©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Lipopolysaccharide Binding Domain in CD14 between Amino Acids 57 and 64 (*)

(Received for publication, November 29, 1994; and in revised form, December 22, 1994)

Todd S.-C. Juan Eric Hailman (1)(§) Michael J. Kelley Leigh A. Busse Elyse Davy Cyril J. Empig (2) Linda O. Narhi Samuel D. Wright (1)(§) Henri S. Lichenstein (¶)

From the  (1)From Amgen, Inc., Thousand Oaks, California 91320, the Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021, and the (2)Department of Biochemistry, University of Southern California, Los Angeles, California 90033

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. Recent limited proteolysis studies have implicated a region in CD14 between amino acids 57 and 64 as being involved in LPS interaction. To specifically assess the importance of this region with respect to LPS binding, we constructed a mutant sCD14 (sCD14) lacking amino acids 57-64. sCD14 was isolated from the serum-free conditioned medium of this cell line, and, in all assays, the purified protein failed to recognize LPS or enable LPS-dependent responses in cells. We also demonstrated that the region between amino acids 57 and 64 is required for binding of a neutralizing CD14 mAb, MEM-18. Native polyacrylamide gel electrophoresis assays were used to demonstrate that MEM-18 and LPS compete for the same binding site on CD14. These data strongly suggest that the region spanning amino acids 57-64 binds LPS and that formation of sCD14bulletLPS complex is required in order for sCD14-mediated responses to occur.


INTRODUCTION

CD14 is a 55-kDa protein which exists as a glycosylphosphatidylinositol-anchored protein found on the surface of leukocytes or as a soluble protein found in serum(1, 2, 3, 4) . CD14 binds one molecule of lipopolysaccharide (LPS) (^1)in a reaction catalyzed by LPS-binding protein (LBP(5) ), an acute phase serum protein(6) . Complexes of LPS with CD14 have been shown to be sufficient to elicit inflammatory responses in leukocytes(5, 7, 8) and endothelial cells(9, 10, 11, 12) . Neutralizing mAbs to CD14 antagonize cellular responses to LPS in vitro(1, 5, 9, 12, 13, 14, 15) , and recent experiments have shown that CD14 mAbs are also effective in vivo(23) . These observations suggest that CD14 may be an important pharmacologic target for diseases mediated by LPS.

In order to better understand how CD14 enables cells to respond to LPS, we have initiated a series of studies aimed at characterizing the interaction between LPS and CD14. To date, we have shown that the amino-terminal 152 amino acids of CD14 binds LPS and enables normal cellular responses to LPS(8) . In our accompanying paper(16) , we demonstrate that LPS protects a region spanning amino acids 57-64 from proteolytic digestion without altering the overall conformation of CD14 (16) . These experiments suggested that amino acids 57-64 were involved in binding LPS.

In this report we analyzed the requirement of amino acids 57-64 for LPS binding by generating a mutant sCD14 protein (sCD14) lacking this region. Here, we show that purified sCD14 no longer binds LPS nor activates cells. In addition, we mapped the epitope of a neutralizing mAb, MEM-18, to the domain specified by amino acids 57-64 and showed that MEM-18 and LPS compete for binding to this site on sCD14.


MATERIALS AND METHODS

Reagents

Recombinant soluble CD14 (rsCD14) and recombinant LBP (rLBP) were constructed and purified as described(5) . Concentrations of all purified proteins were determined with a Micro BCA protein kit (Pierce) according to the manufacturer's specifications. Since full-length rsCD14 terminates at position 348 of the mature protein(5) , we herein refer it as sCD14. The anti-CD14 mAbs used were 3C10 (purified by chromatography on Protein G from the conditioned medium (CM) of ATCC TIB 228), MEM-18 (SANBIO, The Netherlands), My4 (Coulter Immunology), and 60b(17) . Rabbit polyclonal anti-human CD14 antiserum was raised against sCD14 and was prepared by Antibodies, Inc. (Davis, CA). Enzymes for DNA manipulation and polymerase chain reaction were purchased from Boehringer Mannheim. p-Nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate-toluidine salt, and alkaline phosphatase-conjugated goat anti-rabbit IgG were purchased from Bio-Rad.

