MD-2: the Toll ‘gatekeeper’ in endotoxin signalling

https://doi.org/10.1016/j.tibs.2004.04.008Get rights and content

Abstract

Lipopolysaccharide (LPS) from the outer cell wall of Gram-negative bacteria is a potent stimulator of the mammalian innate immune system. The Toll-like receptor 4 (TLR4) pathway triggers the inflammatory responses induced by LPS in a process that requires the interaction of LPS-bound myeloid differentiation-2 (MD-2) with TLR4. Here we propose two possible mechanisms for LPS recognition and signalling that take into account both the structural information available for TLR4 and MD-2, and the determinants of endotoxicity, namely, the acylation and phosphorylation patterns of LPS. In our first model, LPS induces the association of two TLR4–MD-2 heterodimers by binding to two different molecules of MD-2 through the acyl chains of lipid A. In our second model, the binding of LPS to a single TLR4–MD-2 complex facilitates the recruitment of a second TLR4–MD-2 heterodimer. These models contrast with the activation of Drosophila Toll, where the receptor is crosslinked by a dimeric protein ligand.

Section snippets

The basis of endotoxicity

In humans, LPS from E. coli (Figure 2) is one of the most potent stimulators of the immune system [19]. Lipid A is the minimal structure that has complete LPS activity and consists of six C12–14 fatty acids linked to a phosphorylated N-acetylglucosamine dimer. The phosphate groups at the 1 and 6′ positions of the diglucosamine contribute multiple negative charges to lipid A, and these charges are crucial for its activity. It is therefore plausible that proteins require a region of alternating

MD-2 is an essential accessory protein for TLR4

MD-2 is a secreted glycoprotein of 160 amino acids with two heterogeneously occupied but functionally important N-linked glycosylation sites [25]. It is required for LPS signalling through TLR4 – a function that was originally suggested by an inactivating point mutation (C95Y) in a conserved region of MD-2 and subsequently confirmed by the generation of MD-2-null mice, which are unresponsive to LPS 13, 26. MD-2 interacts with the ectodomain of TLR4 in a constitutive manner [27], and TLR4–MD-2

Drosophila and vertebrate Toll: indirect versus direct detection of pathogen patterns

The conserved nature of insect and vertebrate Toll receptors led to the expectation that the molecular mechanism of pathogen recognition would be conserved in evolution. In Drosophila, the presence of fungal and bacterial patterns is detected by circulating binding proteins such as peptidoglycan recognition proteins. These binding proteins activate a serine protease cascade [1], which results in the processing of an inactive precursor form of Spätzle, a protein of the cystine knot family, into

MD-2 is a member of the lipid recognition family

An important step towards answering this question has come from the discovery that MD-2 belongs to a novel family of proteins, the MD-2-related lipid-recognition (ML) family [15]. All members of this family that have been so far characterized biochemically have been shown to bind to lipids. They possess a single-domain architecture that belongs to the all-β class and displays an immunoglobulin-like β-sandwich fold (Figure 4). The 3D structures of members of this family include mite dust

Concluding remarks

We have suggested that the functional form of MD-2 is a receptor-associated monomer, that crosslinking of two TLR4 molecules is sufficient to establish signalling, and that oligomerization is a secondary effect. We have also proposed two possible models to explain how LPS binding to MD-2 causes receptor dimerization to occur. Our models suggest that the function held in common by the LRRs of Drosophila and vertebrate Toll receptors is their ability to make protein–protein interactions with the

Acknowledgements

We thank Kenji Mizuguchi and Bose Kochupurakkal for helpful discussions; David Spring for help with Figure 2; and Tom Blundell and Martin Moncrieffe for carefully reading the article. Work in N.J.G.'s laboratory is supported by the Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC).

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