Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. I. Detection of lipopolysaccharide-binding sites on splenocytes and splenocyte subpopulations

Experiments have been carried out using a unique radio-iodinated, disulfide-reducible, photoactivatable LPS derivative (ASD-LPS) to detect specific LPS-binding proteins on murine splenocytes. Fractionation of LPS-photo-cross-linked, reduced, and solubilized splenocyte extracts on two-dimensional polyacrylamide gels has allowed the identification of an 80-kDa LPS-binding protein with approximate pI of 6.5. This LPS-binding protein is present on partially purified populations of splenic B lymphocytes, T lymphocytes, and macrophages. It is also the dominant LPS-binding protein on the murine 70Z/3 B cell line and the YAC-1 and EL4 T cell lines but is not detectable on the undifferentiated murine Sp2/0 myeloma cell line. Of potential importance is the fact that the 80-kDa protein appears to be indistinguishable when photolabeled extracts of splenocytes from the C3HeB/FeJ (lpsn) and LPS-nonresponder C3H/HeJ (lpsd) mice are compared.     

Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.     

Isolation of a lipopolysaccharide (LPS)-resistant mutant, with defective LPS binding, of cultured macrophage-like cells.

Lipopolysaccharide (LPS)-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line, J774.1. Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without any morphological changes. Assay of 125I-LPS binding to the cell monolayers revealed that one of these LPS-resistant mutants (LR-9) was strikingly defective in LPS-binding activity. Scatchard plot showed that LR-9 cells lacked the high affinity binding sites which were present in J774.1. The high affinity binding was inhibited by addition of excess unlabeled LPS, lipid A, lipid IVA (tetraacyl-beta(1'-6)-linked D-glucosamine disaccharide-1,4'-bisphosphate), and lipid X (2,3-diacylglucosamine 1-phosphate) and sensitive to proteinase K. LPS enhanced O2- generation and the release of arachidonic acid in J774.1 cells but not in LR-9 cells. Other stimulants such as zymosan and 12-O-tetradecanoylphorbol 13-acetate, however, induced the release of arachidonic acid in LR-9 cells as well as in J774.1 cells. LPS-photocross-linked assay allowed the identification of 65- and 55-kDa LPS-binding proteins in the membrane fraction of J774.1 cells. Both of the bands were not detectable in that of LR-9 cells and disappeared by competing with unlabeled LPS or lipid X. These results show that one or both of the two LPS-binding proteins might relate to the specific membrane receptor for LPS.     

Endotoxin-neutralizing antimicrobial proteins of the human placenta

Microbial colonization and infection of placental tissues often lead to adverse pregnancy outcomes such as preterm birth, a leading cause of neonatal morbidity and mortality. The fetal membranes of the placenta, a physical and active barrier to microbial invasion, encapsulate the fetus and secure its intrauterine environment. To examine the innate defense system of the human placenta, antimicrobial peptides were isolated from the fetal membranes of human placenta and characterized biochemically. Two salt-resistant antimicrobial host proteins were purified to homogeneity using heparin-affinity and reversed-phase HPLC. Characterization of these proteins revealed that they are identical to histones H2A and H2B. Histones H2A and H2B showed dose-dependent inhibition of the endotoxin activity of LPS and inhibited this activity by binding to and therefore blocking both the core and lipid A moieties of LPS. Consistent with a role for histones in the establishment of placental innate defense, histones H2A and H2B were highly expressed in the cytoplasm of syncytiotrophoblasts and amnion cells, where the histone proteins were localized mainly to the epithelial surface. Furthermore, culturing of amnion-derived WISH cells led to the constitutive release of histone H2B, and histones H2A and H2B contribute to bactericidal activity of amniotic fluid. Our studies suggest that histones H2A and H2B may endow the epithelium of the placenta with an antimicrobial and endotoxin-neutralizing barrier against microorganisms that invade this immune-privileged site.     

