THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 18, Issue of June 25, pp. 10867-10871,1989 Printed in U.S.A.

Identification of a Lipid A Binding Site in the Acute Phase Reactant Lipopolysaccharide Binding Protein*

(Received for publication, June 1, 1988, and in revised form, March 1, 1989)

Peter S. Tobias$, Katrin Soldau, and Richard J. Ulevitch From the Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037

Lipopolysaccharide (LPS) binding protein (LBP), a recently discovered 60-kDa acute phase protein, is present in the acute phase serum of many species in- cluding human, rabbits, mice, and rats. Using either highly purified LBP from acute phase rabbit serum or unfractionated acute phase rabbit serum as a source of LBP, we examined the binding of LBP to LPS immo- bilized on plastic microtiter plates and to LPS electro- transferred to nitrocellulose after sodium dodecyl sul- fate-polyacrylamide gel electrophoresis. The presence of LBP bound to LPS was detected with goat anti- rabbit LBP and peroxidase-conjugated rabbit anti- goat IgG. LBP was found to bind to a variety of LPS types from both rough and smooth strains of Gram- negative bacteria, to lipid A, and to the tetraacyl glu- cosamine disaccharide diphosphate precursor IVA, but bound very poorly to the diacyl glucosamine phosphate, lipid X. No binding to 3-deoxyoctulosonic acid was observed. Binding affinities for LPS are near los M-’. The data presented here support the concept that LBP contains a binding site for lipid A.

Numerous studies have described the effect of extracellular proteins on the hydrodynamic and endotoxic properties of bacterial lipopolysaccharides (LPS)’ (1-7). However, there are no studies documenting a protein that has a specific binding site for lipid A, the biologically active structure of the LPS molecule. Identification and characterization of lipid A binding sites in proteins could provide much needed infor- mation about LPS-protein interactions that lead to many of the profound biological effects of LPS. Among the potential candidates for such studies is an acute phase protein that we

*This work was supported in part by Grant AI 15136 from the National Institutes of Health and by Contract DAMD17-88-C-8017 from the United States Army Medical Research and Development Command. This is Publication 4664 IMM from the Department of Immunology at the Research Institute of Scripps Clinic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence and reprint requests should be ad- dressed Dept. of Immunology, IMM 12, Research Inst. of Scripps

8215. Clinic, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-

The abbreviations used are: LPS, lipopolysaccharide; LBP, LPS binding protein; APRS, acute phase rabbit serum; SDS-PAGE, so- dium dodecyl sulfate-polyacrylamide gel electrophoresis; Re595-LPS, LPS from S. rninnesota Re595; 55 lipid A, lipid A from E. coli 55; BSA, bovine serum albumin; DADG, 0-[2-amino-2-deoxy-I-(3-hy- droxytetradecanoyl)-~-~-glucopyranosyl]-(l-6)-2-amino-2-deoxy- ~-(3-hydroxytetradecanoyl)-a-~-glucopyranose 1,4’-biphosphate; KDO, 3-deoxyoctulosonic acid HEPES, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid TBS, Tris-buffered saline.

have discovered, purified, characterized, and named lipopoly- saccharide binding protein (LBP) (8). LBP is a 60-kDa gly- coprotein present in acute phase rabbit serum at 10-50 pg/ml and is estimated to be at less than 100 ng/ml in normal rabbit serum. To date, the majority of our studies with LBP have been performed with LPS isolated from the Re595 mutant of Salmonella minnesota; interactions of LBP with lipid A and the influence of core or 0-antigen polysaccharides have not been evaluated.

To address the binding of LBP with LPS we have developed a solid phase binding assay incorporating LPS immobilized on microtiter plates. Using this assay, we find that LBP binds avidly to many different LPS isolates from rough and smooth strains of Gram-negative organisms, to lipid A, and to lipid A precursor IVA (9). In contrast, binding to lipid X (9) and other partial lipid A structures is reduced. Furthermore, interactions between LBP and a number of other negatively charged polymeric substances including DNA, RNA, heparin, phos- pholipids, and lipoteichoic acid are poor to nonobservable. Thus, the data presented here support the concept that there is a binding site in LBP with a high degree of specificity for the lipid A portion of bacterial lipopolysaccharides.

