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(The FASEB Journal. 2006;20:1836-1842.)
© 2006 FASEB

Structural similarity between the hydrophobic fluorescent probe and lipid A as a ligand of MD-2

Mateja Mancek-Keber and Roman Jerala1

Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia

1Correspondence: Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. E-mail: roman.jerala{at}ki.si


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toll-like receptors (TLRs) belong to the family of pattern recognition receptors, as they recognize molecules sharing a broad structural pattern rather than a single defined structure. Bacterial LPS is recognized by MD-2, which is associated with the extracellular domain of TLR4. Understanding the molecular recognition pattern of MD-2 could lead to efficient inhibitors of the excessive LPS signaling needed for early treatment of sepsis. The effect of the acyl chain variability of lipid A on its biological activity indicates that in addition to electrostatic interactions, the recognition must also involve hydrophobic interactions. We show that the fluorescent hydrophobic probe bis-ANS binds to MD-2 with a dissociation constant in the 10 nanomolar range, both to glycosylated and to nonglycosylated MD-2, and requires its native conformation. The binding site of bis-ANS overlaps with the binding site of LPS and is in the proximity of the single tryptophan residue. Furthermore, photoincorporation of bis-ANS by UV light inhibits the ability of MD-2 to confer the LPS responsiveness to the TLR4-transfected HEK293 cell line. Our results show that the structural pattern recognized by MD-2 is defined by the hydrophobic patch and a pair of separated negative charges.—Mancek-Keber, M., Jerala, R. Structural similarity between the hydrophobic fluorescent probe and lipid A as a ligand of MD-2.


Key Words: bacteria • Toll-like receptor • LPS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RECOGNITION OF BACTERIA within the human body rapidly initiates the response of the innate immune system. Molecular components of pathogenic microorganisms, which are distinct from the host, are detected by a number of pattern recognition receptors (1) that recognize the conserved structural motifs of predominantly essential bacterial molecules such as lipid A, flagellin, and peptidoglycans. LPS, one of the most potent inducers of the innate immune system, is signaled through the transmembrane receptor TLR4 (2) . However, TLR4 alone is not sufficient for the recognition of LPS. The extracellular protein MD-2 has been identified as a TLR4 accessory protein that is required for LPS signaling through the receptor (3) . Physical association between MD-2 and the ectodomain of TLR4 as well as the ability of MD-2 to bind LPS are critical for the LPS signaling (4) . Mice without MD-2 are resistant to LPS and survive the endotoxic shock (5) . MD-2 is also important as a chaperone of TLR4, required for its proper glycosylation and transport to the cell surface (6) . MD-2 and TLR4 associate in the endoplasmatic reticulum and are transported to the cell surface as a complex (7) , whereas in MD-2–/– fibroblasts the majority of TLR4 resides in the Golgi apparatus (5) . A multicomponent membrane receptor complex is probably necessary for efficient cell signaling (8) . Mature MD-2 contains 142 amino acid residues and is glycosylated. It forms disulfide-linked dimers and oligomers (7) whose relevance to biological activity is still unclear (7 , 9 , 10) . It was demonstrated that monomeric MD-2 is capable of binding LPS with high affinity and that the addition of the MD-2 conferred LPS responsiveness to cells expressing only TLR4 (4 , 7 , 11) . Mutation of all cysteine residues except 95–105 abolished the formation of oligomers with a concomitant decrease in biological activity (9) . Accumulating evidence indicates that MD-2 determines the species-specific response to ligands. Human MD-2 binds lipid IVA, but in contrast to murine MD-2, this does not lead to cell activation (12) . Although several residues responsible for this species selectivity have been identified recently within MD-2 (13) , Poltorak et al. have shown that TLR4 is also important for the differentiation between LPS partial structures (14) . LPS is an amphiphilic molecule, prone to aggregation, that also plays an important role in signal transduction, although the precise role of aggregated vs. monomeric LPS in its binding to receptors is not yet clear (15 16 17) . We previously reported a model of tertiary structure of MD-2 (18) based on the template structures of proteins belonging to the ML superfamily, which have an apolar pocket formed between two ß-sheets (19) . Binding of lipid ligands to the hydrophobic pocket of ML proteins results in a conformational change and a pocket expansion as shown by the structure of GM2 ganglioside-activating protein (AP) (GM2-activating protein) with and without of the bound GM2 (20 21 22) . The MD-2 model predicts that the edge of the hydrophobic pocket is occupied by basic residues separated by a distance corresponding to the separation between the phosphate groups of the lipid A moiety (18) . The importance of the number, arrangement, and length of the acyl chains of the lipid A moiety on the variability of biological response has been well documented (reviewed in ref. 23 ). Therefore, electrostatic interactions identified by the mutagenesis of basic residues (18 , 24 , 25) of MD-2 are insufficient to differentiate between the LPS chemotypes.

