Lipopolysaccharide Binding Site Research Articles

SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains.

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane β-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic β-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate β-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

Radix Glycyrrhizae extract and licochalcone a exert an anti-inflammatory action by direct suppression of toll like receptor 4

Ethnopharmacological relevanceRadix Glycyrrhizae (GL), a herbal medicine that is widely available, has shown advantages for a variety of inflammatory diseases. Toll like receptor 4 (TLR4) pathway has been shown to play a key role in the progression of inflammation. Aim of the studyThe purpose of this study was to investigate the involvement of TLR4 in the anti-inflammatory mechanism of GL extract and its active constituent on acute lung injury (ALI). Materials and methodsA model of inflammation produced by lipopolysaccharide (LPS) was established in C57BL/6 mice and macrophages derived from THP-1. To screen the active components of GL, molecular docking was used. Molecular dynamics and surface plasmon resonance imaging (SPRi) were used to study the interaction of a specific drug with the TLR4-MD2 complex. TLR4 was overexpressed by adenovirus to confirm TLR4 involvement in the anti-inflammatory activities of GL and the chosen chemical. ResultsWe observed that GL extract significantly reduced both LPS-induced ALI and the production of pro-inflammatory factors including TNF-α, IL-6 and IL-1β. Additionally, GL inhibited the binding of Alexa 488-labeled LPS (LPS-488) to the membrane of THP-1 derived macrophages. GL drastically reduce on the expression of TLR4 and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-κB). Furthermore, molecular docking revealed that Licochalcone A (LicoA) docked into the LPS binding site of TLR4-MD2 complex. MD2-LicoA binding conformation was found to be stable using molecular dynamic simulations. SPRi indicated that LicoA bound to TLR4-MD2 recombinant protein with a KD of 3.87 × 10−7 M. LicoA dose-dependently reduced LPS-488 binding to the cell membrane. LicoA was found to significantly inhibit LPS-induced lung damage and inflammation. Furthermore, LicoA inhibited TLR4 expression, MAPK and NF-κB activation in a dose-dependent manner. The inhibitory effects of GL and LicoA on LPS-induced inflammation and TLR4 signaling activation were partly eliminated by TLR4 overexpression. ConclusionOur findings imply that GL and LicoA exert inhibitory effects on inflammation by targeting the TLR4 directly.

SARS-CoV-2 spike protein aggregation is triggered by bacterial lipopolysaccharide.

SARS‐CoV‐2 spike (S) protein is crucial for virus invasion in COVID‐19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin‐scavenging thrombin‐derived peptide TCP‐25. Aggregation‐prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein–LPS higher‐order oligomers. Collectively, our results provide evidence of LPS‐induced S protein aggregation.

Ginsenoside Ro, an oleanolic saponin of Panax ginseng, exerts anti-inflammatory effect by direct inhibiting toll like receptor 4 signaling pathway

BackgroundPanax ginseng Meyer (P. ginseng), a herb distributed in Korea, China and Japan, exerts benefits on diverse inflammatory conditions. However, the underlying mechanism and active ingredients remains largely unclear. Herein, we aimed to explore the active ingredients of P. ginseng against inflammation and elucidate underlying mechanisms. MethodsInflammation model was constructed by lipopolysaccharide (LPS) in C57BL/6 mice and RAW264.7 macrophages. Molecular docking, molecular dynamics, surface plasmon resonance imaging (SPRi) and immunofluorescence were utilized to predict active component. ResultsP. ginseng significantly inhibited LPS-induced lung injury and the expression of pro-inflammatory factors, including TNF-α, IL-6 and IL-1β. Additionally, P. ginseng blocked fluorescence-labeled LPS (LPS488) binding to the membranes of RAW264.7 macrophages, the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Furthermore, molecular docking demonstrated that ginsenoside Ro (GRo) docked into the LPS binding site of toll like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex. Molecular dynamic simulations showed that the MD2-GRo binding conformation was stable. SPRi demonstrated an excellent interaction between TLR4/MD2 complex and GRo (KD value of 1.16 × 10−9 M). GRo significantly inhibited LPS488 binding to cell membranes. Further studies showed that GRo markedly suppressed LPS-triggered lung injury, the transcription and secretion levels of TNF-α, IL-6 and IL-1β. Moreover, the phosphorylation of NF-κB and MAPKs as well as the p65 subunit nuclear translocation were inhibited by GRo dose-dependently. ConclusionOur results suggest that GRo exerts anti-inflammation actions by direct inhibition of TLR4 signaling pathway.