Site-directed Mutagenesis

A cDNA which encodes mutant sCD14 lacking amino acids 57-64 (sCD14) was constructed using a Transformer site-directed mutagenesis kit (Clontech) according to the protocol specified by the manufacturer. Briefly, mutation primer (5`-TAAAGCGCGTCGATGCGGACACGGTCAAGGCTCTCC-3`) and selection primer (Trans oligo SspI/EcoRV, Clontech) were annealed to a mammalian expression vector (pDSRalpha2) containing the cDNA for sCD14(8) . Primers were extended and ligated using T4 DNA polymerase/T4 DNA ligase for 2 h. The reaction was digested with SspI to linearize unmutated wild-type plasmids, and undigested circular plasmids which contained mutagenized DNA were transformed into Escherichia coli strain DH5alpha. Plasmid DNA was isolated from transformants, and DNA sequence analysis verified the presence of the deletion.

The Transformer site-directed mutagenesis kit was also used to generate mutant cDNAs encoding sCD14 having alanine substituted at various positions between amino acids 59 and 65. For these experiments, the following mutant primers were used: 5`-GATGCGGACGCCGCCCCTAGGCAGTATGCTGACACG-3` for sCD14, 5`-GATGCGGACGCCGACGCGCGGCAGTATGCTGAC-3` for sCD14, 5`-GCGGACGCCGACCCTGCGCAGTATGCTGACAC-3` for sCD14, 5`-GACGCCGACCCGCGAGCGTATGCTGACACGGTC-3` for sCD14, 5`-CGCCGACCCGCGTCAGGCTGCTGACACGGTTCAAG-3` for sCD14, 5`-CCGCGGCAGTATGCTGCCACGGTCAAGGCTCTCC-3` for sCD14, and 5`-GTCGATGCGGACGCCGCCGCGGCGGCGGCTGCTGCCACGGTCAAGGCTCTCCGC-3` for sCD14. Introduction of the appropriate mutation in all cDNAs was confirmed by DNA sequencing.

Transient Expression of Mutant sCD14 Proteins in COS-7 Cells

To express mutant sCD14 proteins, mammalian expression vectors containing mutant sCD14 cDNAs were introduced into COS-7 (ATCC CRL 1651) cells by electroporation. Conditions for electroporation and generation of serum-free CM from transfected COS-7 cells was as described(8) . Expression of mutant sCD14 was analyzed by Western blot, and the concentration of mutant proteins was determined with the aid of a BIAcore biosensor instrument (Pharmacia Biosensor, Piscataway, NJ) using protocols described previously(8) .

Purification of sCD14

The expression vector containing the cDNA encoding sCD14 was stably transfected into Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase as described(5) . A single clone was grown without serum to generate CM containing sCD14. Mutant protein was purified by immunoaffinity chromatography on a column to which mAb 3C10 was coupled to Sepharose 4B (Pharmacia). Briefly, CM was concentrated 20times using a S10Y10 spiral-wound cartridge (Amicon). Concentrated CM was passed over the column pre-equilibrated with phosphate-buffered saline (PBS, Life Technologies, Inc.), and protein was monitored by following the absorbance at 280 nm. The column was washed with PBS until the absorbance reached baseline. The protein was then eluted with 0.1 M glycine-HCl, pH 2.5, into collection tubes containing 0.5 M phosphate, pH 8.0. sCD14-containing fractions were pooled, concentrated, and diafiltered into PBS in a Centriprep-10 (Amicon) concentrator. Purity of the sample was checked by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining or Coomassie Blue staining. Circular dichroism (CD) analysis was performed as described previously (16) for sCD14.

Native PAGE Assay

Variations of a previously described native PAGE assay (5, 8) were used to assess whether unpurified or purified sCD14 preparations bound LPS. For unpurified sCD14 expressed in CM of COS-7 cells, 30 µl of CM was incubated at 37 °C with various amounts of LPS (Salmonella minnesota type Re595, List Biological Laboratories) for 30 min, and mixtures were resolved by native PAGE on 10% gels (Noval Experimental Technologies). Proteins were then transferred to nitrocellulose membranes, and Western blot analysis was performed as described previously (8) using anti-human CD14 polyclonal antibody.