In this study we employed the apolar probe bis-ANS, which is often used to detect hydrophobic binding sites in lipid binding proteins (26) . We found that bis-ANS binds to MD-2 with high affinity. Additional methods of fluorescence quenching, shift of the emission maximum wavelength, and fluorescence resonance energy transfer (FRET) provide further evidence for the hydrophobic ligand binding site of MD-2. Photoincorporated bis-ANS inhibits MD-2-mediated cell signaling. Structural similarity between lipid A and bis-ANS refines the definition of the minimal structural pattern recognized by MD-2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
S-LPS (from S. abortus equi HL83) was prepared by a phenol extraction procedure and was kindly provided by Dr. Brandenburg (Forschungszentrum Borstel, Germany). Before use, LPS was sonicated and subjected to three cycles of heating to 56°C and cooling down to 4°C. Therefore, LPS was a mixture of monomers and aggregates. 1,8-Anilino-naphthalene sulfonic acid (ANS), 1,1'-bis(anilino)-4-,4'-bis(naphthalene)-8,8'-disulfonate (bis-ANS), acrylamide, and sodium iodide were obtained from Sigma (St. Louis, MO, USA).

Cell lines
Human embryonic kidney (HEK) 293 cells (American Type Culture Collection-Nr. CRL-1573) were used to assay the inhibition of MD-2 in LPS cell activation. HEK293 cells were cultured as adherent monolayer at 37°C, 5% CO2, and normal humidity in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, San Diego, CA, USA) supplemented with 10% (v/v) FBS (BioWhittaker, Walkersville, MD, USA).

Preparation of recombinant MD-2
Recombinant MD-2 was produced in Escherichia coli, as described (12) , using solubilization of inclusion bodies, followed by their purification and refolding on reversed phase column chromatography. Biological activity of each batch of MD-2 was tested on HEK293 cells transfected with TLR4 (12) . Glycosylated MD-2 was isolated from supernatants of the HEK293 cell line transfected with MD-2 expression plasmid. HEK293 cells were cultured in DMEM + 10% FBS in 10 cm Petri dishes and transfected with 20 µg of MD-2 expression vector. The next day the medium was changed to 10 ml of DMEM + 2% FBS and incubated for 5 days. Supernatant was collected and dialyzed against PBS with 5 mM imidazole. Supernatant was incubated with Ni-NTA chromatographic resin and MD-2 protein was eluted with 200 mM imidazole in PBS and dialyzed against milliQ water.

Fluorescence spectroscopy
Fluorescence was measured on Perkin Elmer fluorimeter LS 55 (Perkin Elmer, UK). Quartz glass cuvettes (5x5 and 10x5 mm optical path, Hellma Suprasil, Hellma, Müllheim, Germany) were used. Binding of bis-ANS to MD-2 was measured at 20°C using excitation at 385 nm and measuring the emission fluorescence spectra between 420 and 550 nm. Bis-ANS was added to 50 nM MD-2. Each measurement was prepared separately and preincubated with bis-ANS prior to the measurement in order to reach a stable fluorescence. Data were evaluated by nonlinear fit of fluorescence intensities at maximum taking into account ligand depletion using the equation F = Fmax. (Kd+E0+L0-{surd}((Kd+E0+L0)2-4.E0L0))+C with Kd, E0 and L0 for dissociation constant, concentration of MD-2 and ligand, respectively, implemented by the program Origin 6.0 (Microcal, MA, USA). Displacement of bis-ANS was used to determine binding of nonfluorescent LPS to MD-2. 100 nM bis-ANS and 100 nM MD-2 were mixed and incubated to reach stable fluorescence. LPS was added in 10 nM increments (for the calculations, a MW of 10,000 g/mol for LPS was used). Apparent dissociation constant (Kapp) for LPS, which displaced bis-ANS, was obtained by nonlinear fit using the above-mentioned equation and corrected using the displacement equation: KD = Kapp/(1 + [bis-ANS]/KDbis-ANS). The FRET effect was measured between MD-2 tryptophan as an energy donor and bis-ANS as an acceptor. A wavelength of 295 nm was used to excite the tryptophan residue. MD-2 concentration was 1 µM and bis-ANS concentration was varied in the range 0–1.6 µM by increments of 0.2 µM.

