Analysis of TLR4 Polymorphic Variants: New Insights into TLR4/MD-2/CD14 Stoichiometry, Structure, and Signaling

Rallabhandi, Prasad; Bell, Jessica K.; Boukhvalova, Marina S.; Medvedev, Andrei E.; Lorenz, Eva; Arditi, Moshe; Hemming, Val G.; Blanco, Jorge C. G.; Segal, David M.; Vogel, Stefanie N.

doi:10.4049/jimmunol.177.1.322

Cited by 209 publications

(230 citation statements)

References 59 publications

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“…Both horse and human TLR4 show equal surface expression on HEK cells (2.16% horse TLR4 vs 1.29% human TLR4), but there are slight differences in the intracellular levels of TLR4 expression (7.28% for horse TLR4 vs 3.33% for human TLR4). These levels of TLR4 expression are comparable with those seen by others using similar transient transfection techniques (20).…”

Section: Constructssupporting

confidence: 89%

“…MD-2 binds to TLR4 on a lateral surface of the TLR4 solenoid near to the N terminus (18). Interestingly, TLR4 single nucleotide polymorphisms (D299G and T399I) in humans are located far away from the N-terminal MD-2 binding site but reduce LPS responsiveness (19,20). The mechanism for this is unclear, but the mutations could either affect the cooperative binding of LPS or alter the conformational changes that occur during ligand-induced signal transduction.…”

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confidence: 99%

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Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Walsh

Gangloff

Monie

et al. 2008

The Journal of Immunology

115

137

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LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure ( L ipopolysaccharide molecules are complex glycolipids that form the outer layer of the outer membrane of Gramnegative bacteria (1). The lipid A domain of LPS is responsible for cellular activation and consists of a disaccharide to which various substituents, including acyl chains of variable length and number, are attached (2). Escherichia coli lipid A is usually hexa-acylated whereas a tetra-acylated lipid A, lipid IVa, is also produced by E. coli as an intermediate in the lipid A biosynthetic pathway (2). Lipid IVa was originally identified as an inhibitor at the human LPS receptor and was considered a candidate to be developed for clinical use as an endotoxin antagonist. Lipid A signals to host cells through a transmembrane complex consisting of the lipid-binding protein MD-2 and the type 1 receptor TLR4 (1, 3-5). MD-2 is probably the key player in lipid A recognition whereas TLR4, unlike other TLRs, is thought not to participate directly in lipid A binding (5).LPS and lipid A are believed to be recognized by MD-2 following transfer from CD14, which does not participate in the signaling complex (6). Contrary to expectations, ligand binding does not significantly alter the overall structure of MD-2 (7, 8), but the ligands used in the crystallographic studies (lipid IVa and eritoran) are antagonists to human MD-2/TLR4, so it remains unclear what happens to MD-2 and TLR4 upon agonist binding and activation. Active ligands such a lipid A (9) presumably induce structural rearrangements that trigger dimerization of TLR4 and initiate signal transduction (7, 10 -13). Mutagenesis studies identified amino acids 79 -83, 121-124, 125-129 (12), K128, and K132 of human MD-2 as being important in lipid A binding (13), but in the crystal structure of the inactive MD-2-lipid IVa complex, only residues I46, L78, I80, and F121-I124 contact the ligand. The other residues...

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Section: Constructssupporting

confidence: 89%

mentioning

confidence: 99%

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Walsh

Gangloff

Monie

et al. 2008

The Journal of Immunology

115

137

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“…5). Transfection studies show that these TLR4 variants do not exhibit a general defect in the transport of the receptor to the cell surface (39). Similarly, we have shown that the hyporesponsiveness to TLR1 agonists exhibited by the P315L variant is not due to lack of cell surface expression (Fig.…”

Section: Discussionsupporting

confidence: 65%

The Polymorphism P315L of Human Toll-Like Receptor 1 Impairs Innate Immune Sensing of Microbial Cell Wall Components

