RP105 was cloned in our laboratory as the first LRR molecule expressed on lymphocytes, the extracellular LRR of which had similarity to Drosophila
). We have recently isolated MD-1 as a molecule that is physically associated with RP105 on the cell surface (24
). MD-1 itself is a secretory molecule but tethered to the cell surface by coexpressed RP105. MD-1 is likely to interact with LRR of RP105, which constitutes an entire region of the extracellular portion of RP105. As the extracellular LRR of RP105 is similar to Drosophila
Toll or homologous TLRs, we hypothesized that TLRs might also be associated with a molecule like MD-1. The hypothesis prompted us to seek a molecule with similarity to MD-1. A computer search using the MD-1 amino acid sequence retrieved a human cDNA clone from an expressed sequence tag database (Sequence data available from EMBL/GenBank/DDBJ under accession no. AA099571
). The whole nucleotide sequence was determined and is shown in Fig. A. After an in-frame stop codon, the longest open reading frame codes for 160 amino acids, including a 16-residue, NH2
-terminal hydrophobic stretch that may function as a signal peptide. We refer to a mature molecule as MD-2. Significant similarity (23% identity) to MD-1 was observed over the mature polypeptide (Fig. B). Notably, five out of seven cysteines of MD-2 are shared by MD-1. Expression of the MD-2 transcript was examined with Northern hybridization (Fig. ). The size of the transcript was ~0.7 kb, which is consistent with the size of the cDNA clone. The transcript was demonstrable in all human lines studied (Fig. A). Three B lymphoma cell lines (Nalm-6, Daudi, and RPMI8866) showed relatively high expression. We also studied distribution of the MD-2 transcript in mouse tissues, using the mouse MD-2 probe that was cloned in our laboratory (Shimazu, R., and K. Miyake, unpublished data). Mouse MD-2 was similar to a human homologue in the size of mRNA. The transcript was also ubiquitously observed in all mouse tissues examined, among which spleen and kidney showed pronounced expression (Fig. B).
Figure 1 The nucleotide sequence of human MD-2 and its similarity to human MD-1. (A) The nucleotide sequence is shown with a deduced amino acid sequence. The signal peptide and canonical N-glycosylation sites are underlined. The stop codon is denoted by an (more ...)
Figure 2 Expression of the MD-2 transcript. (A) Total RNA (20 μg/lane) from the indicated human cell lines was electrophoresed and hybridized with probes for human MD-2, TLR4, and GAPDH as indicated. Nalm-6, Ramos, Daudi, and RPMI8866 are of B lymphocyte (more ...)
We then studied interaction of MD-2 and RP105 or TLRs. RP105 seemed unlikely to interact with MD-2, as transfection of MD-2 with RP105, contrary to the case with MD-1, did not result in cell surface expression of MD-2 (data not shown). We next studied interaction with TLRs, among which TLR4 is of particular interest, because it most resembled RP105 in the extracellular LRR domain, and cells expressing the TLR4 transcript were also positive for MD-2 mRNA (Fig. A). The expression vector encoding MD-2 was transfected into the Ba/F3 line. Although the precursor was demonstrable inside the cell (Fig. d), MD-2 was not detectable on the cell surface (Fig. a). In sharp contrast, MD-2 appears on the cell surface in a stable line expressing TLR4 (Fig. c) as well as MD-2 (Fig. b) and seemed to be colocalized with TLR4 on a scanning confocal microscope (data not shown). These results are consistent with membrane anchoring of MD-2 via physical association with TLR4.
Figure 3 Membrane anchoring of MD-2 via TLR4. A stable line expressing MD-2 with the flag epitope was stained with the anti-flag mAb, followed by goat anti–mouse IgG–FITC (a and d). Staining with cell permeabilization is shown in d. Another stable (more ...)
To confirm the association, immunoprecipitation experiments were conducted using the transfectants expressing TLR4 and MD-2 with the newly made mAb to TLR4 (HTA125; see Materials and Methods). It should be noted that the HTA125 mAb was established by immunizing cells expressing TLR4 alone and recognizes TLR4 but not MD-2 (Fig. A). Coprecipitation of MD-2 with the HTA125 mAb therefore demonstrates physical interaction of MD-2 with TLR4. We used two different transfectants, one of which expressed the flag epitope on both TLR4 and MD-2 (Fig. B, lanes 1 and 2). The other line, which had the flag epitope on TLR4 but not on MD-2, was used as a control (Fig. B, lane 3). Precipitates were detected with the anti-flag mAb. TLR4 was specifically precipitated from either line (Fig. B, lanes 2 and 3) with HTA125 as a 120-kD band, which is consistent with a previous report (19
). The signal just below presumably represents an intracellular precursor. Another species of ~25–30 kD was detected from the transfectant expressing MD-2 with the flag epitope (Fig. B, lane 2). The size is similar to that of MD-1 (24
) and is within a range expectable from the MD-2 amino acid sequence, consisting of 160 amino acids with two N
-glycosylation sites (Fig. A). On the other hand, this signal was not observed in the control precipitate, in which MD-2 did not bear the flag epitope (Fig. B, lane 3). MD-2 is thus physically associated with TLR4.
Figure 4 MD-2 is coprecipitated with TLR4. (A) Stable transfectants expressing MD-2 alone or with TLR4 were stained with or without cell permeabilization. The intracellular MD-2 precursor was stained with the anti-protein C mAb. The specificity of the anti-TLR4 (more ...)