To assess LPS binding of purified sCD14 preparations, sCD14 or sCD14 were incubated at various concentrations (0, 101, 303, and 909 nM) with 3 µg/ml [^3H]LPS prepared from E. coli K12 strain LCD25 (List Biological Laboratories) in the presence or absence of 16.7 nM rLBP. The reaction was incubated at 37 °C for 30 min and then electrophoresed on native 4-20% polyacrylamide gels. Gels were prepared for fluorography as described previously(5) .

Experiments were also performed to determine whether various CD14 mAbs could compete with LPS for binding to sCD14. In these studies, [^3H]LPSbulletsCD14 complexes were formed by incubating 130 µg/ml sCD14 with 10 µg/ml [^3H]LPS for 15 h at 37 °C in PBS with 1 mM EDTA. Complexes were then diluted 10-fold and incubated for 20 min at 37 °C with 200 µg/ml concentrations of various mAbs in a total volume of 10 µl. Mixtures were then electrophoresed on 8-16% native gels and processed for fluorography as above.

In other experiments, we examined whether rLBP could lower the effective dose of LPS required to competitively inhibit binding of MEM-18 or 3C10 to sCD14. MEM-18 or 3C10 (40 µg/ml) was incubated with sCD14 (2.6 µg/ml) for 10 min at 37 °C. Various concentrations of LPS (from S. minnesota strain R60, List Biological Laboratories) were then added in the presence or absence of rLBP (1 µg/ml) for 20 min at 37 °C in a total volume of 10 µl. Mixtures were then electrophoresed on 8-16% native gels and transferred to nitrocellulose in Tris-glycine buffer with 20% methanol. The nitrocellulose was blocked in PBS with 10% dry milk and incubated with polyclonal antibodies in PBS with 0.1% dry milk. CD14 was detected using a rabbit polyclonal antibody (generous gift of Dr. Pat Detmers) raised against sCD14 and an alkaline phosphatase-conjugated secondary antibody. Bound antibody was detected using p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate-toluidine salt according to the manufacturer's instruction.

Activation of Polymorphonuclear Leukocytes (PMN) by LPS and sCD14

The ability of rLBP and sCD14 or sCD14 to enable PMN adhesion to fibrinogen-coated plates was assessed by previously established protocols(5, 8) . Briefly, PMN were incubated for 10 min with LPS, LBP, and sCD14 and washed, and adhesion to fibrinogen-coated surfaces was measured. When smooth LPS is used in this protocol, adhesion is completely dependent on addition of sCD14(8) .

U373 Bioassays

Growth of U373 cells, activation by COS-7 CM containing sCD14 or by purified sCD14 preparations, and quantitation of IL-6 were performed exactly as described(8) . Briefly, mixtures of sCD14 and LPS were added to monolayers of U373 cells in serum-free medium and incubated for 24 h. IL-6 in the supernatant was then measured by enzyme-linked immunosorbent assay.

BIAcore Analyses of Interactions between sCD14 and anti-CD14 mAbs

Recognition of sCD14 preparations by anti-CD14 mAbs was performed with a BIAcore biosensor instrument. The instrument, CM5 sensor chips, and amine coupling kit were purchased from Pharmacia Biosensor. Briefly, mAb 3C10 (200 µg/ml in 20 mM sodium acetate, pH 3.4) was immobilized to a CM5 sensor chip by amine coupling according to the manufacturer's specifications. The flow cell immobilized with 3C10 was then incubated in succession with four solutions as detailed in the following steps: Step 1, COS-7 CM or 10 µg/ml purified sCD14 for 2 min; Step 2, HBS buffer (10 mM HEPES, pH 7.5, 0.15 M NaCl, 3.4 mM EDTA, 0.005% (v/v) surfactant P20 (Pharmacia Biosensor)) for 2 min; Step 3, 50 µg/ml MEM-18 (in HBS buffer) for 2-3 min; Step 4, HBS buffer for 2 min. All solutions were injected at a flow rate of 5 µl/min. To quantitate the binding of MEM-18 to sCD14 mutants in COS-7 CM, we calculated the relative response unit. The relative response unit was obtained by subtracting the response unit recorded just before injection of MEM-18 from the response unit recorded after injection of MEM-18 and a 2-min wash. Since there are slight differences in the concentrations of sCD14 proteins expressed in CM, and since the signal is linearly related to the concentration of sCD14 under the condition employed, we present binding data as the relative response unit per nanomolar concentration of sCD14 mutants.


