title: MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex.
abstract: We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.
authors.affiliation: Department of Periodontics, University of Washington School of Dentistry, Seattle 98195, USA. scoats@u.washington.edu
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Coats
authors.forename: Thu-Thao T
authors.initials: TT
authors.lastname: Pham
authors.forename: Brian W
authors.initials: BW
authors.lastname: Bainbridge
authors.forename: Robert A
authors.initials: RA
authors.lastname: Reife
authors.forename: Richard P
authors.initials: RP
authors.lastname: Darveau
mesh_terms: D000215:Acylation; D017398:Alternative Splicing; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000502:physiology; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000378:metabolism; D020033:Protein Isoforms; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D020033:Protein Isoforms