Omueti

Mazur²,

Thompson³

et al. 2007

The Journal of Immunology

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As a pattern recognition receptor, TLR1 mediates innate immune responses to a variety of microbial cell wall components including bacterial lipoproteins. We have previously shown that the central region of the extracellular domain of human TLR1, comprising leucine-rich repeat (LRR) motifs 9–12, is required for the sensing of bacterial lipopeptides. In this study, we have investigated three nonsynonymous single nucleotide polymorphisms (SNPs) located in this region of TLR1 by generating these variants and examining receptor function. We have found that a variant of TLR1 based upon the SNP P315L, located in the loop of LRR motif 11 (LRR11), is greatly impaired in mediating responses to lipopeptides and a variety of other bacterial agonists for this receptor. Despite normal cell surface expression, the P315L variant also fails to bind to GD2.F4, a commonly used anti-TLR1 mAb. Although a number of amino acid substitutions at position 315 impair receptor function, the leucine substitution has the strongest deleterious effect. GD2.F4 inhibits agonist-induced activation of TLR1, supporting a crucial role for the loop of LRR11 in receptor function. These results also suggest that the P315L SNP may predispose certain individuals to infectious diseases for which the sensing of microbial cell components by TLR1 is critical to innate immune defense.

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“…38 These findings are supported by recent evidence that both TLR4 299Gly and TLR4 399Ile demonstrate decreased responsiveness to LPS and other TLR4 ligands in an in vitro reconstituted TLR4 signaling apparatus when compared to the TLR4 proteins bearing the major allele of each SNP (TLR4 Asp299 and TLR4 Thr399 ). 47 A construct containing both minor alleles had a significantly lower response to TLR4 stimulation than all other constructs, and all of these differences in responses were noted to be dependent on the stochiometry of TLR4, MD-2 and CD14. Thus, we have several clues that could implicate either or the combination of both minor alleles as being causal for susceptibility to IBD, but additional work is required to reconcile the results of these in vitro and ex vivo experiments and to confirm that no other polymorphism in this large TLR4 haplotype contributes to the susceptibility phenotype.…”

Section: Discussionmentioning

confidence: 94%

The role of the Toll receptor pathway in susceptibility to inflammatory bowel diseases

et al. 2007

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The intestinal flora has long been thought to play a role either in initiating or in exacerbating the inflammatory bowel diseases (IBD). Host defenses, such as those mediated by the Toll-like receptors (TLR), are critical to the host/pathogen interaction and have been implicated in IBD pathophysiology. To explore the association of genetic variation in TLR pathways with susceptibility to IBD, we performed a replication study and pooled analyses of the putative IBD risk alleles in NFKB1 and TLR4, and we performed a haplotype-based screen for association to IBD in the TLR genes and a selection of their adaptor and signaling molecules. Our genotyping of 1539 cases of IBD and pooled analysis of 4805 cases of IBD validates the published association of a TLR4 allele with risk of IBD (odds ratio (OR): 1.30, 95% confidence interval (CI): 1.15-1.48; P ¼ 0.00017) and Crohn's disease (OR: 1.33, 95% CI: 1.16-1.54; P ¼ 0.000035) but not ulcerative colitis. We also describe novel suggestive evidence that TIRAP (OR: 1.16, 95% CI: 1.04-1.30; P ¼ 0.007) has a modest effect on risk of IBD. Our analysis, therefore, offers additional evidence that the TLR4 pathway -in this case, TLR4 and its signaling molecule TIRAP -plays a role in susceptibility to IBD.

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Analysis of TLR4 Polymorphic Variants: New Insights into TLR4/MD-2/CD14 Stoichiometry, Structure, and Signaling

Cited by 209 publications

References 59 publications

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

The Polymorphism P315L of Human Toll-Like Receptor 1 Impairs Innate Immune Sensing of Microbial Cell Wall Components

The role of the Toll receptor pathway in susceptibility to inflammatory bowel diseases

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