RESULTS

LPS Does Not Bind to sCD14 Expressed in COS-7 CM

We have shown (16) that endoproteinase Asp-N cleaves sCD14 before aspartic acid residues 57, 59, and 65, and that these cleavage sites are masked when LPS is complexed to sCD14. This observation suggested that the region between amino acids 57 and 65 could be important for LPS binding. To test this hypothesis, we utilized the technique of site-directed mutagenesis to construct a cDNA which encodes mutant sCD14 (sCD14) lacking amino acids 57-64. A mammalian expression vector containing sCD14 was transiently transfected into COS-7 cells, and serum-free CM was collected. Western blot analysis (data not shown) revealed that expression of sCD14 was comparable to expression of sCD14 in COS-7 transfected cells.

We then used a native PAGE assay (5, 8) to assess whether sCD14 present in COS-7 CM binds LPS. CM containing sCD14 or sCD14 were incubated with increasing amounts of LPS, and the mixtures were electrophoretically transferred to nitrocellulose membranes. sCD14 or sCD14bulletLPS complexes were then detected with anti-CD14 polyclonal antiserum. Fig. 1shows that LPS caused a shift in the electrophoretic mobility of sCD14, and previous studies showed that this shift is caused by binding of LPS to CD14(5) . In contrast, no shift was observed in CM containing sCD14 even at an LPS concentration 5-fold higher then that needed to completely shift sCD14. These results are consistent with the notion that amino acids 57-64 in sCD14 are critical for LPS binding.


Figure 1: COS-7 CM containing sCD14 does not form complexes with LPS. Thirty µl of CM containing sCD14 or sCD14 were incubated with 1 µg (lanes 2 and 6), 2 µg (lanes 3 and 7), or 5 µg (lanes 4 and 8) of Re 595 LPS for 30 min at 37 °C. Lanes 1 and 5 represent CM alone without LPS. Protein mixtures were run on 10% native polyacrylamide gels and then transferred to nitrocellulose membranes. sCD14 was detected using polyclonal anti-human CD14 antiserum.The position of sCD14 alone or LPSbulletsCD14 complexes are indicated by arrows.



Purification and Characterization of sCD14

To further characterize the LPS binding and biological activities of sCD14, it was necessary to purify large quantities of the mutant protein. Therefore, a stable CHO cell line expressing sCD14 was constructed, and mutant protein was purified from the serum-free CM of this cell line. Purified sCD14 migrated with an apparent M(r) of 55,000 when analyzed by reducing SDS-PAGE (data not shown). In order to determine whether the deletion in sCD14 affected protein structure, we analyzed purified sCD14 and sCD14 by CD. Both the far- and near-UV spectra of the sCD14 were within experimental error of sCD14, as was the thermal stability determined by a change in the CD signal with temperature (data not shown). This result suggests that the deletion does not significantly interfere with the folding structure or stability of sCD14. Furthermore, the data also imply that tyrosine at position 63 does not contribute to the spectrum of the native protein and that this residue therefore is located in a flexible or symmetrical environment. This is consistent with the theory that the region between amino acids 57-64 is a flexible bridge connecting two compactly folded domains(16) .

Purified sCD14 Does Not Form a Stable Complex with LPS

To directly address whether sCD14 is capable of binding LPS, we used the native PAGE assay to detect stable complexes between sCD14 or sCD14 and [^3H]LPS. As previously reported(5) , incubation of sCD14 with LPS for 30 min lead to stable complexes (Fig. 3A), and addition of rLBP lowered the concentration of sCD14 required for the complex formation (compare lane 2 of Fig. 2B to lane 2 of Fig. 2A). This is consistent with the previous observation (5) that rLBP accelerates the transfer of LPS to sCD14. In contrast, even the highest concentration of sCD14 did not support complex formation with [^3H]LPS in the absence (Fig. 2A, lanes 5-7) or presence (Fig. 2B, lanes 5-7) of rLBP. These data confirm that sCD14 fails to bind LPS.


Figure 3: sCD14 but not sCD14 mediates responses of PMN to LPS and LBP. Freshly isolated PMN were incubated with ``smooth'' LPS (E. coli 0111:B4, 30 ng/ml), rLBP (1 µg/ml), and the indicated concentrations of sCD14 or sCD14 for 10 min at 37 °C. Cells were washed, and adhesion to fibrinogen-coated wells was measured(5, 22) . Error bars indicate standard deviations of triplicate determinations.




