TLR4–MD-2 complex is negatively regulated by an endogenous ligand, globotetraosylceramide

Abstract

Although endogenous ligands for Toll-like receptor (TLR)4-myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4-MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment. In turn, introduction of A4galt cDNA resulted in the neo expression of Gb4, leading to the reduced expression of LPS-inducible genes. Exogenous Gb4 induced similar effects. As a mechanism for the suppressive effects of Gb4 on LPS signals, specific binding of Gb4 to the LPS receptor TLR4-MD-2 was demonstrated by coprecipitation of Gb4 with recombinant MD-2 and by native PAGE. A docking model also supported these data. Taken together with colocalization of TLR4-MD-2 with Gb4 in lipid rafts after LPS stimulation, it was suggested that Gb4 competes with LPS for binding to TLR4-MD-2. Finally, administration of Gb4 significantly protected mice from LPS-elicited mortality. These results suggest that Gb4 is an endogenous ligand for TLR4-MD-2 and is capable of attenuating LPS toxicity, indicating the possibility for its therapeutic application in endotoxin shock.innate immunity | sepsis

Blood Groups in Infection and Host Susceptibility

“…When challenged with LPS, A4GALT1 Ϫ/Ϫ mice developed severe inflammation and had significantly higher mortality rates than did wild-type mice (70% versus 10%; P ϭ 0.068). In wild-type mice, LPS upregulated A4GALT1 transcription, leading to increased Gb4 synthesis and expression on cells (393). Furthermore, there was recruitment of TLR4-MD2 into Gb4-enriched GEMs.…”

“…Recently, Gb4 was identified as an endogenous ligand for TLR4-MD2 (393). Preliminary evidence came from studies in A4GALT1 knockout mice, which lack all globo-GSLs (p phenotype) but have normal TLR4-MD2 expression.…”

“…LPS with fewer acyl chains can evade or block TLR4 signaling due to altered MD2 binding. This is observed in many pathogens (i.e., Y. pestis) and is the primary mechanism by which lipid A is detoxified in vivo (393). Ceramide, with only two acyl chains, is sufficiently hydrophobic to block LPS-MD2 binding but is unable to induce inflammation via the MD88 pathway (393).…”

Glycosphingolipid–Protein Interaction in Signal Transduction

“…Although the biological and evolutionary meaning of this extensive variability is still largely unclear [17], the glycans on GSLs are in a favourable position to interact with the lumenal portions of membrane proteins and receptors [18]. Notably, indeed, over the last decades, a number of studies have demonstrated how GSLs influence the behaviour and signalling capability of membrane proteins [2,19,20,21,22,23,24,25,26,27,28,29]. …”

“…The specificity of these interactions depends on the precise composition of GSL-glycan sugar residues [20,23,29,89,90,91]. By this virtue, the glycan portion of the GSLs has the potential to interface with specific plasma membrane proteins to modify their activity [2,19,20,21,22,23,24,25,26,27,28,29] by carbohydrate–carbohydrate or protein–carbohydrate interactions [2,29,92]. …”