We next explored a possible role for MD-2 in TLR4-dependent signaling. As shown in Fig. , expression of TLR4 alone conferred the triggering of NF-κB activation in 293T cells, which confirmed previous reports (11
). Interestingly, expression of MD-2 enhanced TLR4-dependent activation of NF-κBs by 2–3 fold. Transfection of MD-1 with TLR4 did not have such an effect. Physical association of MD-2 therefore influences the signaling via the transmembrane TLR4 molecule. Preliminary studies suggested that MD-2 forms a homodimer or a larger complex on the cell surface. Such a complex may have multiple binding sites for TLR4 and facilitate cross-linking of TLR4, leading to higher NF-κB activation. Further studies are underway.
Figure 5 MD-2 enhances ligand-independent signaling via TLR4. A human kidney cell line, 293T, was transfected with expression vectors encoding the molecules indicated. A reporter plasmid for NF-κB activity and an expression vector encoding β-galactosidase (more ...)
TLR4 may be an LPS receptor, as its gene is mutated in low-responder mice C3H/HeJ and C57BL/10ScCr (12
). Transfection of TLR4, however, did not confer LPS responsiveness on recipient cell lines (11
), suggesting a requirement for another molecule that linked TLR4 to LPS signaling. We hypothesized that MD-2 might be such a link, as it interacts with TLR4 and influences the signaling of TLR4. To address this possibility, we studied LPS responsiveness of stable transfectants expressing TLR4 alone or with MD-2 by measuring NF-κB activity (see Materials and Methods). The stable transfectant line that expressed TLR4 alone did not respond to LPS from Escherichia coli
0111:B4, E. coli
055:B5 or Salmonella minnesota
Re595 or to lipid A (Fig. A), which is consistent with the report by Kirschning et al. (11
). The Ba/F3 line, like the 293 cell line (11
), might lack a molecule indispensable for LPS signaling via TLR4. The mRNA of a candidate molecule MD-2 was not expressed in either cell line (data not shown). The stable line expressing TLR4 and MD-2 was then examined. Transfection of MD-2 conferred on the line strong NF-κB responses to either LPS or lipid A at concentrations as low as 0.1 ng/ml (Fig. A). No response was observed to detoxified LPS from which the fatty acid side chains of the lipid A moiety were removed (data not shown). Receptor activity acquired by introducing MD-2 was triggered through TLR4, as the anti-TLR4 mAb HTA125 specifically inhibited the responses (Fig. B). MD-2 thus confers LPS signaling on TLR4.
Figure 6 MD-2 confers LPS signaling on TLR4. (A) Stable transfectants (see Materials and Methods for details) expressing TLR4 alone () or TLR4 and MD-2 (•) were stimulated with LPS from E. coli 055:B5, S. minnesota Re595, or lipid A at the concentrations (more ...)
We found, by reverse transcriptase (RT)-PCR expression of the transcript of TLR2, another LPS receptor in the parental line Ba/F3. TLR3, TLR4, and TLR5 were not detected by RT-PCR or Northern hybridization (data not shown). In spite of the expression of the TLR2 transcript, stable transfectants expressing the NF-κB reporter gene alone (data not shown) or with TLR4 (Fig. A) did not show any significant LPS response. The amount of the cell surface TLR2 protein, if any, may be too small to sense the presence of LPS, or mouse TLR2 may not respond to LPS as effectively as its human counterpart. Taken together with specific inhibition with the anti-TLR4 mAb, LPS responses in stable transfectants expressing TLR4 and MD-2 are mediated by the cell surface complex of TLR4–MD-2 but not by TLR2. The TLR4–MD-2 receptor complex efficiently senses the presence of bacterial endotoxin.
CD14, another LRR molecule capable of binding to LPS, is able to enhance LPS signaling via TLR2 (10
). Mouse CD14 was not demonstrable by cell surface staining of the Ba/F3 line (data not shown), but it is still possible that soluble CD14 in FCS of culture medium contributes to LPS signaling via TLR4–MD-2. Further study is of importance and underway concerning a role of soluble and membrane CD14 in LPS signaling of TLR4–MD-2.
Another finding with the new receptor complex TLR4– MD-2 is that it has broader specificity than that recently described for TLR2 (10
). TLR2 recognizes the LPS from S. minnesota
Re595 much better than that from E. coli
055:B5. On the other hand, the TLR4–MD-2 complex responded equally to the two different types of LPS (Fig. A). LPS is a complex glycolipid composed of hydrophilic polysaccharides of the core and O-antigen structures, as well as a hydrophobic domain called lipid A. Lipid A is a common component, whereas considerable diversity of structure is noted among the O-antigens. Because both TLR2 and TLR4/MD-2 receptors responded well to lipid A (reference 10
and Fig. A), the core and O-antigen from E. coli
055:B5 must selectively affect recognition by TLR2. Studies using stable transfectants expressing each TLR would reveal further difference in recognition specificity of each TLR. MD-2 might associate with other TLR family members and confer the ability to respond to a broader spectrum of pathogens, including gram-positive bacteria and fungi. Such fundamental information concerning innate recognition of pathogens may also suggest new treatments for infectious diseases and endotoxin shock.