Peptidoglycan and lipopolysaccharide bind to the same binding site on lymphocytes

Bacterial cell wall peptidoglycan (PGN) and lipopolysaccharide (LPS), which are both macrophage activators and polyclonal B cell mitogens, were shown to bind to the same dominant 70-kDa 6.5 pI protein on the surface of mouse B lymphocytes. This conclusion was supported by the following results: (a) the PGN- and LPS-binding proteins co-migrated following photoaffinity cross-linking and two-dimensional polyacrylamide gel electrophoresis; (b) cross-linking of PGN to this 70-kDa protein was competitively inhibited by LPS (IC50 = 7.3 microM), LPS from a deep rough mutant (IC50 = 6.9 microM), and lipid A (IC50 = 18-72 microM); (c) cross-linking of LPS to this 70-kDa protein was competitively inhibited by polymeric soluble PGN (IC50 = 0.09 microM) and sonicated high Mr PGN (IC50 = 0.6 microM); (d) cross-linking of both PGN and LPS to this 70-kDa protein was also competitively inhibited by dextran sulfate (IC50 = 115-124 microM); (e) cross-linking of both PGN and LPS to this 70-kDa protein was inhibited by a (GlcNAc)2-specific lectin; and (f) peptide maps of the 70-kDa proteins digested with chymotrypsin, subtilisin, staphylococcal protease V, or papain were identical for PGN- and LPS-binding proteins and unique for each enzyme. Based on competitive inhibition experiments, binding of PGN to the 70-kDa protein was 20-1200 times stronger than the binding of LPS or lipid A on a per mol basis. However, when aggregated micellar structures of LPS or lipid A were considered, the avidities of LPS and PGN binding were similar. These results demonstrate binding of PGN and LPS to the same 70-kDa protein on lymphocytes and suggest that the binding is specific for the (GlcNAc-MurNAc)n backbone of PGN and the (GlcNAc)2 part of lipid A.     

The proteasome as a lipopolysaccharide-binding protein in macrophages: differential effects of proteasome inhibition on lipopolysaccharide-induced signaling events

We have developed a novel LPS probe using a highly purified and homogenous preparation of [(3)H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The alpha-subunit (PSMA1 C2, 29.5 kDa) and the beta-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome's chymotrypsin activity as well as macrophage TNF-alpha secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.     

High-throughput characterization of lipopolysaccharide-binding proteins using mass spectrometry

Lipopolysaccharide (LPS)-binding proteins interact with LPS in human serum and mediate various immune responses. We describe a high-throughput LPS-binding protein profiling platform for discovering unknown LPS-binding proteins and potential inflammatory mediators. As a pull-down method, the LPS molecules were immobilized onto epoxy beads and then directly incubated with human serum to screen LPS-binding proteins. Through the "untargeted" mass spectrometric approach, potential LPS-binding proteins which elicit various immune responses in human serum were identified by a highly sensitive LTQ Orbitrap Hybrid Fourier Transform Mass Spectrometer (LTQ Orbitrap FT MS). Therefore, this mass spectrometry (MS)-based profiling method is straightforward for screening unknown LPS-binding proteins and provides physiologically relevant binding partners in human serum.     

The functional and structural properties of MD-2 required for lipopolysaccharide binding are absent in MD-1

MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4. In vivo studies revealed that MD-1 and MD-2 have roles in responses to LPS. Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown. In this study, we compared the LPS-binding functions of MD-1 and MD-2. LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2. In contrast, binding was not observed for RP105/MD-1-transfected cells. When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS. In contrast, preparations of MD-1 did not contain LPS. When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LPS binding disappeared. Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions. An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site. We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids. A mutation at Phe(191) or Phe(121) strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring. The Phe residues are present in MD-2 and absent in MD-1. Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.     

Characterization of Brucella abortus lipopolysaccharide macrodomains as mega rafts

The lipopolysaccharides (LPS) of intracellular Proteobacteria such as Brucella, Chlamydia, Legionella and Rickettsia, have properties distinct from enterobacterial LPSs. These properties include deficient LPS induction of host cell activation, low endotoxicity and resistance to macrophage degradation. Together these constitute key virulence mechanisms for intracellular survival and replication. We previously demonstrated that B. abortus LPS captured by macrophages was recycled back to the plasma membrane where it was found associated with macrodomains. Furthermore, this LPS interferes with the MHC class II (MHC-II) presentation of peptides to specific T cell hybridomas. Here, we characterized the Brucella LPS macrodomains by microscopy and biochemistry approaches. We show for the first time that LPS macrodomains act as detergent resistant membranes (DRMs), segregating several lipid-raft components, LPS-binding proteins and MHC-II molecules. Brucella LPS macrodomains remain intact for several months in macrophages and are resistant to the disruptive effects of methyl beta-cyclodextrin. Fluorescent anisotropy measurements show that B. abortus LPS is responsible for the formation of rigid surface membrane complexes. In addition, relocalization of MHC-II molecules is observed in these structures. The effects of B. abortus LPS on membrane properties could be responsible for pathogenic effects such as the inhibition of MHC-II-dependent antigen presentation.     

