Lipopolysaccharide Is in Close Proximity to Each of the Proteins in Its Membrane Receptor Complex

Abstract

The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation of Drosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated crosslinking group ( 125 I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex. Bacterial endotoxin (lipopolysaccharide, LPS)1 okonp61 is a complex glycolipid composed of a hydrophilic polysaccharide moiety and a hydrophobic domain known as lipid A (1). LPS is an outer membrane constituent of all Gram-negative bacteria where it has indispensable barrier functions. LPS is also a potent activator of innate immune responses that result in the production of pro-and anti-inflammatory mediators from myeloid lineage and other cell types (2). LPS-induced cell activation depends on the presence of three proteins comprising a multiprotein cell surface receptor complex here termed the LPS receptor complex. One essential protein of the LPS receptor complex is CD14 (3), a 55-kDa glycoprotein present in soluble form (sCD14) in blood or as a membrane-bound form (mCD14) in myeloid lineage cells. This latter form is attached to the outer leaflet of the cell membrane via a glycosylphosphatidylinositol anchor. Multiple lines of biochemical and genetic evidence support the contention that CD14 principally acts to bind LPS and does not participate in signaling directly. Thus, others and we postulated that there must be at least one transmembrane protein that acts in concert with CD14 (2). This putative transmembrane protein is now identified as a member of the mammalian Toll-like receptor (TLR) family and is TLR4 (4, 5). Genetic and biochemical studies suggest that TLR4 plays an important role in LPS signaling under physiological conditions. Positional cloning and sequencing of the lps d locus localized the defect to the tlr4 gene (6). The importance of TLR4 in LPS signaling is further supported by the fact that TLR4-deficient mice are LPS hyporesponsive but resp...

N-Linked Glycosylations at Asn26 and Asn114 of Human MD-2 Are Required for Toll-Like Receptor 4-Mediated Activation of NF-κB by Lipopolysaccharide

“…It is unlikely that the biotinylation reagent we used labeled intercellularly located MD-2 because of the following reasons: 1) This reagent is well characterized (19,20) not to pass through cell membrane due to its strong negative charge (SO 3 Ϫ ); 2) we performed the biotinylation at 4°C to prevent endocytosis, and we stopped the biotinylation reaction by adding glycine before lysing the cells; and 3) we were unable to detect the biotinylation of a cytosolic protein, I B-␣, with this biotinylation reagent. Recently, da Silva Correia et al (34) also observed that MD-2 was clearly detectable on the cell surface without TLR4 expression using FACS analysis when MD-2 was transiently expressed in 293 cells. Therefore, the difference between our result and the result of Shimazu et al (13) seems to be attributable to the difference in the level of MD-2 expression between stable and transient transfection.…”

Identification of Mouse MD-2 Residues Important for Forming the Cell Surface TLR4-MD-2 Complex Recognized by Anti-TLR4-MD-2 Antibodies, and for Conferring LPS and Taxol Responsiveness on Mouse TLR4 by Alanine-Scanning Mutagenesis

“…These findings suggest that both LPS and Taxol physically associate with the TLR4-MD-2 complex. Furthermore, using LPS conjugated to a photo-activated cross-linker, Ulevitch and coworkers (20) observed LPS in close proximity to both TLR4 and MD-2 in the presence of CD14, which is an initial cell surface receptor for LPS. These findings, taken together, suggest that TLR4 and MD-2 constitute the central part of the LPS receptor complex.…”

Toll-Like Receptors in Health and Disease: Complex Questions Remain

“…There is also still controversy as to whether the principal TLR4 activator, LPS, binds to TLR4. Most certainly, there is a close interaction of LPS with CD14, MD-2, CD11b/ CD18, and TLR4, suggesting presentation of LPS to TLR4 by these proteins (12), but others have not found evidence of a direct interaction of LPS with TLR4 (10). Of these accessory molecules, MD-2 plays a critical role in TLR4 expression and signaling, and may mediate interactions between LPS and TLR4 (its essential role is reviewed in Ref.…”