EXPERIMENTAL PROCEDURES

Materials-Normal and acute phase rabbit sera, LBP, and goat antisera to LBP were prepared as described previously (8), as was S. rninnesota Re595 LPS (12). Bovine serum albumin (BSA) conjugates of Re595-LPS, Escherichia coli 55 LPS, precursor IVA, and 0-[2- amino-2-deoxy-N2-(3-hydroxytetradecanoyl)-~-~-glucopyranosyl]- (l-6)-2-amino-2-deoxy-~-(3-hydroxytetradecanoyl)-a-~-glucopyr- anose 1,4’-bisphosphate (DADG) prepared by a published method (10) were obtained from Dr. T. Kirkland (University of California, San Diego, CA). Salmonella typhimurium Rc LPS and the desacyl form derived from it (11) were provided by Dr. R. Munford (Univer- sity of Texas, Dallas, TX). Commercial sources were used to obtain other lipopolysaccharides (List Biologicals, Campbell, CA), synthetic 55 lipid A (LA-15-PP; Daichi Pure Chemicals, Tokyo), lipid X (Lip- idex, Middleton, WI), polymyxin B (Calbiochem), horseradish per- oxidase coupled to rabbit anti-goat IgG (Cooper Biomedical, Inc., Malvern, PA), 4-chloro-1-naphthol (Bio-Rad), 0-phenylenediamine, lipoteichoic acid, salmon testes DNA, yeast transfer RNA, and AMP (Sigma), dextran sulfate (prepared from 500-kDa dextran; Pharmacia, Uppsala, Sweden), heparin (Liquaemin sodium, 140-165 units/mg; Organon Diagnostics, West Orange, NJ), and dimyristoyl phospha- tidic acid (Avanti Polar Lipids, Pelham, AL). Microtiter plates were Dynatech No. 001-010-2201. A mixture of 3-deoxyoctulosonic acid (KDO) mono- and disaccharides were prepared from Re595-LPS by mild acid hydrolysis (19). A portion of the hydrolyzed Re595-LPS was biosynthetically tritiated using [3H]acetate (8), which should have resulted in label principally incorporated into the fatty acids of lipid A. No tritium was found in the KDO preparation, which we interpreted as an absence of LPS and lipid A.

Microtiter Plate-immobilized LPS-Microtiter plates were coated with LPS by incubating 100 pllwell of 30 pg/ml Re595-LPS in 0.1 M Na2C03, 20 mM EDTA, pH 9.6, for 3 h at 37 “C. The LPS solution was flicked out, and the plates were rinsed thoroughly under running water and air-dried overnight. The binding properties of the dried,

10867

10868 LPS Binding Protein Has a Lipid A Binding Site

LPS-coated plates remained unchanged for up to 8 weeks. Excess binding sites were then blocked with 200 pl/well of 10 mg/ml BSA in 50 mM HEPES, 0.15 M NaC1, pH 7.4 (HS buffer) for 30 min at 37°C with agitation, after which the BSA solution was flicked out. LBP samples, APRS, or normal rabbit serum were added in a total volume of 100 pllwell diluted in 1 mg/ml BSA in HS buffer. Binding of LBP was allowed to occur for 3 h at 37 "C. The plate was then rinsed three times with 200 Gl/well of 1 mg/ml BSA in HS buffer with agitation over a 10-min interval. Goat anti-LBP antiserum, 100 pl/well, diluted 2000 X in 1 mg/ml BSA in HS buffer was then incubated in the wells for 2 h at 37 "C. The anti-LBP antiserum was then rinsed out as above and replaced for 1 h at room temperature with 100 pl/well of peroxidase-conjugated rabbit anti-goat IgG diluted 2500 X in 1 mg/ ml BSA. Finally, 100 pl/well o-phenylenediamine solution (4 mM o- phenylenediamine, 2.3 mM H~02, 50 mM citric acid, 133 mM Na2HP04, pH 5.0) was added until sufficient color had developed (5- 15 rnin). The reaction was stopped by addition of 100 pl/well of 5 M HzS04, and the absorbance at 490 nm of each well was quantitated in a microtiter plate reader. Mean absorbances and standard errors of the means were calculated from four to eight replicates of each well. Inhibition of LBP binding to the plates was studied by premixing the compound under study at the desired concentration with 1 pg/ml purified LBP for 10 min at 37 "C before adding the mixture to the microtiter plate. We found that there were significant differences in the utility of different brands of microtiter plates for these experi- ments. Most brands avidly bound LBP, but not LBP . LPS complexes, without any LPS bound to the plate. In one set of experiments, polymyxin B at 10 pg/ml with 1 mg/ml BSA in HS buffer was incubated in the LPS-coated, BSA-blocked wells for 30 min at room temperature. Before addition of LBP to the wells, the polymyxin B solution was removed.

