Specific High Affinity Interactions of Monomeric Endotoxin·Protein Complexes with Toll-like Receptor 4 Ectodomain

Polonca Prohinar,Fabio Re,Richard Widstrom,Desheng Zhang,Athmane Teghanemt,Jerrold P Weiss,Theresa L Gioannini

doi:10.1074/jbc.m609400200

Abstract

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin.protein complexes of high specific radioactivity. The binding of endotoxin.MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD+/-MD-2. These interactions are specific, of high affinity (KD<300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr approximately 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin.MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin.protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.

Highlights

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined
This ordered action implies differences in endotoxin binding specificity, with lipopolysaccharide-binding protein (LBP) having the highest affinity for endotoxin organized at lipid/water interfaces, CD14 for LBP-modified endotoxin-rich interfaces, MD-2 for monomeric endotoxin1⁄7CD14 and TLR4, apparently, for endotoxin presented as a monomeric complex with MD-2 [8]
Harvested control and TLR4 ectodomain (TLR4ECD)-containing culture media were incubated with 1 nM of purified [3H]LOS aggregates (LOSagg), monomeric [3H]LOS1⁄7sCD14, or [3H]LOS1⁄7MD-2-His6

Summary

Introduction

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. The binding of endotoxin1⁄7MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD ؎ MD-2. The ability to assay specific high affinity interactions of monomeric endotoxin1⁄7protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.

Results

Conclusion