Determination of binding stoichiometry
A Job plot was used to determine a binding stoichiometry of bis-ANS to MD-2. A total concentration of MD-2 and bis-ANS was maintained at 4 µM while varying the ratio between MD-2 and bis-ANS. Fluorescence intensities at 490 nm were plotted against [MD-2]/([MD-2]+[bis-ANS]). The maximum provides the stoichiometry of the complex or the number of bis-ANS binding sites on MD-2.

Fluorescent quenching
Solvent accessibility of MD-2 bound bis-ANS was determined using a fluorescence quenching method. Sodium iodide and acrylamide were used as quenchers. Quenching was quantified using the Stern-Volmer equation to obtain the quenching constant (Ksv): (F0-Fbuffer)/(F- Fbuffer) = 1+Ksv[Q], where F0 is the fluorescence without a quencher; F fluorescence in the presence of a given quencher concentration Q, Fbuffer background fluorescence, and [Q] the quencher concentration. (F0-Fbuffer)/(F- Fbuffer) was plotted against the concentration of the quencher in the form of a linear plot. Experiments were performed adding two different quenchers, sodium iodide or acrylamide, in 0.02 M increments to the preincubated mixture of 100 nM MD-2 and 100 nM bis-ANS and measuring a decrease of bis-ANS fluorescence intensity at 490 nm.

Photoincorporation of bis-ANS
Samples of MD-2 (3 µM) were incubated with various concentrations of bis-ANS or with 3 µM bis-ANS in combination with different concentrations of urea and irradiated on ice by UV light (250 nm) for 20 min unless stated otherwise. After irradiation, MD-2 was precipitated using pyrogallol red (Sigma), dissolved in SDS sample buffer, analyzed by SDS-PAGE, and fluorescence of the band corresponding to the size of MD-2 was captured on camera.

Luciferase reporter assay
To determine the activity of recombinant MD-2 with photoincorporated bis-ANS, 6 µM MD-2 was mixed with 0, 1, 2, 5, 10, 20, 50, and 100 x molar excess of bis-ANS and irradiated on ice under UV light for 10 min. HEK293 cells were transiently transfected with NF-{kappa}B-dependent luciferase and constitutive Renilla reporter plasmids, as well as 80 ng of TLR4 expression vector. Twenty-four hours later the transfection media were changed to DMEM + 2% FBS. MD-2/bis-ANS complex (200 nM) was added; after 1 h of incubation, 1 µg/ml of S-LPS was added. After 20 h cells were lysed and analyzed for reporter gene activities using a dual luciferase reporter assay system on a Mithras LB940 luminometer. The data from luciferase activity were normalized using Renilla luciferase readings.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tight binding of ANS and bis-ANS to MD-2
ANS has been used as a fluorescent probe to monitor interaction with the lipid binding site of several lipid binding proteins (26 27 28) . Here it is assessed as a probe for the hydrophobic binding site of MD-2. SDS-PAGE analysis of recombinant MD-2 indicated that after the final chromatographic purification step, MD-2 was in the monomeric form but dimers started to appear within couple of days at 4°C. All experiments with recombinant MD-2 were performed with freshly refolded protein. Intrinsic fluorescence emission of ANS increased with the addition of MD-2, producing a concomitant blue shift of its maximum of fluorescence emission (from 520 nm to 505 nm), characteristic of the hydrophobic environment of the fluorophore in the MD-2 binding site environment (Fig. 1 ). Bis-ANS, essentially a dimer of ANS, binds to MD-2 with even greater affinity. Intrinsic fluorescence of bis-ANS with an emission maximum at 523 nm was considerably enhanced in the presence of MD-2 compared to ANS, with a blue wavelength shift of the fluorescence maximum to 490 nm (Fig. 1) . The dissociation constant of bis-ANS to MD-2 was 25 ± 5 nM (Fig. 2 A). The stoichiometry of the bis-ANS/MD-2 complex was investigated by the method of continuous variation, keeping the total concentration of MD-2 and bis-ANS at 4 µM, and the data were analyzed by a Job plot (data not shown). The fluorescence maximum was reached at 0.55 ± 0.1 mol fraction, which suggests an equimolar ratio of MD-2 to bis-ANS in the complex. Since the native human MD-2 is glycosylated, we also determined the affinity of bis-ANS with glycosylated MD-2 isolated from the supernatant of the human cell line culture overexpressing MD-2. A dissociation constant with a value similar to that of the nonglycosylated MD-2 was found (KD=15±10 nM, Fig. 2B ). This agrees with our previous report showing that glycosylation is not required for the binding of LPS nor for the LPS signaling ability through TLR4 (18 , 29) .


