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Helicobacter pylori is recognized as an etiologic agent of gastroduodenal diseases. Among toxic substances produced by H. pylori, LPS exhibits extremely low endotoxic activity as compared to the typical LPSs, such as that produced by Escherichia coli. We found that the LPS-low-responder stomach cancer cell line MKN28, which expresses Toll-like receptor 4 (TLR4) at extremely low levels, showed similar levels of interleukin-8 (IL-8) induction by H. pylori or E. coli LPS preparations. Weak IL-8 induction by H. pylori LPS preparations was suppressed by expression of a dominant negative mutant of TLR2 but not of TLR4. Data from luciferase reporter analysis indicated that cotransfection of TLR2-TLR1 or TLR2-TLR6 was required for the activation induced by H. pylori LPS preparations. In conclusion, the H. pylori LPS preparations significantly induce an inflammatory reaction via the receptor complex containing TLR2-TLR1 or TLR2-TLR6 but not that containing TLR4. The TLR2-TLR1 complex was preferentially recognized by the H. pylori LPS preparations over the TLR2-TLR6 complex. Whereas the magnitude of response to H. pylori LPS preparation was markedly less than that to E. coli LPS preparation in LPS-high-responder cells strongly expressing TLR4, it was comparable to that of E. coli LPS in low-responder cells expressing negligible amount of TLR4.

MD-2 has been reported to be required for the translocation of the Toll-like receptor 4 (TLR4) to the cell surface. However, the mechanism by which MD-2 promotes TLR4 translocation is unknown. We identified the presence of two forms of TLR4 with different molecular masses (approximately 110 and 130 kDa) when TLR4 was expressed together with MD-2. Expressing TLR4 alone produced only the 110-kDa form. Using a membrane-impermeable biotinylation reagent, we found that only the 130-kDa form of TLR4 was expressed on the cell surface. When a cellular extract prepared from cells expressing TLR4 and MD-2 was treated with N-glycosidase, the two forms of TLR4 converged into a single band whose size was smaller than the 110-kDa form of TLR4. Mutation of TLR4 at Asn 526 or Asn 575 resulted in the disappearance of the 130-kDa form and prevented TLR4 from being expressed on the cell surface without affecting the ability of TLR4 to associate with MD-2. These results indicate that TLR4 is able to undergo multiple glycosylations without MD-2 but that the specific glycosylation essential for cell surface expression requires the presence of MD-2.

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MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn26 and Asn114) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn26 and Asn114. Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-κB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn26 and Asn114, and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.

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A cell surface receptor complex consisting of CD14, Toll-like receptor (TLR4), and MD-2 recognizes lipid A, the active moiety of lipopolysaccharide (LPS). Escherichia coli-type lipid A, a typical lipid A molecule, potently activates both human and mouse macrophage cells, whereas the lipid A precursor, lipid IVa, activates mouse macrophages but is inactive and acts as an LPS antagonist in human macrophages. This animal species-specific activity of lipid IVa involves the species differences in MD-2 structure. We explored the structural region of MD-2 that determines the agonistic and antagonistic activities of lipid IVa to induce nuclear factor-B activation. By expressing human/mouse chimeric MD-2 together with mouse CD14 and TLR4 in human embryonic kidney 293 cells, we found that amino acid regions 57-79 and 108 -135 of MD-2 determine the species-specific activity of lipid IVa. We also showed that the replacement of Thr 57 , Val 61 , and Glu 122 of mouse MD-2 with corresponding human MD-2 sequence or alanines impaired the agonistic activity of lipid IVa, and antagonistic activity became evident. These mutations did not affect the activation of nuclear factor-B, TLR4 oligomerization, and inducible phosphorylation of IB␣ in response to E. coli-type lipid A. These results indicate that amino acid residues 57, 61, and 122 of mouse MD-2 are critical to determine the agonist-antagonist activity of lipid IVa and suggest that these amino acid residues may be involved in the discrimination of lipid A structure. Bacterial lipopolysaccharide (LPS)2 is a constituent of the outer membrane of the cell wall of Gram-negative bacteria and plays a major role in septic shock (1, 2). Engagement of LPS on the host cell results in rapid activation of a number of transcription factors, including NF-B, which leads to production of inflammatory cytokines (3). Significant progress has been made in the identification of cell surface molecules that recognize LPS and transmit its signal to intracellular components. CD14, Toll-like receptor 4 (TLR4), and MD-2 participate in this molecular event and all of these molecules are necessary for cells to respond to picomolar concentrations of LPS (4, 5). A recent report (6) has suggested that sequential interactions of LPS with each of these molecules are required for optimal molecular recognition. LPS is first opsonized by the serum LPS-binding protein and then transferred to a CD14 molecule. This LPS-CD14 complex is further recognized by MD-2 to generate an LPS-MD-2 complex that produces TLR4-dependent cell stimulation. It has also been reported that MD-2 is necessary for TLR4 to undergo proper glycosylation and trafficking to the cell surface (7-9).Without MD-2, TLR4 is not able to reach the plasma membrane and resides predominantly in the Golgi apparatus. Thus, MD-2 is considered to play an important role for transferring LPS from CD14 to TLR4 and for correct cellular distribution of TLR4.MD-2 also plays an important role for discriminating lipid A structure. The lipid A portion has been identif...

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