Functional Activity of MD-2 Polymorphic Variant Is Significantly Different in Soluble and TLR4-Bound Forms: Decreased Endotoxin Binding by G56R MD-2 and Its Rescue by TLR4 Ectodomain

Abstract

MD-2 is an essential component of endotoxin (LPS) sensing, binding LPS independently and when bound to the ectodomain of the membrane receptor TLR4. Natural variation of proteins involved in the LPS-recognition cascade such as the LPS-binding protein, CD14, and TLR4, as well as proteins involved in intracellular signaling downstream of LPS binding, affect the cellular response to endotoxin and host defense against bacterial infections. We now describe the functional properties of two nonsynonymous coding polymorphisms of MD-2, G56R and P157S, documented in HapMap. As predicted from the MD-2 structure, the P157S mutation had little or no effect on MD-2 function. In contrast, the G56R mutation, located close to the LPS-binding pocket, significantly decreased cellular responsiveness to LPS. Soluble G56R MD-2 showed markedly reduced LPS binding that was to a large degree rescued by TLR4 coexpression or presence of TLR4 ectodomain. Thus, cells that express TLR4 without MD-2 and whose response to LPS depends on ectopically produced MD-2 were most affected by expression of the G56R variant of MD-2. Coexpression of wild-type and G56R MD-2 yielded an intermediate phenotype with responses to LPS diminished to a greater extent than that resulting from expression of the D299G TLR4 polymorphic variant.

Lipopolysaccharide activates NF-κB by TLR4-Bcl10-dependent and independent pathways in colonic epithelial cells

“…The TLRs are able to recognize the pathogen-associated molecular pattern of LPS produced by bacteria or other pathogens and trigger the innate immune response (1). Other endogenous factors, including MD-2 and the lipoprotein receptor, may be required for interaction with TLR4 (18,25). We have reported that the sulfated polysaccharide carrageenan, which has the unusual ␣-1,3-galactosidic link that is the epitope for the anti-Gal antibody (2,12), is recognized by TLR4 and induces an innate immune response mediated by Bcl10, as well as an ROS-mediated inflammatory response (3,4).…”

A TIR Domain Variant of MyD88 Adapter-like (Mal)/TIRAP Results in Loss of MyD88 Binding and Reduced TLR2/TLR4 Signaling

“…Notable among them is a study by Khor et al (9), who reported that individuals heterozygous for the polymorphism S180L in Mal are protected against pneumococcal disease, bacteremia, malaria, and tuberculosis (9). Within the coding region of a gene, SNPs can alter the characteristics and affect the ability of a protein to function (10). Interestingly, Mal is the most polymorphic of all adapter proteins, harboring at least eight nonsynonymous variants in its coding region.…”

Therapeutic Targeting of Toll-Like Receptors for Infectious and Inflammatory Diseases and Cancer

“…Studies in the MD-2 knockout mice have been very much more limited compared with the TLR4 knockout mice. Three human polymorphisms have been described: T35A (Hamann et al, 2004), C1625G in the MD-2 promoter (Gu et al, 2007), and G56R (Vasl et al, 2008). The promoter polymorphism may be linked to increased susceptibility to complications such as organ dysfunction and sepsis after major trauma (Gu et al, 2007), whereas the other polymorphisms have yet to show any disease association.…”

“…not respond to LPS (Nagai et al, 2002). A number of MD-2 polymorphisms have been identified that alter LPS binding and/or activation (Hamann et al, 2004;Gu et al, 2007;Vasl et al, 2008). LPS interaction with MD-2/TLR4 involves at least two other proteins.…”