Figure 2: sCD14 does not form stable complexes with [^3H]LPS. Various concentrations of sCD14 (lanes 2-4) or sCD14 (lanes 5-7) were incubated without rLBP (A) or with 16.7 nM rLBP (B) as described under ``Materials and Methods.'' Lane 1 contains LPS in the absence of additional protein. Mixtures were run on 4-20% native polyacrylamide gels and processed for fluorography. Positions of uncomplexed LPS, and complexes between LPS and sCD14 are indicated.



sCD14Delta57-64 Does Not Enable Cellular Responses to LPS

The lack of interaction between sCD14 and LPS suggested that the mutant protein would be impaired in its ability to enable cellular responses to LPS. We tested this hypothesis by using two previously characterized assays (5, 8, 9) which measure sCD14 function. In the first assay, we examined whether sCD14 could enable LPS-induced adhesion of PMN to fibrinogen. Fig. 3shows that 100 ng/ml sCD14 enabled a strong adhesive response of PMN to smooth LPS and rLBP. However, no response was seen even when 10,000 ng/ml sCD14 was added.

We also examined the ability of sCD14 to support responses of U373 cells to LPS. Addition of as little as 5 ng/ml sCD14 enabled strong IL-6 production in response to LPS (Fig. 4), confirming previous reports(8, 9) . In contrast, sCD14 failed to support LPS-dependent IL-6 production even at a concentration of 80 ng/ml. These findings confirm that residues 57-64 are crucial to the biological function of sCD14.


Figure 4: sCD14 does not induce IL-6 production in U373 cells. U373 cells were treated with various concentrations of sCD14 or sCD14 in the presence or absence of LPS (10 ng/ml) for 24 h. IL-6 levels were determined as described(8) . Data presented are means ± S.D. from four readings.



An Epitope for mAb MEM-18 Is Localized to Amino Acids 57-64

The importance of amino acids 57-64 for LPS binding suggested that this domain could be the site of interaction between neutralizing mAbs and CD14. Since mAb 3C10 was used to withdraw sCD14 from CM, we reasoned that 3C10 must recognize an epitope outside amino acids 57-64. This was confirmed in a BIAcore analysis in which we observed a signal indicative of binding upon adding sCD14 to immobilized 3C10 (Fig. 5A, compare signal at 0 s to that at 480 s). We used further BIAcore analysis to test whether a different neutralizing mAb, MEM-18, could interact with the bound sCD14. We previously showed that MEM-18 binds to sCD14 captured by immobilized 3C10(8) . However, no binding of MEM-18 was observed to sCD14 (Fig. 5A, compare signal at 480 s to that at 800 s). To confirm this result, we used Western blot analysis. While MEM-18 bound immobilized sCD14, it failed to bind sCD14 (Fig. 5B). Parallel studies performed with polyclonal anti-CD14 confirmed the presence of sCD14 on the nitrocellulose membrane. These results suggest that MEM-18 recognizes an epitope in the region between amino acids 57 and 64.


Figure 5: mAb MEM-18 does not recognize sCD14. A, immobilization of mAb 3C10 to a sensor chip and injection of solutions at various ``Steps'' are detailed under ``Materials and Methods.'' Wash indicates a washing step using HBS buffer. Binding of purified proteins to mAbs was assessed by measuring the change in response unit (RU) after a 2-min wash. B, various amounts (2 ng for lanes 1, 2, 3, and 5; 10 ng for lanes 4 and 6) of purified sCD14 (lanes 1, 3, and 4) or sCD14 (lanes 2, 5, and 6) were electrophoresed on 4-20% SDS-PAGE, and proteins were transferred to nitrocellulose membranes. For detection of sCD14 protein, polyclonal anti-CD14 antiserum (lanes 1 and 2) or mAb MEM-18 (lanes 3-6) were incubated with the filters for 1 h. Immune complexes were detected by enhanced chemiluminescence (ECL, Amersham) as described by the manufacturer.