Electroblotted LPS-Duplicate samples of 2 p g of each LPS were electrophoresed in a 15% acrylamide gel containing SDS (13). Sub- sequently, one set of samples was silver-stained (14), and one set was electrotransferred to a sheet of nitrocellulose (15, 16). Excess mem- brane binding sites were blocked by soaking the membranes in 30 mg/ml BSA, 50 mM Tris, 0.2 M NaC1, pH 7.4 (TBS), for 15 min at 37 "C. Membranes were then soaked overnight in approximately 1 ml/cmz membrane area of APRS with gentle stirring at room tem- perature to permit LBP binding. The membranes were then washed for 5 min with each of three changes of TBS before incubation in rat anti-LBP antiserum diluted 1000 X in TBS with 1 mg/ml BSA for 3 h. After rinsing as described above, the membranes were incubated in a 1000 X dilution of peroxidase-conjugated goat anti-rat IgG in TBS with 1 mg/ml BSA for 1 h. Again, after extensive rinsing, the membranes were stained with hydrogen peroxide and 4-chloro-l- naphthol using procedures described by the supplier.

RESULTS

Binding of LBP to LPS-coated Surface-The specificity and dependence of the microtiter plate system on LBP binding to LPS was first established using purified LBP. When any one of the components of the assay was omitted, the color devel- oped in the microtiter well was less than 0.15 absorbance unit as compared with more than 0.8 absorbance unit when all components were present. Additionally, heat-denatured (9O"C, 20 min) LBP yielded less than 0.1 absorbance unit when it was used in place of active LBP. When polymyxin B was incubated in the LPS-coated wells prior to addition of the LBP, the absorbance was reduced 47%. Fig. 1 shows the curves obtained when serial dilutions of purified LBP, APRS, and normal rabbit serum were used as LBP sources. These data show that color development in the wells is due to the interaction of LBP with immobilized LPS and that the ab- sorbance is dependent on the concentration of active LBP available to bind to the LPS. From the data of Fig. 1, the LBP activity in the APRS sample was more than 300 x the activity in the normal rabbit serum sample and equivalent to approximately 48 pg/ml LBP by comparison with the purified LBP used.

Other studies not shown were performed to establish the time- and dose-dependence of the various steps in the assay. In summary, these results showed (i) that concentrations of

-2 -1 0 1 2 Serum (%) or LBP (pg/ml)

FIG. 1. Concentration dependence of LBP binding to LPS- coated microtiter plate. See "Experimental Procedures" for details. W, dilution of 1 pg/ml purified LBP; 0, dilution of APRS; 0, dilution of normal rabbit serum.

Re595-LPS higher than 30 pg/ml would not lead to more LPS bound to the microtiter plate and that binding of LPS to the well was at a plateau after 2 h, (ii) that the binding of LBP to the plates is complete after 3 h at 37 "C, and (iii) that the dilutions of antisera used and the time periods of their incu- bation in the wells were optimal.