Functional activity of MD-2 polymorphic variant is significantly different in soluble and TLR4-bound forms: decreased endotoxin binding by G56R MD-2 and its rescue by TLR4 ectodomain

MD-2 is an essential component of endotoxin (LPS) sensing, binding LPS independently and when bound to the ectodomain of the membrane receptor TLR4. Natural variation of proteins involved in the LPS-recognition cascade such as the LPS-binding protein, CD14, and TLR4, as well as proteins involved in intracellular signaling downstream of LPS binding, affect the cellular response to endotoxin and host defense against bacterial infections. We now describe the functional properties of two nonsynonymous coding polymorphisms of MD-2, G56R and P157S, documented in HapMap. As predicted from the MD-2 structure, the P157S mutation had little or no effect on MD-2 function. In contrast, the G56R mutation, located close to the LPS-binding pocket, significantly decreased cellular responsiveness to LPS. Soluble G56R MD-2 showed markedly reduced LPS binding that was to a large degree rescued by TLR4 coexpression or presence of TLR4 ectodomain. Thus, cells that express TLR4 without MD-2 and whose response to LPS depends on ectopically produced MD-2 were most affected by expression of the G56R variant of MD-2. Coexpression of wild-type and G56R MD-2 yielded an intermediate phenotype with responses to LPS diminished to a greater extent than that resulting from expression of the D299G TLR4 polymorphic variant.     

Heat shock protein 60: specific binding of lipopolysaccharide

Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [(3)H]LPS. [(3)H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335-366 of HSP60. Clone 4B9/89 also strongly inhibited [(3)H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331-380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351-370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354-365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.     

Identification and characterization of heparan sulfate-binding proteins from human lung carcinoma cells

The heparan sulfate proteoglycan/heparin-binding proteins of the human lung carcinoma cell line LX-1 have been identified, partially purified, and characterized. Analysis of the binding of [3H]heparin to membranes isolated from LX-1 cells indicated the presence of two classes of binding sites, with Kd values of approximately 2 x 10(-10) and 4 x 10(-8) M and corresponding Bmax values of 1 x 10(5) and 2 x 10(7) binding sites/cell. Binding was also observed with isolated heparan sulfate chains and with intact heparan sulfate proteoglycan isolated from two different cell types. With each ligand, binding was inhibited by addition of unlabeled heparin. The binding proteins were extracted from LX-1 cell membranes in detergent solution, and two size classes of binding proteins were identified by overlaying transblots of electrophoretically separated proteins with radioactive ligands. These two classes of binding proteins were shown to contain doublets with estimated molecular masses of approximately 16 kDa (HSBP1A and HSBP1B) and approximately 32 kDa (HSBP2A and HSBP2B). The proteins were partially purified by heparin-Sepharose chromatography and shown to bind heparin and heparan sulfate proteoglycan. By amino acid composition, N-terminal amino acid sequence, and reactivity with antibody, HSBP1A was shown to be very similar to histone 2B; HSBP1B may also be related to histone 2A. HSBP2A and HSBP2B, however, did not react with antibodies to the major histones and had compositions different from one another and from HSBP1.     

The histone H3/H4.N1 complex supplemented with histone H2A-H2B dimers and DNA topoisomerase I forms nucleosomes on circular DNA under physiological conditions

We have fractionated the whole cell extract of Xenopus oocytes (oocyte S-150) and isolated the endogenous components required for DNA supercoiling and nucleosome formation. Histone H2B and the three oocyte-specific H2A proteins were purified as free histones. Histones H3 and H4 were purified 100-fold in a complex with the acidic protein N1. In the presence of DNA topoisomerase I or II, histone H3/H4.N1 complexes supercoil DNA in a reaction that is inhibited by Mg2+, and this inhibition is relieved by NTPs. The supercoiling reaction induced by H3/H4.N1 complexes is enhanced by free histone H2A-H2B dimers, which by themselves do not supercoil DNA. Nuclease digestions and protein analyses indicate that H3/H4.N1 complexes form subnucleosomal particles containing histones H3 and H4. Nucleosomes containing 146-base pair DNA and the four histones are formed when histones H2A and H2B complement the reaction.     

