MD-2 and TLR4 N-Linked Glycosylations Are Important for a Functional Lipopolysaccharide Receptor

Abstract

The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn 26 and Asn 114 , whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn 526 or Asn 575 failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.A family of related genes encoding the 10 known Toll-like receptors (TLR) 1 is involved in innate immune responses in mammals (1-4). Engagement of TLRs by specific products of the pathogen, i.e. bacterial endotoxin or lipopolysaccharide (LPS) (5-7), bacterial DNA (8), outer membrane lipoproteins and lipopeptides (9 -11), and bacterial flagellin protein (12), results in activation of innate immune responses. Among these substances LPS is considered to be the prototypic activator (13,14). The functional membrane receptor for LPS is comprised of at least three proteins, CD14, TLR4, and MD-2. Binding of LPS to TLR4 and MD-2 is enhanced by CD14 and the plasma protein LPS-binding protein (LBP) (15).Although there have been a number of detailed structurefunction analyses for both , only limited information is available for MD-2 or TLR4. The mature human MD-2 and TLR4 sequences deduced from cDNA contain 160 and 839 amino acids residues, respectively. Both proteins have potential N-linked glycosylation sites. Sequence analysis suggests MD-2 has two potential N-linked glycosylation sites, whereas the ectodomain of TLR4 contains nine. Herein we describe the properties of MD-2 and TLR4 mutants lacking the potential N-linked glycosylation sites. We compared the mutated proteins with the parent...

Genetic regulation of host responses to Salmonella infection in mice

“…TLR4 appears to interact directly with LPS with the cooperation of LBP (LPS-binding protein) and coreceptors CD14 and MD-2. [90][91][92] Ultimately, LPS sensing by TLR4 leads to the activation of the transcription factor NF-kB and the MAP kinases, JNK and p38, through the activation of two known signaling pathways: a common TLR signaling MyD88-TOLLIP-IRAK-TRAF6 pathway (reviewed in Ref. 71 ) and an MyD88-independent pathway involving the adapter protein TIRAP.…”

Structure-Activity Relationship of Synthetic Toll-like Receptor 4 Agonists

“…HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 10 g/ml streptomycin (all from Invitrogen). HeLa cells were plated at 1 ϫ 10 5 cells/well in a 12-well plate and transfected with 50 ng of IL-8 promoter-driven luciferase reporter and 10 ng of the indicated plasmids (23). The human CD14 cDNA used in this study was described previously (24).…”

“…Eighteen hours after transfection, cells were stimulated for 6 h, lysed, and then analyzed for luciferase and ␤-galactosidase activity. A ␤-galactosidase construct was used for the normalization of enzyme activity (23). 293 cells were stably transfected with TLR2, then transiently transfected with TLR1 and TLR6, and exposed to heat-killed Staphylococcus aureus at a concentration of 1 g/ml.…”

“…CD14 is a glycosylphosphatidylinositol-linked protein to which LPS has been shown to be transferred via lipopolysaccharide-binding protein circulating in the serum (31). We have previously demonstrated that HeLa cells directed to express TLR4, MD-2, and CD14 following transient transfection respond to LPS (23). We have used a luciferase reporter plasmid containing a portion of the IL-8 promoter, known to be readily induced by LPS via the activation of NF B, as readout in this assay.…”

C1 inhibitor: Biologic activities that are independent of protease inhibition

“…The LPS-CD14 complex then initiates intracellular signalling by binding to Toll-like receptor 4 (TLR4) (Aderem and Ulevitch, 2000). At least one other protein, MD-2, is associated with CD14 and TLR4 on the cell surface; together, these make up the LPS receptor (da Silva Correia and Ulevitch, 2002;Juan et al, 1995;Stelter et al, 1997;Stelter et al, 1999;Viriyakosol and Kirkland, 1995;Viriyakosol et al, 2000). The binding of C1 inhibitor to LPS (or lipid A) was inhibited by fetal bovine serum, which contains LBP, and by an LBP peptide that includes the binding site for LBP (Liu et al, 2003).…”