Structural regions of MD-2 that determine the agonist-antagonist activity of lipid IVa

Abstract

A cell surface receptor complex consisting of CD14, Toll-like receptor (TLR4), and MD-2 recognizes lipid A, the active moiety of lipopolysaccharide (LPS). Escherichia coli-type lipid A, a typical lipid Amolecule, potently activates both human and mouse macrophage cells, whereas the lipid A precursor, lipid IVa, activates mouse macrophages but is inactive and acts as an LPS antagonist in human macrophages. This animal species-specific activity of lipid IVa involves the species differences in MD-2 structure. We explored the structural region of MD-2 that determines the agonistic and antagonistic activities of lipid IVa to induce nuclear factor-κB activation. By expressing human/mouse chimeric MD-2 together with mouse CD14 and TLR4 in human embryonic kidney 293 cells, we found that amino acid regions 57-79 and 108-135 of MD-2 determine the species-specific activity of lipid IVa. We also showed that the replacement of Thr57, Val61, and Glu 122 of mouse MD-2 with corresponding human MD-2 sequence or alanines impaired the agonistic activity of lipid IVa, and antagonistic activity became evident. These mutations did not affect the activation of nuclear factor-κB, TLR4 oligomerization, and inducible phosphorylation of IκBα in response to E. coli-type lipid A. These results indicate that amino acid residues 57, 61, and 122 of mouse MD-2 are critical to determine the agonist-antagonist activity of lipid IVa and suggest that these amino acid residues may be involved in the discrimination of lipid A structure. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.