Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain

Hannah Aucott,Peter Lundback,Agnieszka Sowinska,Helena Erlandsson Harris

doi:10.1186/s10020-018-0021-x

Hannah Aucott, Peter Lundback + Show 2 more

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https://doi.org/10.1186/s10020-018-0021-x

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Abstract

BackgroundHigh mobility group box 1 (HMGB1) protein is a central endogenous inflammatory mediator contributing to the pathogenesis of several inflammatory disorders. HMGB1 interacts with toll-like receptors (TLRs) but contradictory evidence regarding its identity as a TLR2 ligand persists. The aim of this study was to investigate if highly purified HMGB1 interacts with TLR2 and if so, to determine the functional outcome.MethodsFull length or C-terminal truncated (Δ30) HMGB1 was purified from E.coli. Binding to TLR2-Fc was investigated by direct-ELISA. For the functional studies, proteins alone or in complex with peptidoglycan (PGN) were added to human embryonic kidney (HEK) cells transfected with functional TLR2, TLR 1/2 or TLR 2/6 dimers, macrophages, whole blood or peripheral blood mononuclear cells (PBMCs). Cytokine levels were determined by ELISA.ResultsIn vitro binding experiments revealed that Δ30 HMGB1, lacking the acidic tail domain, but not full length HMGB1 binds dose dependently to TLR2. Control experiments confirmed that the interaction was specific to TLR2 and could be inhibited by enzymatic digestion. Δ30 HMGB1 alone was unable to induce cytokine production via TLR2. However, full length HMGB1 and Δ30 HMGB1 formed complexes with PGN, a known TLR2 ligand, and synergistically potentiated the inflammatory response in PBMCs.ConclusionsWe have demonstrated that TLR2 is a receptor for HMGB1 and this binding is negatively regulated by the C-terminal tail. HMGB1 did not induce functional activation of TLR2 while both full length HMGB1 and Δ30 HMGB1 potentiated the inflammatory activities of the TLR2 ligand PGN. We hypothesize that Δ30 HMGB1 generated in vivo by enzymatic cleavage could act as an enhancer of TLR2-mediated inflammatory activities.

Highlights

High mobility group box 1 (HMGB1) protein is a central endogenous inflammatory mediator contributing to the pathogenesis of several inflammatory disorders
Our results reveal that binding of HMGB1 to TLR2 is negatively regulated by the acidic C-terminal tail domain, with truncated HMGB1 lacking the tail domain displaying dosedependent binding to TLR2-Fc in contrast to full length HMGB1
Since a number of recent studies have revealed that HMGB1 binding to TLR4 is strictly regulated by cysteine redox modifications, we tested the binding of both fr- and ds-HMGB1 isoforms

Summary

Introduction

High mobility group box 1 (HMGB1) protein is a central endogenous inflammatory mediator contributing to the pathogenesis of several inflammatory disorders. High mobility group box 1 (HMGB1), originally described as a nuclear DNA-binding protein, was rediscovered as an endogenous inflammatory mediator released from dying and activated cells in response to infectious and sterile stimuli in the late 90s (Wang et al 1999). HMGB1 has since been identified as a pathogenic mediator during sepsis, arthritis, cancer, drug-induced liver injury and stroke, among other diseases, and Structurally, HMGB1 expresses 215 amino acids organized into two DNA binding domains (boxes A and B) and a 30 amino acid long unstructured C-terminal acidic tail comprised of repeating glutamic and aspartic acid residues. Biophyscial studies have shown that the tail interacts with residues in both boxes and the linker regions (Stott et al 2010; Watson et al 2007; Knapp et al 2004). Binding of the tail to the boxes has been reported to modulate the interaction with other molecules including DNA and to regulate posttranslational modifications (Lee and Thomas 2000; Muller et al 2001; Sheflin et al 1993; Stros et al 1994; Pasheva et al 2004)

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