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Separate Functional Domains of Human MD-2 Mediate Toll-Like Receptor 4-Binding and Lipopolysaccharide Responsiveness ¹

Fabio Re^2,* and Jack L. Strominger^*,

^* Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, MA 02115; and Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cellular responses to LPS are mediated by a cell surface receptor complex consisting of Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4. Site-directed mutagenesis was used to identify the regions of human MD-2 involved in its ability to bind TLR4 and confer LPS responsiveness. A separate region of MD-2 was found to mediate each function. MD-2 binding to TLR4 was dependent on Cys⁹⁵ and Cys¹⁰⁵, which might form an intramolecular disulfide bond. Hydrophilic and charged residues surrounding this area, such as R90, K91, D100, and Y102, also contributed to the formation of the TLR4-MD-2 complex. A different region of MD-2 was found to be responsible for conferring LPS responsiveness. This region is not involved in TLR4 binding and is rich in basic and aromatic residues, several of which cooperate for LPS responsiveness and might represent a LPS binding site. Disruption of the endogenous MD-2-TLR4 complex by expression of mutant MD-2 inhibited LPS responses in primary human endothelial cells. Thus, our data indicate that MD-2 interaction with TLR4 is necessary but not sufficient for cellular response to LPS. Either of the two functional domains of MD-2 can be disrupted to impair LPS responses and therefore represent attractive targets for therapeutic interventions.z

	Introduction

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The LPS derived from the outer membrane of Gram-negative bacteria is among the most powerful stimulators of innate immune responses. Extremely low concentrations of LPS can be sensed by immunocompetent cells, such as macrophages and dendritic cells, leading to the release of proinflammatory mediators and effector molecules.

Cellular responses to LPS are mediated by a cell surface receptor composed of at least three proteins: CD14, Toll-like receptor 4 (TLR4) ³ and MD-2 (reviewed in Refs. 1 and 2). A fourth protein, the LPS-binding protein, is also required for optimal responses (3). TLR4 is one of the 10 members of the TLR family of receptors that mediates response to a wide variety of microbial products (reviewed in Refs. 4 and 5). These receptors are type I transmembrane proteins characterized by leucine-rich repeats in their extracellular portion and by the signaling Toll-IL-1R domain in their cytoplasmic region. Several groups demonstrated that TLR4 is the signaling subunit of the LPS receptor using both genetic (6, 7, 8) and biochemical approaches (9). To function as an LPS receptor, however, TLR4 must interact with an additional protein, MD-2. MD-2 is a secreted protein that is retained on the cell surface through interaction with the extracellular portion of TLR4 (9). Despite its absolute requirement for LPS signaling (9, 10, 11), it is still unclear what role MD-2 plays in LPS signaling. In MD-2-deficient mice, LPS responses are abrogated and the TLR4 molecule appears to be preferentially retained in the endoplasmic reticulum/Golgi compartment rather than on the cell surface (11). Thus, one function of MD-2 might be the correct targeting of TLR4 to the cell membrane. The existence of an anti-TLR4 mAb that recognizes TLR4 only as a complex with MD-2 (12) suggests that MD-2 could induce a conformational change in TLR4 that might be required for proper transport of TLR4 to the cell surface. However, addition of recombinant MD-2 protein to the culture medium of cell lines that express only TLR4 and do not respond to LPS render them LPS responsive (10, 13, 14), arguing that subcellular targeting of TLR4 might not be the sole function of MD-2.

MD-2 is able to form disulfide-linked multimers, adding a further level of complexity (14, 15). It is still unclear what function the MD-2 multimers might perform but since only monomeric MD-2 can bind TLR4 and confer LPS responsiveness (15), the involvement of MD-2 multimers in the signaling process seems unlikely. Several experimental approaches indicated that MD-2’s role in the LPS response is not only structural and that this molecule might directly contribute to the recognition of the LPS molecule. MD-2 is responsible for the species-specific action of LPS mimetic substances, such as Taxol (16), and of particular types of LPS (17, 18). In addition, MD-2, TLR4, and CD14 can be chemically cross-linked to the LPS molecule (19), indicating that they are in close proximity to the agonist. Finally, MD-2 can directly bind LPS in some experimental settings (13). Interestingly, a region of human MD-2 spanning aa 119–132 contains several features common to other LPS-binding proteins (20). This region is rich in basic and aromatic residues that were proposed to interact with the negatively charged and hydrophobic pattern of the LPS molecule. A synthetic peptide derived from this sequence was shown to be able to bind LPS and, remarkably, was also shown to possess antimicrobial activity (20).

