Species-specific activation of TLR4 by hypoacylated endotoxins governed by residues 82 and 122 of MD-2.

Alja Oblak,Roman Jerala,Jörg Hermann Fritz

doi:10.1371/journal.pone.0107520

Abstract

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation.

Highlights

Immediate and effective innate immune response is crucial for the prevention of microbial colonization or dissemination in mammalian host
We show that human TLR4 can be potently activated with hexaacylated lipid A in combination with either human or equine MD-2 (Fig. 1A)
Equine TLR4 appears to be most adept at recognizing lipid IVa, since it can be activated in combination with either equine or mouse MD-2 (Fig. 1C)

Summary

Introduction

Immediate and effective innate immune response is crucial for the prevention of microbial colonization or dissemination in mammalian host. The most important and so far the most studied group of innate immune receptors are the Toll-like receptors (TLRs) They recognize distinctive microbial molecules, such as double stranded RNA, flagellin or endotoxins [1]. Endotoxins (i.e. lipopolysaccharides, LPS) are the main constituents of the cell envelope of most Gram-negative bacteria (with few exceptions [2,3,4]) They are composed of a highly variable polysaccharide and a lipid A moiety, which is the active principle of endotoxin that is recognized by the TLR4/ MD-2 receptor complex. Lipid A is typically composed of a glucosamine disaccharide modified with two phosphates and comprising four primary glucosamine-linked hydroxyacyl chains and two secondary acyl chains [5] This type of hexaacylated endotoxin can be found in the majority of Gram-negative bacteria, ranging from soil and plant inhabitants to mammalian mucosal pathogens. Pseudomonas aeruginosa [7] can produce weakly activating pentaacylated endotoxin and Yersinia pestis and Helicobacter pylori can produce a tetraacylated form of lipid A [8,9], which cannot activate the human TLR4/MD2, and can avoid activation of the immune response

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Conclusion