Figure 1
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  		Figure 1. Addition of MD-2 increases fluorescence of hydrophobic dyes ANS. Fluorescence emissions spectra were determined for ANS and bis-ANS both without and in the presence of added MD-2. Left panel: ANS (2 µM), dashed line; ANS with added MD-2 (200 nM), solid line; right panel: bis-ANS (100 nM), dashed line; bis-ANS with added MD-2 (200 nM), solid line.


Figure 2
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  		Figure 2. Fluorescence titration of bis-ANS binding to MD-2. A) increase of the fluorescence of bis-ANS by the addition of MD-2. bis-ANS was added to 50 nM MD-2, preincubated to reach stable fluorescence, and measured. Curve indicates the best fit according to the dissociation constant of 25 ± 5 nM. B) fluorescence of bis-ANS added to the glycosylated MD-2, curve indicates the best fit with a dissociation constant of 15 ± 10 nM. The data are representative of more than three separate experiments performed using different MD-2 preparations.

Overlap with LPS binding site and characteristics of bis-ANS binding site
For the biological relevance of the bis-ANS binding site of MD-2, the most important question was whether this hydrophobic site overlaps with the LPS binding site. To assess this, LPS was titrated into the mixture of bis-ANS and MD-2, which resulted in the decrease of fluorescence intensity, indicating the displacement of bis-ANS from the complex with MD-2. The affinity of LPS for MD-2 was ~ 5-fold tighter than that of bis-ANS ( Fig. 4 ). Solvent exposure of bis-ANS, bound to MD-2, was investigated by a fluorescence quenching experiment of MD-2-bound bis-ANS. The iodide ion was used to quench the solvent accessible chromophore with a quenching constant Ksv of 2.5 M–1. Addition of the nonpolar quencher acrylamide, which can penetrate the nonpolar interior of the protein, gave a significantly higher Ksv value (5.0 M–1), implying that the bound bis-ANS was located in the nonpolar pocket, with limited accessibility to the solvent (Fig. 5 ). MD-2 contains a single tryptophan residue (Trp23) and the emission spectrum of the tryptophan overlaps with the absorption spectrum of bis-ANS, allowing investigation of the proximity between the two fluorophores using a fluorescence resonance energy transfer (FRET) effect. Upon addition of bis-ANS, the intrinsic fluorescence of the tryptophan residue decreased with the simultaneous increase of bis-ANS fluorescence, indicating the proximity of the tryptophan residue and bis-ANS binding site of MD-2 (Fig. 3 ).


Figure 3
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  		Figure 3. Fluorescence resonance energy transfer between MD-2 and bis-ANS. FRET between the tryptophan residue of MD-2 and bound bis-ANS was observed with excitation of tryptophan at 295 nm and measuring the fluorescence emission spectra, indicating close proximity between the molecules. Concentration of bis-ANS added to 1 µM MD-2 was from 0 to 1.6 µM in 0.2 µM increments. Arrows indicate the direction of the increasing concentrations of bis-ANS, causing a decrease of tryptophan and increase of bis-ANS fluorescence.


Figure 4
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  		Figure 4. Overlap of LPS and bis-ANS binding sites. LPS, added to 100 nM of preincubated MD-2/bis-ANS complex, competed with bis-ANS for binding to MD-2 resulting in a decrease of bis-ANS fluorescence. Curve indicates the best fit to the data with the apparent dissociation constant of 21 ± 5 nM, resulting in KD of 5 nM for binding of LPS to MD-2. The data are representative of three separate experiments performed using different MD-2 preparations.


