Multimeric MD-2 as an LPS decoy Recognition of Gram-negative bacteria lipopolysaccharide (LPS) is mediated by a cell surface receptor complex constituted by Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4 and that is able to bind directly to LPS. MD-2 exists in both monomeric form and as disulfide-linked multimer. We found that only MD-2 monomer binds to TLR4. Moreover, only MD-2 monomer confers LPS responsiveness to TLR4 expressing cell lines. Thus, monomeric MD-2 is a functional partner of TLR4 in LPS recognition while the function of MD-2 multimer remains unclear. The present grant proposal will test the hypothesis that MD-2 multimer can act as an LPS decoy. In order to demonstrate this property we will have to prove that multimeric MD-2, despite its inability to bind to TLR4 and confer LPS responsiveness, is still able to bind LPS. Specifically we will: 1. Test MD-2 multimer's ability to directly bind LPS using biochemical and biophysical methods. We will also use site-directed mutagenesis to identify the region of MD-2 that is responsible for LPS recognition 2. Analyze whether addition of purified MD-2 multimer to cultures of human dendritic cells, endothelial cells, and TLR4/MD-2 expressing cell lines inhibits their responses to LPS. 3. Study the regulation of MD-2's expression and multimer formation by pro-inflammatory and antiinflammatory stimuli. These studies will augment our understanding of the molecular aspects of LPS recognition and cell activation and will characterize mechanisms to inhibit LPS action that might be exploited for therapeutic intervention. ? ?
| Teghanemt, Athmane; Re, Fabio; Prohinar, Polonca et al. (2008) Novel roles in human MD-2 of phenylalanines 121 and 126 and tyrosine 131 in activation of Toll-like receptor 4 by endotoxin. J Biol Chem 283:1257-66 |
| Prohinar, Polonca; Re, Fabio; Widstrom, Richard et al. (2007) Specific high affinity interactions of monomeric endotoxin.protein complexes with Toll-like receptor 4 ectodomain. J Biol Chem 282:1010-7 |
| Re, Fabio; Strominger, Jack L (2004) IL-10 released by concomitant TLR2 stimulation blocks the induction of a subset of Th1 cytokines that are specifically induced by TLR4 or TLR3 in human dendritic cells. J Immunol 173:7548-55 |