Thus, although the mechanistic aspects of the interaction among TLR4, MD-2, and LPS are still obscure, the existing data would suggest the existence of two functional domains in MD-2, one responsible for TLR4 binding and another that mediates the interaction with the agonist. In this study, using site-directed mutagenesis, we report the identification of MD-2 residues that are important for each function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells

HeLa cells were grown in DMEM supplemented with 10% FCS. The HeLa-TLR4 cell line was previously described (15). Pooled HUVEC were purchased from Clonetics (Walkersville, MD). HUVEC were grown on gelatin-coated Petri dishes and maintained for <10 passages in human endothelial-serum-free medium basal growth medium (Invitrogen, San Diego, CA) supplemented with 20% FCS (Invitrogen), 10 ng/ml human recombinant epidermal growth factor (Intergen, Purchase, NY), 15 ng/ml human recombinant basic fibroblast growth factor (Intergen), and 1 µg/ml heparin (Sigma-Aldrich, St. Louis, MO).

Expression vectors

The expression vector for soluble forms of TLR4 (Flag-sTLR4) was described previously (15). MD-2-Flag-His was expressed using the vector pBOS-MD-2-Flag-His kindly provided by Dr. K. Miyake (Saga Medical School, University of Tokyo, Tokyo, Japan).

Site-directed mutagenesis

All mutations (Cys to Ala) were introduced into pBOS-MD-2-Flag-His using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s recommendations and were confirmed by sequencing. Primers sequences are available upon request.

TLR4/MD-2 complex purification by nickel resin chromatography

HeLa cells (7 x 10⁶) were transfected by electroporation (250 V, 950 µF) with 30 µg of a plasmid mixture (20 µg of Flag-sTLR4 and 10 µg of MD-2-Flag-His) in Dulbecco’s PBS/1.25% DMSO. Cells were replated in 10-cm plates in FCS-containing medium to allow recovery and cell adhesion. After 12 h, plates were rinsed in PBS and 6 ml of serum-free medium 293 SFM (Invitrogen) was added. Media were collected 24–48 h later. To capture histidine-tagged protein complexes, filtered supernatants (typically 5 ml) were incubated overnight with ProBond nickel resin (Invitrogen), washed in PBS, and the resin was resuspended in Laemmli sample buffer, boiled, and analyzed by SDS-PAGE and immunoblot.

Luciferase assay

HeLa-TLR4 were transiently transfected in six-well plates using Superfect reagent (Qiagen, Valencia, CA) with 0.66 µg of endothelial leukocyte adhesion molecule (ELAM)-luciferase, 0.66 µg of pBOS-MD-2-FLAG-His, 0.16 µg of pcDNA-CD14 (kindly provided by Dr. D. Golenbock, University of Massachusetts, Worcester, MA), and 0.16 µg of CMV--galactosidase (-Gal). For HUVEC transfection, cells were briefly trypsinized, washed in DMEM with 10% FCS, and resuspended in ice-cold PBS with 1.25% DMSO at a concentration of 2 x 10⁷ cells/ml. Three hundred microliters of the cell suspension was electroporated with a total of 30 µg of plasmid DNA (7 µg of ELAM-luciferase, 3 µg of CMV-CD14, 1 µg of CMV--Gal, and 20 µg of pBOS-MD-2-Flag-His or CMV-enhanced green fluorescence protein) at 250 V, 960 µF, using a Bio-Rad (Hercules, CA) electroporator. Cells were replated on gelatin-coated six-well plates. Four hours after electroporation, cell culture medium was aspirated and cell debris were removed by PBS rinsing. Fresh complete medium was added and cells were used for experiments 36 h later. Luciferase assay was performed using Promega (Madison, WI) reagents according to the manufacturer’s recommendations. Efficiency of transfection was normalized by measuring -Gal in cell lysates.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MD-2’s domain of interaction with TLR4

We have previously developed a biochemical assay to characterize in detail the interaction between MD-2 and the extracellular portion of TLR4 (15). Using this assay, the interaction between these two proteins was shown to occur in solution and the ability of a panel of MD-2 mutants to bind TLR4 was analyzed. Mutation of two of the seven cysteine residues present in MD-2, Cys⁹⁵ and Cys¹⁰⁵, was found to disrupt the ability of MD-2 to bind TLR4 (Fig. 1A and Ref.15). These mutations also completely abrogated the ability of MD-2 to confer LPS responsiveness to a HeLa-TLR4 (Fig. 1B), a cell line that stably expresses TLR4 but not MD-2 and therefore is LPS unresponsive (15). Mutation of the remaining five cysteines did not affect MD-2 binding to TLR4 and decreased their ability to render the cell line LPS responsive by 40–70%. Thus, Cys⁹⁵ and Cys¹⁰⁵ may form an intramolecular disulfide bond that creates a tertiary structure that is required for MD-2-TLR4 interaction. The analysis of the MD-2 amino acid sequence reveals that the majority of the residues that separate Cys⁹⁵ and Cys¹⁰⁵ and several others that surround this area are highly charged or hydrophilic, suggesting that this part of the molecule might form a loop that is exposed to the aqueous phase and that might offer a proper surface for electrostatic interaction with TLR4. To test this hypothesis, a number of the hydrophilic and charged residues that span this region of the molecule were mutagenized and the ability of the mutant proteins to bind TLR4 was tested. As shown in Fig. 2A, mutations D99A-D100N-D101A, D100G, Y102A-S103A, R90A-K91A, or Y102A (data not shown) resulted in MD-2 molecules that, although expressed at wild-type levels, lacked TLR4 binding ability. Mutations R96A, S98G, or in charged residues that are more distantly located from this region, such as K55A–K58A, did not affect the ability of MD-2 to bind TLR4 (data not shown). TLR4 binding was also not affected by mutation of the glycosylation sites N26A and N114A (data not shown).

