Structural Regions of MD-2 That Determine the Agonist-Antagonist Activity of Lipid IVa

Masashi Muroi,Ken-Ichi Tanamoto

doi:10.1074/jbc.m509193200

Masashi Muroi, Ken-Ichi Tanamoto

Open Access

https://doi.org/10.1074/jbc.m509193200

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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-kappaB 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-kappaB, TLR4 oligomerization, and inducible phosphorylation of IkappaBalpha 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.

Highlights

Without MD-2, TLR4 is not able to reach the plasma membrane and resides predominantly in the Golgi apparatus
Responsiveness to Lipid A Molecules in HEK293 Cells Expressing CD14, TLR4, and MD-2—We first attempted to confirm the involvement of MD-2 in the animal species-specific activity of lipid IVa in HEK293 cells, which only respond to lipid A for the activation of NF-␬B when CD14, TLR4, and MD-2 molecules are present
To examine the antagonistic activity of lipid IVa, HEK293 cells expressing mouse CD14, TLR4, and either mouse MD-2 or human MD-2 were stimulated with compound 506 in the presence of increasing concentrations of lipid IVa (Fig. 2)

Summary

Introduction

Without MD-2, TLR4 is not able to reach the plasma membrane and resides predominantly in the Golgi apparatus. To examine the antagonistic activity of lipid IVa, HEK293 cells expressing mouse CD14, TLR4, and either mouse MD-2 or human MD-2 were stimulated with compound 506 in the presence of increasing concentrations of lipid IVa (Fig. 2).

Results

Conclusion