In an attempt to further characterize the MEM-18 epitope, we constructed a series of cDNAs encoding sCD14 having alanine substituted at various positions between amino acids 59 and 65. Table 1summarizes the corresponding amino acid changes for each mutant construct. Mammalian expression vectors containing each mutant cDNA were transiently transfected into COS-7 cells, and expression of mutant protein in CM was monitored by Western blot analysis. No differences in expression of mutant sCD14 proteins were observed in COS-7 CM (data not shown). Therefore, we performed BIAcore analyses to test the ability of each CM containing mutant sCD14 to bind MEM-18. Immobilized mAb 3C10 recognized each of the constructs, but sCD14, sCD14, sCD14, and sCD14 were not recognized by MEM-18 (Table 1). Binding of MEM-18 was not affected if Arg or Asp was mutated, and substitution of alanine at Pro partially inhibited MEM-18 binding. In summary, we have demonstrated that MEM-18 recognizes an epitope which is minimally comprised of residues Asp, Gln, and Tyr.



LPS Competes for the Same Site on CD14 as MEM-18

The localization of the MEM-18 epitope to a region we have implicated in LPS binding suggests that LPS should compete with MEM-18 for binding to sCD14. To demonstrate this, we measured the ability of MEM-18 to bind preformed LPSbulletsCD14 complexes (Fig. 6A) and the ability of LPS to bind preformed sCD14bulletMEM-18 complexes (Fig. 6B). sCD14 (Fig. 6B) and complexes of [^3H]LPS and sCD14 (Fig. 6A, lane 2) showed mobility characteristic of a 50-kDa protein, and this mobility was not affected by an irrelevant antibody (anti-CD18, Fig. 6A, lane 7). Addition of anti-CD14 mAbs MEM-18, My4, 60b, or 3C10 each caused a quantitative ``supershift'' in the mobility of sCD14 to a position consistent with a 250-kDa complex of IgG with two molecules of sCD14 (Fig. 6B and data not shown). We further observed that a subset of these mAbs (MY4, 60b, and 3C10) also shifted the mobility of the [^3H]LPSbulletsCD14 complexes (Fig. 6A). These observations indicate that MY4, 60b, and 3C10 bind to LPSbulletsCD14 complexes and therefore do not compete with LPS for a binding site. The implications of these and related studies will be the subject of a separate report. (^2)In contrast, MEM-18 failed to shift the mobility of [^3H]LPS in LPSbulletsCD14 complexes (Fig. 6A, lane 6) using conditions that caused complete shifting of sCD14 to the higher molecular weight position in the absence of LPS (Fig. 6B and data not shown). This observation indicates that sCD14 cannot simultaneously bind MEM-18 and LPS.


Figure 6: LPS competes with mAb MEM-18 for binding to sCD14. A, [^3H]LPSbulletsCD14 complexes were formed as described under ``Materials and Methods.'' Complexes were then diluted 10-fold and incubated with buffer (lane 2) or mAbs MY4, 3C10, MEM-18, or IB4 (lanes 3-7, respectively). The same concentration of [^3H]LPS was run in the absence of sCD14 for comparison (lane 1). Mixtures were run on 8-16% gels and processed for fluorography. Free [^3H]LPS, [^3H]LPSbulletsCD14, and [^3H]LPSbulletsCD14bulletmAb complexes are indicated. B, purified sCD14 was incubated with mAb MEM-18 (lanes 3-11) or 3C10 (lanes 12-13) in the absence of rLBP (lanes 3-7 and 12) or in the presence of rLBP (lanes 8-11 and 13) at a concentration of 16.7 nM. Increasing concentrations of LPS (0.25 µg/ml for lanes 4 and 8; 1 µg/ml for lanes 5 and 9; 5 µg/ml for lanes 6 and 10; 25 µg/ml for lanes 7, 11, and 13) were used to competitively inhibit binding of MEM-18 to sCD14. MEM-18 (lane 2) and 3C10 (lane 14) were run without sCD14 to demonstrate the specificity of the antibody used for blotting. Protein mixtures were run on 8-16% native gels and processed for Western blot analysis as indicated under ``Materials and Methods.'' Complexes of sCD14bulletLPS and sCD14bulletmAb are indicated.