View larger version (36K): [in this window] [in a new window]   FIGURE 1. MD-2’s Cys95 and Cys105 are required for TLR4 binding and LPS responsiveness. A, HeLa cells were cotransfected with expression vectors for Flag-sTLR4 and the indicated MD-2-Flag-His mutant constructs. Protein complexes were purified from the culture supernatants by nickel resin chromatography, separated by SDS/PAGE, and detected by immunoblot with anti-Flag mAb. B, HeLa-TLR4 was transfected with the ELAM-luciferase reporter vector, CMV-CD14, and the indicated MD-2-Flag-His mutant constructs. Cells were stimulated for 6 h with LPS and luciferase was measured in cell lysates. Results are representative of three experiments.

View larger version (41K): [in this window] [in a new window]   FIGURE 2. Identification of MD-2’s domain of interaction with TLR4. The experiments were performed as described in Fig. 1 using the mutants shown.

These results thus identify the region of MD-2 that is responsible for its interaction with TLR4. They also demonstrate that to confer LPS responsiveness MD-2 must bind to TLR4.

MD-2’s domain responsible for LPS responsiveness

It has been suggested that a region of MD-2 (aa119–132) rich in basic and aromatic residues might be able to interact with the LPS molecule (20). To test whether this region of MD-2 is indeed the putative LPS binding site, several of the basic and aromatic residues were mutagenized, either singularly or in different combinations, and the ability of the mutant proteins to bind TLR4 and confer LPS responsiveness was examined. Single mutations (data not shown) or double mutations in any of these residues did not affect the ability of MD-2 to bind TLR4 (Fig. 3A). Even a quadruple mutant completely retained the ability to form a complex with TLR4. However, the ability of these mutants to render HeLa-TLR4 LPS responsive was progressively affected as the mutations accumulate (Fig. 3B). As expected for mutations that would decrease the affinity of MD-2 for the LPS molecule, the effect on LPS responsiveness was more pronounced as the agonist concentration was lowered. Thus, response to maximal doses of LPS was reduced 80% by mutation F121G-K122A, 20% by mutation K125A-F126A, and 83% by mutation Y131G-K132A. However, the response of these mutants was totally impaired at suboptimal LPS concentrations that are nevertheless still able to elicit a powerful response through the wild-type MD-2. The combination of the four mutations F121G-K122A-Y131G-K132A completely disrupted the response to LPS even at the highest LPS doses. Thus, although still able to bind TLR4 to the same extent as the wild-type MD-2, the mutant F121G-K122A-Y131G-K132A is totally LPS unresponsive. These experiments indicate that this region of MD-2 contains several residues that might cooperate to create the LPS binding site. These results also suggest that MD-2 interaction with TLR4 is necessary but not sufficient for proper TLR4 responses to LPS.

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Identification of MD-2’s domain responsible for LPS responsiveness. The experiments were performed as described in Fig. 1 using the mutants shown.

The complex TLR4/MD-2 is believed to form in the endoplasmic reticulum/Golgi compartment. The hypothesis that some of the MD-2 mutants might compete with the endogenous MD-2 protein for binding to TLR4, and therefore act as dominant negatives and abrogate LPS responses, was examined. For these experiments, we used primary HUVEC that express TLR4 and MD-2 and are LPS responsive (21, 22). Transfection of the LPS-unresponsive MD-2 mutant F121G-K122A-Y131G-K132A greatly diminished the ability of HUVEC to respond to LPS (Fig. 4). On the other hand, transfection of the mutant Y102A–S103A, which is unable to bind TLR4, did not affect LPS responses. Both mutant proteins were expressed at comparable levels (data not shown). These data are consistent with the notion that mutant F121G-K122A-Y131G-K132A which is still able to bind TLR4, when overexpressed, might displace the endogenous MD-2 from the TLR4 molecule and therefore render the cells LPS unresponsive. This phenomenon does not occur when mutant Y102A-S103A is overexpressed due to its inability to bind TLR4.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Disruption of the endogenous MD-2-TLR4 complex by mutant MD-2 inhibit LPS responses in HUVEC. HUVEC were transfected with the ELAM-luciferase reporter vector, CMV-CD14, and the indicated MD-2-Flag-His mutant constructs or CMV-enhanced green fluorescent protein. Cells were stimulated for 6 h with LPS and luciferase was measured in cell lysates.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The MD-2’s domain of interaction with TLR4 is dependent on Cys⁹⁵ and Cys¹⁰⁵. A putative disulfide bond between these residues would create a tertiary structure that might be exposed to the aqueous phase and available for the interaction with TLR4. Several residues comprised or surrounding this area, such as D100, Y102, R90, and K91, are important for interaction with TLR4.

