlcNac], or Fucose), or biotinylated Lewis-X/Y (LeX/Y) before performing the fluorescent bead adhesion assay as described in (A). Salp15 binding was set at 100%.
(D) Salp15 interacts specifically with DC-SIGN. The fluorescent bead adhesion assay was performed using the Raji-cell line transfected with DC-SIGN. Mannan and EDTA was used to determine specificity for C-type lectins.
(E) Glycosylation of Salp15 is important in the interaction with DC-SIGN. A DC-SIGN-Fc binding ELISA was performed on Salp15, deglycosylated Salp15 (by incubation with PGNaseF), and mannan (positive control). For Salp15 and mannan DC-SIGN-Fc binding could be blocked by preincubation with mannan (significance determined by a two-sided unpaired Student _t_-test).
(F) DC-SIGN is the major receptor on immature DCs for Salp15. Both a cell-surface biotinylated and non-biotinylated DC lysate were incubated with Salp15-coupled protein A-Sepharose beads. The biotinylated immunoprecipitate was analysed on SDS-PAGE, transferred to a nitrocellulose membrane, and detected by streptavidin-PO. The non-biotinylated immunoprecipitate was analyzed by SDE-PAGE, transferred to a nitrocellulose membrane, and detected by anti-DC-SIGN antibodies. As controls, anti-DC-SIGN protein A-Sepharose beads and anti-V5-protein A-Sepharose beads were used.
Adhesion experiments and ELISAs are representative of at least three independent experiments. Error bars, where depicted, represent the SE of duplicates or triplicates within one experiment. A _p_-value < 0.05 was considered statistically significant. (*) 0.01 < p < 0.05; two asterisks (**) 0.001 < p < 0.01. The immunoprecipitations are representative of two experiments.