Heparin changes the conformation of high-mobility group protein 1 and decreases its affinity toward receptor for advanced glycation endproducts in vitro

Abstract

High-mobility group protein 1 (HMGB1) has been identified as a late-acting mediator of inflammation. The receptor for advanced glycation end products (RAGE) is the main receptor and mediates the cytokine activity of HMGB1. Since HMGB1 also exhibits heparin-binding activity, we investigated whether heparin interferes with HMGB1/RAGE interaction and prevents the cytokine activity. We used fluorescence spectrometry, circular dichroism spectrometry and SPR biosensor technique to evaluate the effect. After treatment of HMGB1 with different concentrations of heparin (0, 50, 100 and 1000 U/L), the fluorescence peak values of HMGB1 increased and the emission wavelength showed red shifts; further, the secondary structure of HMGB1 showed a marked change in that the content of β-pleated sheet reduced while that of α-helix increased. The equilibrium dissociation constants (K(D)) were determined by SPR technique; K(D)=4.5 × 10(-9)mol/L for heparin and HMGB1 and K(D)=9.77 × 10(-8)mol/L for HMGB1 and RAGE, respectively. Heparin and RAGE had no interaction. The amount of HMGB1 and RAGE bound forms reduced after treatment with heparin. ELISA revealed that addition of heparin inhibited the TNF-α and IL-6 released by macrophages RAW264.7 and HUVEC; 10 U/L and 50 U/L of heparin showed the most marked inhibitory effect in RAW264.7 cells and in HUVEC, respectively. In conclusion, heparin can combine with HMGB1 and affect the affinity of HMGB1/RAGE by changing the conformation of HMGB1. And this effect was independent of heparin concentration, so that a low dose of heparin was sufficient to achieve the best anti-inflammatory effect in our test.