Binding Specificity of LBP-To study the binding specific- ity of LBP, we examined a battery of test substances that included a variety of LPS preparations from rough and smooth organisms, partial lipid A structures, and substances that are not structurally similar to LPS but are charged polymers or amphipathic compounds. Compounds used in this series of experiments were selected to explore the roles of three aspects of LPS structure: namely, (i) whether the structural variation in the core and/or 0-antigen among dif- ferent LPS isolates would influence the binding of LBP, (ii) whether a substructure of LPS (e.g. lipid A) could be identified for which LBP had affinity, and (iii) whether negatively charged polymers and aggregates having little basic structural similarity to LPS would bind to LBP. Examples of the results obtained are shown in Fig. 2, A-C, and a complete tabulation is provided in Table I. The numerical results presented in Table I are the concentrations necessary to reduce the binding of 1 pg/ml LBP to the plate by 50%, i.e. EDSO. In many instances, the structure of the inhibitor is either not fully defined or the inhibitor is heterogeneous preventing a calcu- lation of a molar concentration for ED50. A qualitative analy- sis of these data indicates that an intact lipid A moiety is essential for the strongest inhibition. Thus, as the lipid A moiety is dissected in the series lipid A, IVA, DADG, and lipid X, the ED50 values steadily increased. In addition, when S. typhimurium Rc LPS is enzymatically deacylated to a product analogous to IVA, the ED50 value also increases. By contrast, removing the 0-antigen and the core oligosaccharide in the LPS from S. minnesota wild type, R60, R5, and Re595 has no systematic effect and certainly does not diminish the affinity for LBP for these molecules. The KDO moiety of Re595-LPS, presented as a mixture of the KDO mono- and disaccharides, was completely ineffective as an inhibitor. Large negatively charged molecules such as DNA, RNA, and heparin were inhibitory only at high concentrations, whereas lipoteichoic acid and dextran sulfate were fairly good ligands for LBP. AMP, as a representative monomer of DNA and RNA, was also ineffective.

Intentionally complexing 55 lipid A and Re595 to BSA had little effect on their ability to bind to LBP. Qualitatively this means either that the LBP-LPS interaction is much stronger

LPS Binding Protein Has a Lipid A Binding Site 10869

A TABLE I 100 Binding parameters

Test substance EDso 0 In C

nRlm1 nM Re595 sugars >200,000 >840,000" AMP >1,000,000 >2,800,000 Lipid X 2,800 3,900

2-

s Dimyristoyl phosphatidic acid 2,500 3,900

; 50

DADG. BSA 540 5'70 IVA. BSA 190 135 S. typhimurium Rc (desacyl) 266 78 55 lipid A (BSA) '79 40 55 lipid A (synthetic) 40 20 55 lipid (isolated) 79 40 Re595. BSA 26 12 Re595 13 6

0 -9 -a -7 -6 -5

Log [Inhibitor] (M)

B E. coli D31m4 (Re) 13 6 100 S. typhimurium Rc 22 7

S. minnesota R5 (Rc) 67 0 S. minnesota R60 (Ra) 12 C S. minnesota wild type 69 VI

n E. coli 01 11:B4 50 a 50 E. coli K12 mm294 18

E. coli 055.B5 48 5 S. marscesens 25

S. typhimurium 107 Vibrio cholerae 50 Klebsiella pneumoniae 53 RNA >300,000 >850,000b DNA >300,000 >850,000b Heparin 21,000 Lipoteichoic acid 480

C - Dextran sulfate 73 Molarity of the constituent 3-deoxyoctulosonic acid. Molarity of the constituent monomers.