The ability to confer LPS responsiveness maps to a different portion of MD-2 that is not involved in TLR4 binding. This area is rich in basic and aromatic residues. Although single mutations in various residues contained in this area affect LPS responsiveness to different degrees, the combination of multiple mutations progressively impair this function with mutant F121G-K122A-Y131G-K132A being completely unresponsive while still able to bind TLR4. Thus, MD-2 interaction with TLR4 is necessary but not sufficient for TLR4-mediated responses to LPS.

A similar conclusion was reached by a recently published study in which a saturating alanine scanning mutagenesis of mouse MD-2 was conducted (23). The mutant proteins were tested for their ability to increase reactivity to an anti-TLR4 Ab that recognizes TLR4 only when complexed with MD-2 and for conferring responsiveness to LPS and Taxol, a substance that has LPS mimetic activities in mouse but not human cells. Although this study failed to identify any particular region in mouse MD-2 important for each function, several of the residues that were found in the present study to be involved in the ability of human MD-2 to bind TLR4 and confer LPS responsiveness were also found to play a similar role in mouse MD-2. Specifically, mutation of Cys⁹⁵, Tyr¹⁰², and Cys¹⁰⁵ disrupted the ability to form the TLR4-MD-2 complex also in mouse cells. Surprisingly, mutation of Cys³⁷ and Cys¹⁴⁸ disrupted the mouse TLR4-MD2 complex but not the human’s. Although highly homologous, human and murine TLR4 and MD-2 show species specificity (16, 17, 18). It is possible that the interaction between the two proteins has different structural requirements in the two species. Another possible reason for the discrepancies between our study and that of Kawasaki et al. (23) is probably due to the different methods used to study the TLR4-MD2 complex formation: our study relies on an in vitro biochemical assay to detect a direct interaction between the two proteins. Kawasaki et al. (23) monitored the increase in the reactivity to an anti-TLR4 Ab that recognizes TLR4 only when complexed to MD-2 rather than analyzing a direct TLR4-MD-2 interaction.

The residues responsible for LPS responsiveness also seem to be scattered throughout the mouse MD-2 molecule rather than being concentrated in one discreet region as our study indicates to occur in the human protein. The study of Kawasaki et al. (23), although more exhaustive than ours, analyzed only single mutations. In contrast, our study showed that single mutations are not sufficient to completely disrupt LPS responsiveness and the putative LPS binding site was identified only by analyzing multiple mutations. Interestingly, some of the mouse MD-2 residues that affected LPS responsiveness did not affect responses to Taxol, suggesting that the two ligands interact with different regions of the mouse protein. This observation would suggest that also in human MD-2 additional domains might exist that mediate responsiveness to other TLR4 agonists, such as heat shock protein 60 (24), respiratory syncytial virus protein F (25), and fibronectin extradomain A (26).

The identification of the regions of MD-2 involved in the interaction with TLR4 and LPS is a prerequisite for the design of specific inhibitors of LPS actions. Our study identifies two distinct strategies that can be used to block LPS responses: disruption of the MD-2-TLR4 complex or inhibition of MD-2 interaction with LPS.

	Acknowledgments

 
We are grateful to Dr. K. Miyake and Dr. D. Golenbock for providing pBOS-MD-2-Flag-His and CMV-CD14 vectors, respectively, and to L. Steele and C. Stipp for critically reading this manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI-4952402 (to J.L.S.) and AI-05466501 (to F.R.).

² Address correspondence and reprint requests to Dr. Fabio Re, Dana Farber Cancer Institute, 44 Binney Street, Dana-1414, Boston, MA, 02115. E-mail address: fabio_re{at}dfci.harvard.edu

³ Abbreviations used in this paper: TLR4, Toll-like receptor 4; -Gal, -galactosidase; ELAM, endothelial leukocyte adhesion molecule.

Received for publication May 2, 2003. Accepted for publication September 5, 2003.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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