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23679870[PMID] - PMC - NCBI

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1.
Figure 5

Figure 5. Solution structure of RAGE oligomer. From: Stable RAGE-Heparan Sulfate Complexes are Essential for Signal Transduction.

(a) Raw SAXS scattering curves of the monomeric V-C1 domain, the hexameric V-C1 domain/dodecasaccharide complex and the hexameric sRAGE/dodecasaccharide complex. (b) P(r) pair function analysis of the scattering curves. Profiles are colored as in (a). (c) The GASBOR generated ab initio model of hexameric V-C1 is shown in gray and is superimposed on the crystal structure of V-C1 hexamer shown in green. (d) An ab initio model of hexameric sRAGE superimposed with the crystal structure of V-C1 hexamer (green) and the structure of C2 domain (PDB: 2ENS, salmon) is shown.

Ding Xu, et al. ACS Chem Biol. ;8(7):1611-1620.
2.
Figure 2

Figure 2. Heparin-derived dodecasaccharides induce a stable oligomer of RAGE. From: Stable RAGE-Heparan Sulfate Complexes are Essential for Signal Transduction.

(a) Mixtures of sRAGE and heparin-derived decasaccharides or dodecasaccharides were resolved by SEC. (b) Binding of dodecasaccharides was monitored by isothermal calorimetry by titrating sRAGE with dodecasaccharide. (c) Cartoon representation of RAGE V-C1 domain (PDB-id: 3CJJ). Amino residues demonstrated to interact with heparan sulfate are shown in stick representation (gray). (d) Binding of wild-type and various RAGE mutants to 35S-labeled endothelial heparan sulfate as determined by filter binding assay (n = 3, error bars represent the standard deviation). The extent of binding was quantified by dividing the counts retained by the membrane by the total input counts.

Ding Xu, et al. ACS Chem Biol. ;8(7):1611-1620.
3.
Figure 1

Figure 1. Endothelial heparan sulfate is required for RAGE signaling. From: Stable RAGE-Heparan Sulfate Complexes are Essential for Signal Transduction.

Erk1/2 phosphorylation of primary human endothelial cells was measured by immunoblotting before (lane 1) and after stimulation by RAGE ligands (lanes 2–12). In lane 2, endothelial cells were stimulated for 15 min with a mutated form of HMGB1 that does not bind heparan sulfate. Cells were stimulated with wild-type HMGB1 (lanes 3 and 4), S100B (lanes 5 and 6), AGE-BSA (lanes 7 and 8), S100A8/A9 (lanes 9 and 10) or S100A12 (lanes 11 and 12). Selected samples were treated with heparin lyases I, II and III for 15 min prior to adding ligand as indicated.

Ding Xu, et al. ACS Chem Biol. ;8(7):1611-1620.
4.
Figure 3

Figure 3. A hydrophobic dimeric interface is stabilized by heparin. From: Stable RAGE-Heparan Sulfate Complexes are Essential for Signal Transduction.

(a) Cartoon diagram of the V-C1 dimer (salmon and green) showing the proposed dimeric interface (gray surface) deduced from the crystal structure of hRAGE V-C1 (pdb-id: 3CJJ). The relevant hydrophobic residues are shown in stick representation. (b) Mutants of sRAGE were incubated with oligosaccharide at 1:1 molar ratio and the mixtures were resolved by SEC. (c) The proposed heparan sulfate binding cleft of the dimer. Monomers are shown in green or salmon. Amino acid residues that bind to heparan sulfate are shown in stick representation on the corresponding monomer. A space-filling model of the dodecasaccharide is modeled into the binding cleft (carbon in cyan, oxygen in red, nitrogen in blue and sulfate in yellow). The hydrophobic interface between the dimers is represented as a grey molecular surface. (d) Binding of dodecasaccharide to wild-type, V35A or V78A-L79A RAGE V-C1 domain was assessed by Isothermal calorimetry.

Ding Xu, et al. ACS Chem Biol. ;8(7):1611-1620.
5.
Figure 4

Figure 4. Crystal structure of the RAGE hexamer. From: Stable RAGE-Heparan Sulfate Complexes are Essential for Signal Transduction.

(a) Stereo view of the cartoon representation of the crystal structure of the hexameric mRAGE V-C1 domain. The three dimmers are colored in green and cyan, yellow and orange, and salmon and magenta, respectively. Dodecasaccharides (in sticks) were modeled into the heparan sulfate binding clefts based on the observed sugar density in the crystal structure. The C-terminal strands, involved in the strand-swap, from neighboring molecules in the crystal are displayed to give a canonical view of the C1 domains. (b) The central part of the hexamer consisting of the V-domains is shown. Residues involved in ligand binding (K52, R98 and K110) are shown as ball and stick model and are located at the surface of the hexamer. The aspartic acids at position 73 of each monomer are oriented inward at the core of the hexamer. (c) D73A mutant or wild-type sRAGE were incubated with oligosaccharide at 2:1 molar ratio and resolved by SEC.

Ding Xu, et al. ACS Chem Biol. ;8(7):1611-1620.
6.
Figure 6

Figure 6. Targeting the oligomerization interface with a mAb blocks RAGE signaling. From: Stable RAGE-Heparan Sulfate Complexes are Essential for Signal Transduction.

(a) Binding of anti-RAGE rabbit mAb H4 to immobilized wild-type mRAGE V-C1 domain, I77A-L78A (mouse equivalent of human V78A-L79A), and L84A-L85A (mouse equivalent of human F85AL86A) mutants were determined by ELISA. (b) Postulated binding model of H4 to RAGE. Fab fragment (PDB-id: 3GRW) of IgG is shown as surface representation with light chain in green and heavy chain in yellow. V-C1 is shown as cartoon in salmon. Also shown are the hydrophobic dimerization interface (gray surface), heparan sulfate binding residues (sticks) and ligand binding residues (spheres). (c) Binding of biotinylated mRAGE V-C1 domain to immobilized HMGB1 or S100b was measured in the absence or presence of anti-RAGE rabbit polyclonal Ab (polyAb) or H4 (both at 5 µg/ml). The percentage of inhibition was determined by comparing the absorbance value to standard binding curves of mRAGE V-C1 to HMGB1 or S100b. n = 3, the error bar represents SD. (d) Immunoblot analysis of Erk1/2 and phosphoErk1/2 in human endothelial cells after stimulation with HMGB1 or S100b. Cells were pre-incubated with non-specific rabbit IgG, H4, or anti-RAGE polyAb at 5 µg/ml for 30 min prior to stimulation.

Ding Xu, et al. ACS Chem Biol. ;8(7):1611-1620.

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