In

0

Log [Inhibitor] olg/ml)

100 - - - - -

0 VI C

VI

0

A B C O E F G H SDS gel A B C O E F G H 1)

- j: -

blot n g 50-

s " -. - " . ."". .- - - - -

o 1 I I I I I I I

-4 -3 -2 -1 0 1 2 3 Log [Inhibitor] (pg/rnl)

$i

FIG. 2. Inhibition of LBP binding to LPS-coated microtiter p1ate.y Axis scale refers to color developed in the absence of inhibitor (=lOO%) or in the absence of LBP ( = O % ) . A, inhibitors are lipid X w (+), DADG.BSA (O), 55 lipid A (B), Re595 (O), and Re595.BSA

stoichiometry in the LpS.LBp complex with Kd = M-* and FIG. 3. Detection Of LPs after polyacrylamide gel electro- (0). Inset, data are for Re595, the line is calculated for one-to-one

[LBp] = 1.25 x M. B, inhibitors are E. coli K12 mm294 (O), S. phoresis using LBP. h i t panel, silver-stained SDS gel; right Panel, minnesota wild type (e), Serratia marSc-Sens (B), and s. typhimurium LBP stain of LPS blotted onto nitrocellulose membrane from SDS (0). C, inhibitors are DNA (0), heparin (0), lipoteichoic acid (B), gel. A, s. minnesota Re595 (Re); & s. minnesota R5 (Rc); CY s. and dextran sulfate (0). minnesota wild type; D, E. coli Olll:B4; E, K. pneumoniae; F, S.

than any BSA-LPS interaction or that BSA and LBP bind to different parts of the LPS in a noninteractive manner. cellulose following SDS-PAGE is shown in Fig. 3. Comparison

LBP ~ i d i ~ to ~ p s Subunits Revealed by SDS-pAGE-It of the LBP-stained electrotransfer and the duplicate silver-

with respect to molecular compos~t~on, each isolate containing that LBP binds to both the smaller lipid A-rich molecules of a of molecules with markedly different lipid A/ LPS as well as to molecules of LPS that have large 0-antigen carbohydrate ratios (14, 18). The microtiter plate binding polysaccharides attached to lipid A* assay does not provide any information about the selectivity of LBP binding to the heterogeneous LPS subunits that are present in a single type of LPS isolate. Because SDS-PAGE A recent paper (25) from this laboratory lists a number of resolves the various molecular species of LPS within a single known and suspected LPS binding proteins that may be LPS preparation (14), we utilized this technique to provide homologues of LBP. For none of these, however, is there data additional information about the binding specificity of LBP. which quantitatively describe the protein's binding specificity. The ability of LBP to stain LPS electrotransferred to nitro- The data presented here support the concept that LBP has a

1 !

typhimurium; C, V. cholerae; H , lipid A from E. coli K12 D31m4.

is well appreciated that most LPS isolates are heterogeneous stained gel reveals VeV similar Patterns. These data show

DISCUSSION

10870 LPS Binding Protein Has a Lipid A Binding Site

binding site for the lipid A moiety of lipopolysaccharides. We did observe that Re forms of LPS were somewhat better bound than lipid A (see Table I), and this could indicate some favorable interaction between LBP and the KDO residues attached to lipid A in the Re595-LPS. However, KDO itself was not inhibitory, and thus the difference probably reflects only a difference in aggregation between Re595-LPS and lipid A. The presence of the carbohydrate core and 0-antigen has no obvious effect on the LBP binding reaction. Thus, for the present, we hypothesize only that LBP has a binding site which recognizes lipid A. Importantly, LBP is capable of binding the lipid A moiety when LPS is in a variety of different physical environments including immobilized on microtiter plates, electrotransferred to nitrocellulose mem- branes after SDS-PAGE, in defined buffers, and in serum (8).

The compounds selected for study were chosen with three objects in mind (i) to explore the minimal structural require- ments for binding to LPS; (ii) to explore whether LBP had some general affinity for large negatively charged molecules that might resemble an LPS micelle simply by virtue of the negative charge; and (iii) to explore whether LBP exhibited selectivity in binding LPS isolated from different Gram- negative bacteria. The compounds whose structures are known and whose ED50 values can be expressed in molar terms are ranked in the first part of Table I.

