Tetraacylated Lipid A and Paclitaxel-Selective Activation of TLR4/MD-2 Conferred through Hydrophobic Interactions

Abstract

LPS exerts potent immunostimulatory effects through activation of the TLR4/MD-2 receptor complex. The hexaacylated lipid A is an agonist of mouse (mTLR4) and human TLR4/MD-2, whereas the tetraacylated lipid IVa and paclitaxel activate only mTLR4/MD-2 and antagonize activation of the human receptor complex. Hydrophobic mutants of TLR4 or MD-2 were used to investigate activation of human embryonic kidney 293 cells by different TLR4 agonists. We show that each of the hydrophobic residues F438 and F461, which are located on the convex face of leucine-rich repeats 16 and 17 of the mTLR4 ectodomain, are essential for activation of with lipid IVa and paclitaxel, which, although not a structural analog of LPS, activates cells expressing mTLR4/MD-2. Both TLR4 mutants were inactive when stimulated with lipid IVa or paclitaxel, but retained significant activation when stimulated with LPS or hexaacylated lipid A. We show that the phenylalanine residue at position 126 of mouse MD-2 is indispensable only for activation with paclitaxel. Its replacement with leucine or valine completely abolished activation with paclitaxel while preserving the responsiveness to lipid IVa and lipid A. This suggests specific interaction of paclitaxel with F126 because its replacement with leucine even augmented activation by lipid A. These results provide an insight into the molecular mechanism of TLR4 activation by two structurally very different agonists.

Bordetella pertussis Lipid A Recognition by Toll-like Receptor 4 and MD-2 Is Dependent on Distinct Charged and Uncharged Interfaces

“…Similar to Resman et al (7,8), we observed species-specific differences in the ability of human and mouse TLR4 mutants to respond to E. coli LPS and lipid IVA. F440A or F463A mutation of human TLR4 caused a more severe phenotype: the complete or near-complete loss in the ability to activate NFB in response to both E. coli LPS and lipid IVA, whereas F438A or F461A mutants of mouse TLR4 could still partially respond to E. coli LPS (Fig.…”

“…Additionally, because one of the acyl chains on hexa-acylated E. coli lipid A sticks out of the pocket and lies on the surface of MD-2, this also promotes dimerization via interaction with uncharged amino acids in TLR4* (4,5). Uncharged amino acids in TLR4* (Phe-440 and Phe-463 in human TLR4 and Phe-438, Phe-461 in mouse TLR4) have also been proposed to engage MD-2 and support receptor activation (5,7,8).…”

Hydroxysafflor Yellow A Alleviates Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome in Mice

“…17) To investigate the antagonizing effect of HSYA on the specific receptor binding LPS, we used a human lung epithelial cell line (A549) and human umbilical vein cell line (Eahy926). Alveolar epithelial cells and capillary endothelial cells are the two most common cells in lung tissue.…”

The molecular mechanism of species-specific recognition of lipopolysaccharides by the MD-2/TLR4 receptor complex

“…2014,2008). Up to now most mutants of MD-2 closely followed this lipid IVa/taxol specificity, however we recently characterized a mutation of murine MD-2 (mMD-2) that retains strong activation by lipid IVa yet is no longer activated by taxol (Resman et al, 2014).…”