Insights from molecular docking and molecular dynamics on the potential of vitexin as an antagonist candidate against lipopolysaccharide (LPS) for microglial activation in neuroinflammation

BackgroundNeuroinflammation has been identified to be the key player in most neurodegenerative diseases. If neuroinflammation is left to be unresolved, chronic neuroinflammation will be establish. Such situation is due to the overly-activated microglia which have the tendency to secrete an abundance amount of pro-inflammatory cytokines into the neuron microenvironment. The abundance of pro-inflammatory cytokines will later cause toxic and death to neurons. Toll-like receptor 4 (TLR4)/MD-2 complex found on the cell surface of microglia is responsible for the attachment of LPS and activation of nuclear factor-κB (NF-κB) downstream signalling pathway. Albeit vitexin has been shown to possess anti-inflammatory property, however, little is known on its ability to bind at the binding site of TLR4/MD-2 complex of microglia as well as to be an antagonist for LPS.ResultsThe present study reveals that both vitexin and donepezil are able to bind at the close proximity of LPS binding site located at the TLR4/MD-2 complex with the binding energy of − 4.35 and − 9.14 kcal/mol, respectively. During molecular dynamic simulations, both vitexin and donepezil formed stable complex with TLR4/MD-2 throughout the 100 ns time length with the root mean square deviation (RMSD) values of 2.5 Å and 4.0 Å, respectively. The root mean square fluctuation (RMSF) reveals that both compounds are stable. Interestingly, the radius of gyration (rGyr) for donepezil shows notable fluctuations when compare with vitexin. The MM-GBSA results showed that vitexin has higher binding energy in comparison with donepezil.ConclusionsTaken together, the findings suggest that vitexin is able to bind at the binding site of TLR4/MD-2 complex with more stability than donepezil throughout the course of 100 ns simulation. Hence, vitexin has the potential to be an antagonist candidate for LPS.

Synthesis and evaluation of the anti-inflammatory activity of novel 8-quinolinesulfonamide derivatives as TLR4/MD-2 inhibitors with efficacy in adjuvant-induced arthritis

In this study, a series of 8-quinolinesulfonamidederivatives was synthesized, and their anti-inflammatory activity was evaluated. Among them, compound 3l was found to be the best anti-inflammatory agent, with IC50 values of 2.61 ± 0.39, 9.74 ± 0.85, and 12.71 ± 1.34 μM against NO, TNF-α and IL-1β production respectively. And 3l could significantly prevent lipopolysaccharide (LPS)-induced expression of inflammatory mediators (iNOS and COX-2). Molecule docking results showed that 3l could bind to the LPS binding site of toll-like receptor 4 (TLR4)/MD-2, and 3l was then identified as TLR4/MD-2 inhibitor by co-immunoprecipitation (co-IP) and cellular thermal shift assay (CTESA). Preliminary mechanism studies indicated that 3l could prevent TLR4 from being activated by disrupting TLR4/MD-2 heterodimerization and TLR4 homodimerization, thereby blocking the activation of the NF-κB/MAPK signaling pathway. Furthermore, observation of rat foot swelling, joint pathology and serum inflammatory cytokine levels proved that compound 3l had a significant therapeutic effect on adjuvant-induced arthritis (AIA) in rats in vivo. These results indicated that compound 3l is a potential anti-inflammatory agent, from which more effective anti-inflammatory drugs could be developed.

Lipopolysaccharide removal affinity matrices based on novel cationic amphiphilic peptides

Lipopolysaccharide (LPS) is one of the most challenging contaminants in biopharmaceutical industries. Cationic amphiphilic peptides (CAPs) -based affinity matrices can be potent tools for LPS removal in such situations. In this study, two newly designed CAPs derived from the LPS binding site of factor C of horseshoe crab S3E3 and S3E3A were immobilized chemo-selectively on diaminodipropylamine (DADPA) and iodoacetyl functionalized Sepharose beads. Both peptides were immobilized via their carboxyl or sulfhydryl (thiol) groups by amide or thioether bonds, respectively. The generated four affinity matrices were used to remove LPS from bovine serum albumin (BSA). The effects of different influential factors including pH, NaCl, Ethylenediaminetetraacetic acid (EDTA), and LPS concentrations on LPS removal efficiency and protein recovery were investigated by Plackett Burman (PB) method. Statistical analysis revealed that immobilized S3E3 removed LPS more effectively than immobilized S3E3A. Increasing pH and LPS concentration had negative effects on LPS removal efficiency and protein recovery. Increasing NaCl concentration improved protein recovery but reduced LPS removal efficiency. Other factors such as EDTA and type of buffer had no significant effect on the measured responses.