The effectiveness of these molecules as inhibitors correlates well with their structural integrity as LPS molecules. The most effective are the Re forms of LPS and the Rc form from S. typhimurium, followed by lipid A, compound IVA, and the desacyl form of S. typhimurium, none of which has fatty acyl groups on the hydroxymyristoyl residues, DADG, which has no ester-linked fatty acids, and lipid X and dimyristoyl phos- phatidic acid as the least effective molecules with any struc- tural similarity to LPS. These data form a clear correlation between an intact structure and the ED50 value. However, lipid A and LPS, and presumably DADG and lipid X, are highly aggregated (20, 21) with documented effects of aggre- gation on the biological properties (17). Unfortunately, there is no way at present to quantitatively separate variability in aggregation state and the changes in ED509 both of which derive from structural changes in the molecules being studied. Keeping in mind the changes in structure among this group of molecules and making the reasonable assumption that more fatty acid tails result in a higher degree of aggregation, one must postulate either that LBP has a binding site that rec- ognizes intact lipid A or that more highly aggregated particles are better ligands for LBP.

Three arguments suggest that the latter hypothesis, that the more highly aggregated LPS particles are better LBP ligands, is incorrect. The first argument centers on the stoi- chiometry of the LBP-LPS reaction. In the inhibition exper- iments, LBP was used at 1 pg/ml or 16 nM. The ED50 for the intact LPS are 6-7 nM, suggesting a one-to-one molecular stoichiometry in the LBP.LPS complex. It is difficult to reconcile such a stoichiometry with LBP binding to a large aggregate of LPS. The second argument is that other large negatively charged molecules, e.g. RNA, DNA, and heparin, are not particularly good ligands for LBP. The fact that lipoteichoic acid and dextran sulfate are fairly good ligands may explain why Bio-Rex 70, a carboxylated acrylic ion exchange resin (Bio-Rad), is so useful in the purification of LBP (8). The third argument is that the smooth form isolates of LPS tested are all excellent ligands for LBP. Given the structural variation among this group, it is difficult to imagine that LBP would bind to large aggregates of these molecules with such uniformity. Thus, the data presented here are all

consistent with the view that LBP requires, a t a minimum, an intact lipid A structure for optimal binding.

As noted above, the ED50 values for the most avidly bound LPS suggest a 1:l molecular stoichiometry of LPS-LBP bind- ing. The inset to Fig. 2A shows an excellent agreement be- tween the data for Re595-LBP binding and a calculated curve based on a one-to-one stoichiometry, KO = lo-' M and [LBP]

There are several consequences of LBP binding to LPS that should be noted. From the avidity and stoichiometry of the binding, our estimate that LBP is present in serum at roughly 10-50 pg/ml after an acute phase response, and the data of Table I, LBP would complex more than 99.9% of 1.5- 2 pg/ml of any of the S form LPS present in serum in the absence of any other interaction for the LPS. The only higher affinity interaction in serum for LPS of which we are aware is that with high density lipoproteins because high density lipoprotein. LPS complexes do form even in acute phase serum. Also of consequence is the fact that LPS bound to LBP is apparently monomeric LPS, and a variety of the endotoxic activities of LPS are known to depend on the physical state of LPS (17, 22, 23). We have documented the fact that Re595-LPS bound to LBP binds to high density lipoproteins more slowly than uncomplexed Re595-LPS (2). Others (26) have implicated an acute phase protein in cata- lyzing the binding of E. coli 0113 LPS to lipoprotein, although the identity of this factor has yet to be determined. LBP is a candidate for this latter effect, and studies are currently in progress to evaluate this possibility.

Other than lipid A directed antibodies, there are only a few lipid A binding proteins known (25). For one such protein, the neutrophil LPS acyloxyhydrolase, a K , of 6 X M has been reported (11). Whether the ability of these proteins to bind lipid A derives from a conserved binding site is difficult to evaluate a t present, although we have observed that the amino-terminal sequences of LBP and bactericidal/permea- bility-increasing protein (24) show significant sequence simi- larity. Further study of these proteins should reveal the struc- tural features required of a lipid A binding site.

= 1.25 X lo-' M.

1.

2.

3.

4.

5.

6. 7.

8.

9.

10.

11.

12.

13. 14.

15.

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