Figure 1.
Langerin ECD interaction onto heparin.
Surface was functionalized with heparin 6 kDa. 100 µL Langerin ECD at 500 nM are injected onto the surface in a Ca2+ containing running buffer. Two modes of surface regenerations are tested, 1: Injection of 30 µL of 50 mM EDTA. 2: Injection of 50 µL of 350 mM MgCl2.
Figure 2.
Comparison of heparin and gp120 binding mode to Langerin.
A) Overlay of sensorgram showing Langerin CRD interaction onto gp120YU2 functionalized surface in Ca2+ buffer. Langerin CRD concentration range is from 400 µM to 12,5 µM with 2 times serial dilution. B) Overlay of sensorgram showing Langerin ECD interaction onto gp120YU2 functionalised surface in calcium buffer. The concentration range of Langerin ECD is from 2 µM to 7,8 nM with 2 times serial dilution. C) SPR sensorgram of Langerin ECD interaction onto gp120YU2 functionalised surface in calcium and EDTA buffer. Langerin ECD injection has been performed at 500 nM concentration of protein. D) SPR binding analysis of gp120 interaction as a function of Langerin ECD concentration. E) Overlay of sensorgram showing Langerin CRD interaction onto biotinylated 6 kDa heparin functionalised surface in calcium buffer. Langerin CRD concentration was from 100 µM to 1.6 µM with 2-fold serial dilution. F) Overlay of sensorgram showing of Langerin ECD interaction onto biotinylated 6 kDa heparin functionalised surface in calcium buffer. The concentration range of Langerin ECD is from 1 µM to 0,49 nM with 2 times serial dilution. G) SPR titration experiment of Langerin ECD interaction onto biotinylated 6 kDa heparin functionalised surface in EDTA buffer. The concentration range of Langerin ECD is from 8 µM to 0,488 nM with 2 times serial dilution. H) Overlay of sensorgram showing Langerin ECD on 6kDa heparin surface in calcium buffer () and in EDTA buffer (←).
Figure 3.
Interaction properties of Langerin to different glycosaminoglycans.
(A-C) Structure of glycosaminoglycan disaccharides. Disaccharide units for heparin/HS (A), CS/DS (B) or KS (C). R = COCH3 or SO3H; X = H or SO3. The star indicates presence of glucuronic or iduronic acid C-5 epimers. D) SPR inhibition experiment of Langerin ECD/heparin interaction by 15 kDa heparin. Langerin ECD concentration was 100 nM and the heparin concentration range was from 7.8 nM to 2 µM with 2-fold serial dilution factor. E) IC50 values obtained with heparin, heparin treated with HSulf2 and HS. F) Histogram representation of CS/DS IC50. SPR inhibition experiment of Langerin ECD/heparin interaction by 15 kDa heparin. Langerin ECD concentration was fixed at 100 nM and the heparin concentration range was from 7.8 nM to 2 µM with 2-fold serial dilution factor. C) Histogram representation of GAGs IC50.
Table 1.
Disaccharide analysis of GAGs.
Figure 4.
Three-dimensional model of langerin ECD and potential heparin docking sites.
The human Langerin ECD is represented by its Connolly surface, color-coded according to the molecular electrostatic potential (from blue for negative to red for positive areas). The most probable regions for interactions with heparin are indicated with colored boxes populated with methylsulfate docking solutions, represented in space fill (5A: top view; 5B: side view).
Figure 5.
Heparin decasaccharide in complex with Langerin.
Predicted binding mode of the heparin decasaccharide (displayed in space fill -A- and capped stick -B-) in complex with Langerin. Langerin is represented with its Connolly surface color-coded according to the electrostatic potential (from blue for negative to red for positive electrostatic areas). Amino acids mediating the main interactions with the decasaccharide are labeled in white.
Table 2.
Details about the geometries of the heparin decasaccharide in complex with Langerin.
Figure 6.
2-N-sulfated, 6-O-sulfated α-D-glucopyranoside (A) and 2-O-sulfated β-L-idopyranoside monomers in its 1C4 (B) and 2SO (C) ring shapes were considered for building heparin fragments for docking calculations. Glycosidic linkages are also indicated, defined as φ = O5i – C1i – O1i – C4j and ψ = C1i – O1i – C4j – C5j.
Table 3.
Model of Heparin-Langerin complex: amino acids mediating the main interactions with the decasaccharide.