Heparan Sulfate Is Essential for High Mobility Group Protein 1 (HMGB1) Signaling by the Receptor for Advanced Glycation End Products (RAGE)

Ding Xu,Jeffrey D. Esko,Danyin Song,Jeffrey Young

doi:10.1074/jbc.m111.299685

Ding Xu, Jeffrey D. Esko + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.299685

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Abstract

In a proteomic search for heparan sulfate-binding proteins on monocytes, we identified HMGB1 (high mobility group protein B1). The extracellular role of HMGB1 as a cytokine has been studied intensively and shown to be important as a danger-associated molecular pattern protein. Here, we report that the activity of HMGB1 depends on heparan sulfate. Binding and competition studies demonstrate that HMGB1 interacts with CHO and endothelial cell heparan sulfate. By site-directed mutagenesis, we identified a loop region that connects the A-box and B-box domains of HMGB1 as responsible for heparan sulfate binding. HMGB1-induced Erk1/2 and p38 phosphorylation is abolished when endothelial heparan sulfate is removed or blocked pharmacologically, resulting in decreased HMGB1-induced endothelial sprouting. However, mutated HMGB1 that lacks the heparan sulfate-binding site retained its signaling activity. We show the major receptor for HMGB1, receptor for advanced glycation end products (RAGE), also binds to heparan sulfate and that RAGE and heparan sulfate forms a complex. Our data establishes that the functional receptor for HMGB1 consists of a complex of RAGE and cell surface heparan sulfate.

Highlights

The cytokine function of the danger-associated molecular pattern protein HMGB1 is mediated through receptor for advanced glycation end products (RAGE)
We focused on the heparin-binding properties of HMGB1 as a paradigm for future studies of the other proteins discovered in this proteomic screen
We show that cell surface biotinylation coupled with heparin affinity chromatography tags many traditionally nuclear and cytoplasmic proteins

Summary

Background

The cytokine function of the danger-associated molecular pattern protein HMGB1 is mediated through RAGE. RAGE is expressed at high level in alveolar epithelial cells as well as leukocytes, endothelial cells, neurons, and tumor cells [9] It binds to HMGB1 as well as other ligands, including S100 proteins, AGEs, and amyloids, through its two N-terminal IgGlike domains [9, 10]. The inflammatory response and migration of target cells mediated by HMGB1-RAGE involve activation of MAPKs, such as Erk1/2 and p38, and NF-␬B nuclear translocation [8, 9] Both HMGB1 and RAGE have been reported to bind heparin, a property that can be exploited for protein purification. The abbreviations used are: DAMP, danger-associated molecular pattern protein; RAGE, the receptor for advanced glycation end products; RFU, relative fluorescence units; sRAGE, soluble RAGE; HMVEC-c, human cardiac microvascular endothelial cells

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