To confirm and extend this observation, complexes of MEM-18 and sCD14 were first formed. These complexes showed a mobility characteristic of a 250-kDa protein, confirming the efficacy of MEM-18 in the supershift assay (Fig. 6B, lane 3). Addition of increasing doses of LPS to these complexes caused dissociation of the sCD14 from the MEM-18 in a dose-dependent fashion. Moreover, the efficacy of LPS in disassociating sCD14 from MEM-18 was enhanced by rLBP, a protein that catalytically hastens the binding of LPS to sCD14(5) . LPS did not cause disassociation of sCD14 from 3C10 (Fig. 6B), MY4, or 60b (data not shown), confirming that these mAbs do not compete with LPS for binding to sCD14. These results further confirm that MEM-18 and LPS bind sCD14 in a competitive fashion and may thus recognize overlapping sites.

CD14 Has an Amphipathic Domain between Amino Acids 53 and 63

It has been hypothesized (18) that Limulus anti-LPS factor (LALF,(19) , LBP, and bactericidal/permeability-increasing (BPI (20) ) proteins possess amphipathic domains which are involved in binding LPS. Since the hydrophobic moment (µ(21) ) is directly proportional to amphipathicity, we calculated µ throughout CD14 and identified the region having the highest µ. Table 2compares this region to analogous regions in LALF, LBP, and BPI. The region (amino acids 53-63) having the highest µ in CD14 overlaps the site we have identified as being critical for LPS binding. This region was similar to LALF, LBP, and BPI with respect to its overall pattern of alternating hydrophilic and hydrophobic residues. However, the amphipathic domain in CD14 did differ significantly from the other proteins with respect to its net charge.




DISCUSSION

In this report, we provide compelling evidence that the region between amino acids 57 and 64 of sCD14 is essential for proper binding of LPS. Deletion of this region abolished the ability of sCD14 to bind LPS in the presence or absence of rLBP. Furthermore, an epitope recognized by neutralizing mAb MEM-18 was mapped to this region, and we showed that this mAb competes with LPS for binding to sCD14. These data are consistent with our previous finding (8) that localized an LPS binding site to the amino-terminal 152 amino acids of sCD14 and our accompanying paper (16) which demonstrates that LPS protects a region spanning amino acids 57-64 from cleavage by endoproteinase Asp-N protease.

Recently, an LPS binding motif has been proposed for LALF(18) . Identification of the putative binding site was based on the observation that in the crystal structure of LALF, there is an extended amphipathic loop which has sequence similarity to polymixin B, an antibiotic that binds lipid A. We observed that the region of highest amphipathicity in CD14 overlapped our proposed LPS binding domain. Interestingly, the amphipathic domain in CD14 has a net negative charge, distinguishing it from analogous domains in LALF, LBP, and BPI. These data support the theory that amphipathic domains are involved in LPS binding and also imply that amphipathicity may be more critical to LPS binding than net charge. However, more data are required to confirm whether amphipathic domains in LALF, LBP, and BPI truly bind LPS.

Our data also demonstrate the biological consequences of impairing LPS binding to sCD14. sCD14 was inactive in enabling PMN and U373 responses to LPS. These results suggest that binding of LPS to sCD14 is a prerequisite for the biological activity of CD14. This conclusion is consistent with the finding (5) that binding of LPS to sCD14 is temporally correlated with biological activity.

While the above studies have identified a region of sCD14 involved in one function of sCD14 (binding of LPS), additional sites may also play important roles. rLBP catalyzes movement of LPS into sCD14, and recognition of sCD14 by rLBP may involve a separate site. sCD14bulletLPS complexes interact with cells and with high density lipoprotein particles, (^3)suggesting yet another site. Experiments to map these sites are currently in progress.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by United States Public Health Grant AI-30556.

To whom correspondence and reprint requests should be addressed. Tel.: 805-447-3064; Fax: 805-499-9452.

(^1)
The abbreviations used are: LPS, lipopolysaccharide; BPI, bactericidal/permeability-increasing protein; CHO, Chinese hamster ovary; CM, conditioned medium; IL-6, interleukin-6; LALF, Limulus anti-LPS factor; LBP, LPS-binding protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMN, polymorphonuclear leukocyte; r, recombinant; mAb, monoclonal antibody; sCD14, soluble CD14.

(^2)
E. Hailman and S. D. Wright, manuscript in preparation.

(^3)
Wurfel, M., Hailman, E., and Wright, S. D. (1995) J. Exp. Med., in press.


ACKNOWLEDGEMENTS

We thank Dr. Mark Zukowski for critical reading of the manuscript and Dr. Rashid Syed for calculating hydrophobic moments. We also thank Viki Jacobsen for her technical support.


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