SpBark Suppresses Bacterial Infection by Mediating Hemocyte Phagocytosis in an Invertebrate Model, Scylla paramamosain.

Scavenger receptors are cell surface membrane-bound receptors that typically bind multiple ligands and promote the removal of endogenous proteins and pathogens. In this study, we characterized a novel scavenger receptor-like protein, namely, SpBark. SpBark was upregulated in hemocytes after challenges with bacteria, suggesting that it might be involved in antibacterial defense. SpBark is a type I transmembrane protein with four extracellular domains, including three scavenger receptor cysteine-rich domains (SRCRDs) and a C-type lectin domain (CTLD). Western blot assay showed that SpBark CTLD possessed a much stronger binding activity to tested microbes than the three SRCRDs. It also exhibited apparent binding activities to lipopolysaccharide (LPS) and acetylated low-density lipoprotein (ac-LDL), whereas the other SRCRDs showed much lower or no binding activities to these components. Agglutination activities were observed in the presence of Ca2+ by incubating microorganisms with SpBark CTLD instead of SRCRDs. These results suggested that SpBark CTLD was the major binding site for ac-LDL and LPS. Coating Vibrio parahemolyticus with SpBark CTLD promoted bacterial clearance in vivo. This finding indicated that SpBark might participate in the immune defenses against Gram-negative bacteria through a certain mechanism. The promotion of bacterial clearance by SpBark was further determined using SpBark-silenced crabs injected with V. parahemolyticus. SpBark knockdown by injection of SpBark dsRNA remarkably suppressed the clearance of bacteria in hemolymph. Meanwhile, it also severely restrained the phagocytosis of bacteria. This finding suggested that SpBark could modulate the phagocytosis of bacteria, and the promotion of bacterial clearance by SpBark was closely related to SpBark-mediated phagocytosis activity. The likely mechanism of bacterial clearance mediated by SpBark was as follows: SpBark acted as a pattern recognition receptor, which could sense and bind to LPS on the surface of invading bacteria with its CTLD in hemolymph. The binding to LPS made the bacteria adhere to the surface of hemocytes. This process would facilitate phagocytosis of the bacteria, resulting in their removal. This study provided new insights into the hemocyte phagocytosis mechanisms of invertebrates and the multiple biological functions of Bark proteins.

Modeling of Specific Lipopolysaccharide Binding Sites on a Gram-Negative Porin.

Protein-lipopolysaccharide (LPS) interactions play an important role in providing a stable outer membrane to Gram-negative bacteria. However, the LPS molecules are highly viscous, and sampling LPS motions is thus challenging on a microsecond time scale in simulations. To this end, we introduce a new protocol to randomly allow the LPS molecules to self-assemble around the protein and thereby reduce the starting bias in the simulations. Here we present all-atom molecular dynamics simulations of the OmpE36 porin in an outer membrane model which sum up to a simulation time of more than 20 μs and identify the geometrical properties of the first LPS shell and the role of calcium ions in LPS binding to the protein. The simulations reproduce LPS binding to the porin observed in a recently determined crystal structure but not as compact as in the crystal structure. In addition, the influence of the outer membrane environment on the protein dynamics was analyzed. Our findings highlight the role of divalent cations in stabilizing the binding between proteins and LPS molecules in the outer membrane of Gram-negative bacteria.

A G-protein-coupled chemoattractant receptor recognizes lipopolysaccharide for bacterial phagocytosis.

Phagocytes locate microorganisms via chemotaxis and then consume them using phagocytosis. Dictyostelium amoebas are stereotypical phagocytes that prey on diverse bacteria using both processes. However, as typical phagocytic receptors, such as complement receptors or Fcγ receptors, have not been found in Dictyostelium, it remains mysterious how these cells recognize bacteria. Here, we show that a single G-protein-coupled receptor (GPCR), folic acid receptor 1 (fAR1), simultaneously recognizes the chemoattractant folate and the phagocytic cue lipopolysaccharide (LPS), a major component of bacterial surfaces. Cells lacking fAR1 or its cognate G-proteins are defective in chemotaxis toward folate and phagocytosis of Klebsiella aerogenes. Computational simulations combined with experiments show that responses associated with chemotaxis can also promote engulfment of particles coated with chemoattractants. Finally, the extracellular Venus-Flytrap (VFT) domain of fAR1 acts as the binding site for both folate and LPS. Thus, fAR1 represents a new member of the pattern recognition receptors (PRRs) and mediates signaling from both bacterial surfaces and diffusible chemoattractants to reorganize actin for chemotaxis and phagocytosis.

Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages.

Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-β-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-β knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.

Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts.

Lipopolysaccharide (LPS) from Gram-negative bacteria is recognized as a microbe-associated molecular pattern (MAMP) and not only induces an innate immune response in plants, but also stimulates the development of characteristic defense responses. However, identification and characterization of a cell surface LPS-receptor/binding site, as described in mammals, remains elusive in plants. As an amphiphilic, macromolecular lipoglycan, intact LPS potentially contains three MAMP-active regions, represented by the O-polysaccharide chain, the core and the lipid A. Binding site studies with intact labeled LPS were conducted in Arabidopsis thaliana protoplasts and quantified using flow cytometry fluorescence changes. Quantum dots (Qdots), which allow non-covalent, hydrophobic labeling were used as a novel strategy in this study and compared to covalent, hydrophilic labeling with Alexa 488. Affinity for LPS-binding sites was clearly demonstrated by concentration-, temperature-, and time-dependent increases in protoplast fluorescence following treatment with the labeled LPS. Moreover, this induced fluorescence increase was convincingly reduced following pre-treatment with excess unlabeled LPS, thereby indicating reversibility of LPS binding. Inhibition of the binding process is also reported using endo- and exocytosis inhibitors. Here, we present evidence for the anticipated presence of LPS-specific binding sites in Arabidopsis protoplasts, and furthermore propose Qdots as a more sensitive LPS-labeling strategy in comparison to the conventional Alexa 488 hydrazide label for binding studies.

The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen

Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.

Functional Characterization of E. coli LptC: Interaction with LPS and a Synthetic Ligand

Lipopolysaccharide (LPS), the main cell-surface molecular constituent of Gram-negative bacteria, is synthesized in the inner membrane (IM) and transported to the outer membrane (OM) by the Lpt (lipopolysaccharide transport) machinery. Neosynthesized LPS is first flipped by MsbA across the IM, then transported to the OM by seven Lpt proteins located in the IM (LptBCFG), in the periplasm (LptA), and in the OM (LptDE). A functional OM is essential to bacterial viability and requires correct placement of LPS in the outer leaflet. Therefore, LPS biogenesis represents an ideal target for the development of novel antibiotics against Gram-negative bacteria. Although the structures of Lpt proteins have been elucidated, little is known about the mechanism of LPS transport, and few data are available on Lpt–LPS binding. We report here the first determination of the thermodynamic and kinetic parameters of the interaction between LptC and a fluorescent lipo-oligosaccharide (fLOS) in vitro. The apparent dissociation constant (Kd) of the fLOS–LptC interaction was evaluated by two independent methods. The first was based on fLOS capture by resin-immobilized LptC; the second used quenching of LptC intrinsic fluorescence by fLOS in solution. The Kd values by the two methods (71.4 and 28.8 μm, respectively) are very similar, and are of the same order of magnitude as that of the affinity of LOS for the upstream transporter, MsbA. Interestingly, both methods showed that fLOS binding to LptC is mostly irreversible, thus reflecting the fact that LPS can be released from LptC only when energy is supplied by ATP or in the presence of a higher-affinity LptA protein. A fluorescent glycolipid was synthesized: this also interacted irreversibly with LptC, but with lower affinity (apparent Kd=221 μM). This compound binds LptC at the LPS binding site and is a prototype for the development of new antibiotics targeting LPS transport in Gram-negative bacteria.

Conformationally Constrained Lipid A Mimetics for Exploration of Structural Basis of TLR4/MD-2 Activation by Lipopolysaccharide

Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein, we report on design, synthesis, and biological activities of a series of conformationally confined Lipid A mimetics based on β,α-trehalose-type scaffold. Replacement of the flexible three-bond β(1→6) linkage in diglucosamine backbone of Lipid A by a two-bond β,α(1↔1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a β–GlcN(1↔1)α–GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4.