Noncovalent interactions of poly(adenosine diphosphate ribose) with histones.

Covalent linkage of ADP-ribose polymers to proteins is generally considered essential for the posttranslational modification of protein function by poly(ADP-ribosyl)ation. Here we demonstrate an alternative way by which ADP-ribose polymers may modify protein function. Using a highly stringent binding assay in combination with DNA sequencing gels, we found that ADP-ribose polymers bind noncovalently to a specific group of chromatin proteins, i.e., histones H1, H2A, H2B, H3, and H4 and protamine. This binding resisted strong acids, chaotropes, detergents, and high salt concentrations but was readily reversible by DNA. When the interactions of variously sized linear and branched polymer molecules with individual histone species were tested, the hierarchies of binding were branched polymers greater than long, linear polymers greater than short, linear polymers and H1 greater than H2A greater than H2B = H3 greater than H4. For histone H1, the target of polymer binding was the carboxy-terminal domain, which is also the domain most effective in inducing higher order structure of chromatin. Thus, noncovalent interactions may be involved in the modification of histone functions in chromatin.     

Localization of testis-variant histones in rat testis chromatin.

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.     

Effect of histone acetylation on the formation and removal of B(a)P chromatin adducts.

The modification of core histone proteins in mouse 10T1/2 cells and human lung epitheloid (A549) cells by B(a)PDE in vivo and in vitro was found to be similar. Only histones H2A and H3 were extensively modified. Also other proteins, possibly A24 protein and the minor histone H1 species seem to be binding relatively high levels of this ultimate carcinogen. Butyrate treatment which causes hyperacetylation of the core histones, did not change the specificity of B(a)PDE binding to core histones, nor did it affect the initial level of DNA modification. The acetylated species of histone H3 were all accessible to B(a)PDE, suggesting that these epsilon-amino-groups of the lysine residues are not the targets of the B(a)PDE. The rate of removal of B(a)P-DNA adducts was not affected by butyrate treatment in either normal human or XP fibroblasts. Furthermore the B(a)P-core histones were not preferentially removed from normal human fibroblast chromatin during a 24 h post-treatment incubation.     

Inhibition of Adherence of Actinobacillus pleuropneumoniae to Porcine Respiratory Tract Cells by Monoclonal Antibodies Directed Against LPS and Partial Characterization of the LPS Receptors

Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia. We have previously identified the lipopolysaccharides (LPS) as the major adhesin of A. pleuropneumoniae involved in adherence to porcine respiratory tract cells. In the present study, adherence of A. pleuropneumoniae to porcine tracheal frozen sections was inhibited by homologous monovalent Fab fragments produced from monoclonal antibodies 5.1 G8F10 and 102-G02 directed, respectively, against the A. pleuropneumoniae serotype 1 or serotype 2 O-antigens. These results confirm the important role played by LPS in adherence of A. pleuropneumoniae and suggest that these adhesins might represent good vaccine candidates. We also investigated the presence of A. pleuropneumoniae receptors in tracheal cell preparations from piglets of four different breeds. Using Far-Western binding assays, we identified proteins recognized by whole cells of A. pleuropneumoniae reference strains for serotype 1 and 2, and local isolates belonging to the same serotypes, and also recognized by extracted LPS from both reference strains. We confirmed the proteinaceous nature of these LPS-binding molecules by their staining with Coomassie brilliant blue, sensitivity to proteinase K digestion, resistance to sodium m-periodate oxidation, and their inability to stain with glycoprotein-specific reagents. Four low-molecular-mass bands (14–17 kDa) seemed to correspond to histones. We also identified proteins at Mr 38,500 that could represent putative receptors for A. pleuropneumoniae LPS in swine respiratory tract cells. Received: 16 April 1999 / Accepted: 1 July 1999     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

Glycosylation, ADP-ribosylation, and methylation of Tetrahymena histones

We have examined some of the postsynthetic modifications that occur in macronuclear histones from Tetrahymena thermophila. When purified macronuclei are incubated with [32P]NAD+, histones H1, H2A, H2B, and H3 are ADP-ribosylated. Furthermore, histones H1, H2A, H2B, and H3 contain fucose and mannose residues as evidenced by the incorporation of [3H]fucose and by the specific binding to these proteins of gorse seed lectin and concanavalin A. Finally, our studies on incorporation of methyl groups into histones show that histone H2A, together with the related nonhistone protein A24, is methylated in Tetrahymena.