Figure 5
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  		Figure 5. Protection of fluorescence quenching of bis-ANS bound to MD-2. Iodide ion (filled circles) and acrylamide (open squares) were added to the preincubated mixture of MD-2 and bis-ANS. Data are shown as Stern-Volmer plot with lines indicating fit to KSV of 2.5 M–1 and 5.0 M–1 for iodide and acrylamide, respectively.

Photoincorporation of bis-ANS
UV irradiation activates bis-ANS and causes the formation of a covalent bond between bis-ANS and the molecule in its immediate vicinity (30) . Irradiation of bis-ANS/MD-2 mixture with UV light caused cross-linking of bis-ANS to MD-2, which remains associated after precipitation and separation using SDS-PAGE (Fig. 6 ). Bis-ANS interacted with monomeric MD-2 and formed a complex with monomeric MD-2, which is demonstrated by the mobility on SDS-PAGE without the reducing agent. The amount of cross-linked product increased with the amount of added bis-ANS (Fig. 6A ). Photoincorporation increased for up to 20 min of irradiation, then slowly started to decrease with the concomitant appearance of aggregated products (data not shown). Addition of LPS inhibited photoincorporation of bis-ANS (data not shown). Bis-ANS only bound to the native conformation of MD-2, since the addition of urea to the mixture of MD-2 and bis-ANS decreased the extent of bis-ANS photoincorporation (Fig. 6B ). 2 M GdnHCl also prevented photoincorporation, although the secondary structure of MD-2, determined by the circular dichroism, is preserved under those conditions (data not shown). The reason for this inhibition is most likely a partial unfolding of the protein or a preferential binding of guanidinium groups to the protein, but not ionic strength, since a buffer solution at the same ionic strength (2M NaCl) did not inhibit photoincorporation (data not shown).


Figure 6
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  		Figure 6. Photoincorporation of bis-ANS into MD-2. 3 µM MD-2 was incubated with bis-ANS under varying conditions, irradiated under UV light for 20 min, then separated on SDS-PAGE and detected by fluorescence. A) Concentration dependence of bis-ANS photoincorporation into MD-2. B) Addition of urea decreased bis-ANS photoincorporation.

Bis-ANS inhibits the LPS signaling activity of MD-2
Competition of bis-ANS with LPS for binding to MD-2 would be expected to inhibit the signaling activity of LPS. However, bis-ANS is toxic to cell cultures at higher concentration, making the simple assay of the inhibition of the biological activity of MD-2 difficult. To avoid the toxicity issues and be sure that bis-ANS bound to MD-2, a cell assay was performed with the addition of MD-2 to TLR4 transfected HEK293 cell line, using MD-2 with photoincorporated bis-ANS. Inhibition of LPS signaling was concentration dependent and the addition of 20 µM bis-ANS almost completely abolished the NF-{kappa}B activation, as determined by the luciferase reporter assay (Fig. 7 ). As a control, recombinant MD-2 without bis-ANS, which had been exposed to the same photoincorporation conditions, efficiently conferred LPS responsiveness to TLR4 transfected HEK293 cells.


