FIGURE 3.

Interaction of the RAGE peptides with GAGs and inhibition of the binding of a soluble form of recombinant RAGE to immobilized CS-E polysaccharides by RAGE-derived peptides and CS-E oligosaccharides. A and B, the binding activity of two peptides from RAGE with various GAG species was analyzed by ELISA, in which authentic commercial GAGs (CS-A, CS-E, and heparin (Hep)) were included. Biotinylated GAGs (0.5 μg each) were individually immobilized to wells of a streptavidin-coated plastic plate and processed for incubation with the two RAGE-derived peptides, CKGAPKKPPQQLEWKLNTGRTEA (Cys-38–Ala-60) and GTFRCRATNRRGKEVKSNYRVRVY (Gly-94–Tyr-117) (5 μg each). The amounts of GAGs immobilized onto the plate were comparable (∼0.1 μg) as estimated by a quantitative analysis using a dye 1,9-dimethylmethylene blue (49). Bound peptides were visualized by subsequent incubation with antisera against the respective peptide followed by alkaline phosphatase-linked goat anti-rabbit IgG/IgM (diluted 5000-fold). Enzymatic activity was measured using p-nitrophenylphosphate as a substrate at 415 nm. Negative controls received no primary antibody. Bars, mean ± S.E. (n = 3). Note that each antiserum recognized the respective peptide bound to GAGs (supplemental Fig. S5). C and D, shown is inhibition of the binding of soluble RAGE to immobilized CS-E and heparin by two RAGE-derived peptides, Cys-38–Ala-60 and Gly-94–Tyr-117. The recombinant RAGE-Fc chimera (20 ng) was added with or without either peptide to the CS-E- and heparin (∼0.1 μg)-coated microtiter plates. The amounts of both peptides were 50, 100, 200, 500, and 1000 ng (bars from the left). Bound RAGE protein was quantified using Protein G-alkaline phosphatase as described under “Experimental Procedures.” E, inhibition of the binding of soluble RAGE to immobilized CS-E polysaccharides by CS-E oligosaccharides is shown. The recombinant RAGE-Fc chimera (50 ng) was added with or without the indicated amounts of CS-E hexa-, deca-, tetradeca-, or polysaccharides to the CS-E-coated microtiter plates. Bound RAGE protein was quantified using protein G alkaline phosphatase as described under “Experimental Procedures.” The mean absorbance (at 415 nm) for binding of RAGE to CS-E in the absence of inhibitor was 1.46, and this value was taken as 100% (positive control). All other values are expressed as percentages of this control quantity. Values and the S.E. were obtained from the average of two separate experiments.