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 A molecule, 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-B 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 Thr 57 , Val 61 , 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-B, TLR4 oligomerization, and inducible phosphorylation of IB␣ 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. Bacterial lipopolysaccharide (LPS)2 is a constituent of the outer membrane of the cell wall of Gram-negative bacteria and plays a major role in septic shock (1, 2). Engagement of LPS on the host cell results in rapid activation of a number of transcription factors, including NF-B, which leads to production of inflammatory cytokines (3). Significant progress has been made in the identification of cell surface molecules that recognize LPS and transmit its signal to intracellular components. CD14, Toll-like receptor 4 (TLR4), and MD-2 participate in this molecular event and all of these molecules are necessary for cells to respond to picomolar concentrations of LPS (4, 5). A recent report (6) has suggested that sequential interactions of LPS with each of these molecules are required for optimal molecular recognition. LPS is first opsonized by the serum LPS-binding protein and then transferred to a CD14 molecule. This LPS-CD14 complex is further recognized by MD-2 to generate an LPS-MD-2 complex that produces TLR4-dependent cell stimulation. It has also been reported that MD-2 is necessary for TLR4 to undergo proper glycosylation and trafficking to the cell surface (7-9).Without MD-2, TLR4 is not able to reach the plasma membrane and resides predominantly in the Golgi apparatus. Thus, MD-2 is considered to play an important role for transferring LPS from CD14 to TLR4 and for correct cellular distribution of TLR4.MD-2 also plays an important role for discriminating lipid A structure. The lipid A portion has been identif...

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