Figure 7
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  		Figure 7. Inhibition of the LPS signaling activity by bis-ANS. 6 µM MD-2 was photoincorporated with various concentrations of bis-ANS and 200 nM MD-2. The complex was added to HEK293 cells transfected with TLR4, luciferase reporter plasmids, and control plasmids. After 1 h of incubation, S-LPS was added. Responsiveness to LPS was determined by the NF-{kappa}B-responsive luciferase reporter plasmid, which was normalized using constitutively active Renilla luciferase after 24 h.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MD-2, appropriately called the "Toll’s gatekeeper" (31) , is the final LPS binding protein of the extracellular endotoxin recognition cascade before the signal is transduced across the cell membrane, and as such is essential for the cellular LPS responsiveness. This protein could be the "Achilles’ heel" of the innate response to LPS and a target for a pharmacological intervention in endotoxemia, as already realized (32 , 33) . Until now, mostly electrostatic interactions between LPS and MD-2 have been studied through mutations of basic residues of MD-2 (18 , 24 , 25) , although the importance of the acylation pattern is the basis for lipid A antagonism (34) . A structural model of MD-2, based on the template of the ML superfamily members (12) and the binding of lipid ligands to the other ML members, suggests that the hydrophobic ligand binding site is located between the two ß-sheets. Binding of lipids to MD-2 homologues GM2-activating protein (22) , Der p2 (35) , and NPC2 (36) causes a conformational change involving an expansion of the protein, which is also likely to be the case for MD-2 and could be the necessary step in triggering the conformational change that could be transmitted to TLR4, leading to the association of its intracellular TIR domains. To prove the existence of a hydrophobic binding site, we have used hydrophobic fluorescent probes ANS and bis-ANS. The binding of these dyes to a hydrophobic surface leads to an increase in their fluorescence intensity and to a blue shift of the maximum fluorescence, making the dyes useful for studying the internal structure, polarity and ligand binding properties of proteins (27 , 30 , 37 , 38) . The affinity of ANS for partially folded states of proteins is in the 10 micromolar range (39) , which is several orders of magnitude weaker than the affinity levels found in this report. High affinity of MD-2 for bis-ANS probably reflects a similarity in the structural pattern between LPS and bis-ANS, comprising a pair of separated negative charges and a hydrophobic patch (Fig. 8 ). It has been shown that the nature of protein-bis-ANS interaction, in contrast to ANS, is driven primarily by hydrophobic interactions (37) . Photoincorporation of bis-ANS at higher ionic strength indicates that electrostatic interactions do not dominate the ligand binding. Fluorescence quenching, shift of the emission maximum wavelength, and FRET effect data indicate that bis-ANS is buried and partially protected from the solvent. In the MD-2 structural model based on Der p 2, the cavity between the ß-sheets occupies ~ 360 Å3, whereas the pocket size based on the structure of the open conformation of the GM2-activating protein increases to almost 3000 Å3. This pocket size would have ample space to accommodate the 1780 Å3 large lipid A moiety. We observed that the ligand could bind to Der p 2 only in the presence of a reducing agent, which allows expansion of the protein core (unpublished results) and that the affinity of Der p 2 for LPS was low (29) ; therefore, GM2-activating protein with its largest binding pocket may be a more appropriate structural template for MD-2.


Figure 8
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  		Figure 8. Comparison of the structure of lipid A from enteric bacteria and bis-ANS, indicating the pattern of similar arrangement of hydrophobic region and a pair of separated negatively charged groups that match the distance between the basic clusters on the surface of MD-2 structural model (18) .

A decrease in the bis-ANS fluorescence on addition of LPS shows that the binding site for bis-ANS overlaps with the binding site for LPS. There was no requirement for LBP and CD14 for binding of LPS to MD-2 in this concentration range, in agreement with results published by Viriyakosol et al. (4) . Based on the effect of denaturants, we conclude that binding of bis-ANS requires the native conformation of MD-2. We have also shown that bis-ANS binds to the MD-2 produced in mammalian cells, ruling out differences due to glycosylation, which may nevertheless be important for TLR4/MD-2 complex trafficking inside the cell (6 ,40) . Bis-ANS is toxic to mammalian cells; therefore, we used the photoincorporation approach in order to provide the proof-of-principle for the inhibition of endotoxemia using a nonlipid A-based molecule, since bis-ANS is the first nonlipid A-based type of a molecule shown to compete with LPS for binding to human MD-2. Monomeric molecules may have important advantages for therapy in comparison to the molecules based on lipid A, which exhibit various aggregation properties that affect its interactions with receptors as well as pharmacological properties. Weaker affinity and antagonistic activity of bis-ANS for MD-2 might be explained by the incomplete match of the two anilino-naphthalene rings of bis-ANS with the hydrophobic pocket, since those groups occupy a significantly smaller volume than acyl chains of the LPS, resulting in an insufficient expansion of the protein. In conclusion, our results contribute to the understanding of the structural pattern of ligand recognized by the LPS receptor MD-2, which can assist the development of inhibitors designed to prevent excessive stimulation in inflammatory states, such as Gram-negative sepsis.


   ACKNOWLEDGMENTS
 
We thank Robert Bremsak for his excellent technical help and Dr. Kensuke Miyake for the MD-2 clone. This project was financed by the Slovenian Research Agency. We thank Dr. Michael Galsworthy for careful reading and corrections of the manuscript.

Received for publication February 7, 2006. Accepted for publication April 10, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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