Structure of Escherichia coli OmpF porin from lipidic mesophase

Outer membrane protein F, a major component of the Escherichia coli outer membrane, was crystallized for the first time in lipidic mesophase of monoolein in novel space groups, P1 and H32. Due to ease of its purification and crystallization OmpF can be used as a benchmark protein for establishing membrane protein crystallization in meso, as a "membrane lyzozyme". The packing of porin trimers in the crystals of space group H32 is similar to natural outer membranes, providing the first high-resolution insight into the close to native packing of OmpF. Surprisingly, interaction between trimers is mediated exclusively by lipids, without direct protein-protein contacts. Multiple ordered lipids are observed and many of them occupy identical positions independently of the space group, identifying preferential interaction sites of lipid acyl chains. Presence of ordered aliphatic chains close to a positively charged area on the porin surface suggests a position for a lipopolysaccharide binding site on the surface of the major E. coli porins.

Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.

Evolutionary conservation of the lipopolysaccharide binding site of β2-glycoprotein I

β₂-Glycoprotein I (β₂GPI) is a highly abundant plasma protein and the major antigen for autoantibodies in the antiphospholipid syndrome. Recently, we have described a novel function of β₂GPI as scavenger of lipopolysaccharide (LPS). With this in mind we investigated the conservation of β₂GPI in vertebrates and set out to identify the binding site of LPS within β₂GPI. The genome sequences of 42 species were surveyed. Surface plasmon resonance (SPR) was performed with peptides to characterise the binding site of β₂GPI for LPS. β₂GPI could be identified in most tested vertebrates with a high overall amino acid homology of 80% or more in mammals. SPR revealed that a synthesised peptide (LAFWKTDA) from domain V of β₂GPI was able to compete for binding of β₂GPI to LPS. The AFWKTDA sequence was completely conserved in all mammals. The peptide containing the LPS binding site attenuated the inhibition by β₂GPI in a cellular model of LPS-induced tissue factor expression. Other important sites, such as the binding site for anionic phospholipids and the antiphospholipid antibody binding epitope, were also preserved. β₂GPI is highly conserved across the animal kingdom, which suggests that the function of β₂GPI may be more important than anticipated.

The second anti-lipopolysaccharide factor (EsALF-2) with antimicrobial activity from Eriocheir sinensis

The anti-lipopolysaccharide factor (ALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724 bp, consisting of an open reading frame (ORF) of 363 bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53–65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional α-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonella anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris, indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture.

Xanthohumol and Related Prenylated Flavonoids Inhibit Inflammatory Cytokine Production in LPS-Activated THP-1 Monocytes: Structure-Activity Relationships andIn SilicoBinding to Myeloid Differentiation Protein-2 (MD-2)

Xanthohumol (XN) is a prenylated chalcone-type flavonoid found in hops and beer. Our objective of this study was to determine the anti-inflammatory activities of XN, isoxanthohumol (IX), and 15 related prenylated chalcones and flavanones, as well as their structure-activity relationships. The anti-inflammatory activities of the flavonoids were measured by their ability to inhibit lipopolysaccharide (LPS)-induced cytokine production in human monocytic THP-1 cells. The position, number, and length of the prenyl groups had a marked influence on the inhibitory activity of the prenylfavonoids towards MCP-1 and IL-6 production. The α,β-unsaturated carbonyl moiety present in chalcones such as XN was not an absolute requirement for inhibitory activity, as the saturated XN derivative, tetrahydroxanthohumol (TX), showed inhibitory activity comparable to XN. With the aim to determine the mechanism of the observed anti-inflammatory effects, cellular protein levels of Toll-like receptor 4 (TLR4) were measured by Western blot 24 h following coexposure of THP-1 cells to LPS and either XN, TX, or IX. Only XN reduced the cellular TLR4 protein content. Therefore, an additional hypothesis was developed for an anti-inflammatory mechanism that involves the TLR4 coreceptor myeloid differentiation protein-2 (MD-2), which provides the actual binding site for LPS. Molecular docking studies showed that the complementarity of prenylated flavonoids with the hydrophobic MD-2 pocket (indicating goodness of fit) directly predicted their relative ability to inhibit MCP-1 and IL-6 production. In conclusion, prenylated flavonoids may suppress LPS-induced TLR4 activation at least partly by interfering with LPS binding to the TLR4 coreceptor MD-2, and XN (but not other prenylflavonoids) exerts an additional anti-inflammatory effect by downregulating the cellular TLR4 protein content.