Sialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation

Voltage-Gated Metal-Enhanced Fluorescence II: Effects of Fluorophore Concentration on the Magnitude of the Gated-Current

Abstract In this letter we report further findings on the ability of an applied direct current to modulate MetalEnhanced Fluorescence (MEF). Fluorophores in closeproximity to just-continuous silver films (JCS) show significantly enhanced fluorescence intensities. However, when a current is applied to the films, the enhanced fluorescence can be gated in a manner that depends on both the fluorophore concentration, the magnitude of the applied current and the extent of the protein mono to multi-layer surface coverage. Our results are consistent and indeed further support our previous hypothesis and model that fluorophore-metal near-field interactions can be influenced by an applied direct current

3 Enthalpy-entropy compensation: the role of solvation

complete understanding of EEC be obtained. Faced with such large, and compensating, changes in the enthalpies and entropies of binding, the best approach to engineering elevated affinities must be through the addition of ionic links, as they generate increased entropy without affecting the enthalpy. Keywords Enthalpy-entropy compensation (EEC) · Solvation water · Hydration · Proteins · DNA Enthalpy-entropy compensation Efforts to establish structure-activity relationships (SARs) and improve the affinity of drugs for target proteins typically involve thermodynamic measurements on a panel of modified forms of the lead compound, principally using isothermal titration calorimetry (ITC). It is frequently observed that whereas the Gibbs energy of binding, i.e., the binding constant, remains largely unchanged in consequence of the addition/subtraction of chemical groups, there are substantial variations in the component enthalpies and entropies. If ∆G remains the same, it follows that changes in ∆H and T∆S compensate one another. In fact, enthalpy-entropy compensation (EEC) is a widely observed phenomenon and is typically explained by assuming that if a molecular change in the ligand leads to more and/or tighter van der Waals contacts and H-bonds with the substrate (giving a more negative ∆H), this inevitably leads to reduced mobility/flexibility in either or both components of the interaction, i.e., a reduction in the overall conformational entropy, and that change compensates the enthalpy decrease. However, the amount of water hydrating the system can also change and if any of this water is tightly bound, its contribution to the enthalpy and entropy of binding will also be Abstract Structural modifications to interacting systems frequently lead to changes in both the enthalpy (heat) and entropy of the process that compensate each other, so that the Gibbs free energy is little changed: a major barrier to the development of lead compounds in drug discovery. The conventional explanation for such enthalpy-entropy compensation (EEC) is that tighter contacts lead to a more negative enthalpy but increased molecular constraints, i.e., a compensating conformational entropy reduction. Changes in solvation can also contribute to EEC but this contribution is infrequently discussed. We review long-established and recent cases of EEC and conclude that the large fluctuations in enthalpy and entropy observed are too great to be a result of only conformational changes and must result, to a considerable degree, from variations in the amounts of water immobilized or released on forming complexes. Two systems exhibiting EEC show a correlation between calorimetric entropies and local mobilities, interpreted to mean conformational control of the binding entropy/free energy. However, a substantial contribution from solvation gives the same effect, as a consequence of a structural link between the amount of bound water and the protein flexibility. Only by assuming substantial changes in solvation-an intrinsically compensatory process-can a more * Colyn Crane-Robinso

c0jm04311g 6179..6185

Over the last decade Metal-Enhanced Fluorescence (MEF) has emerged as the next generation of fluorescence spectroscopy, i.e. near-field fluorescence. However, in contrast to our collective knowledge and understanding of classical far-field fluorescence, we know relatively little. MEF is a consequence of the near-field interactions of fluorophores (dipoles) with the surface plasmons generated in plasmon supporting materials, where the optical properties of the metal afford for a wavelength dependence of MEF. In this paper we show that we are not limited to the properties of the individual metals for MEF, but in fact, surface deposits of mixed metals can create new dephased plasmon resonance bands, not present in the individual metals themselves. Subsequently, mixed metal substrates (MMS) offer significant opportunities for the multifarious and forever growing applications of MEF

Inhibition of endogenous sialidase activity suppressed LPS-induced NFκB activation in HEK cells.

<p>HEK293T cells were infected with recombinant adenoviruses expressing human Neu1 (Ad-Neu1) or Neu3 (Ad-Neu3), or with virus containing empty vector (Ad-GFP) and sialidase activity from cell lysates was assayed using 4-MUNANA as substrate in the absence or presence of the sialidase inhibitor 2-DN (250 µg/ml) (A). TLR4/CD14/MD2-transfected HEK293T cells were further infected with Ad-Neu1, Ad-Neu3 or Ad-GFP, for 2 days, stimulated with LPS (1 ng/ml) for 16 h, and evaluated for luciferase activity (B). TLR4/CD14/MD2-transfected HEK293T cells were incubated with 2-DN (250 µg/ml) or KDO (250 µg/ml) for 2 days, and stimulated with different concentrations of LPS for 16 h prior to analysis of cell lysates for luciferase activity (C). Results shown are representative of data from at least 3 independent experiments, each with similar results. ND: not done.</p

Sialylation of TLR4 and MD2.

<p>Recombinant human TLR4-His/MD2-His proteins expressed in the mammalian NS0 cell line were separated by SDS-PAGE and analyzed on immunoblot for binding to lectin SNA, MAAII and to anti-His antibody (A). Recombinant human TLR4-His/MD2-His expressed in NS0 cells (lane 1) or recombinant human MD2-His expressed in <i>E. coli</i> (lane 2) were separated by SDS-PAGE and probed by lectin blot with SNA (left) and on immunoblot with anti-His antibody (right) (B). Recombinant human CD14 (lane 1) were separated by SDS-PAGE and probed by lectin blot with SNA (left) and MAAII (right). The highly sialylated glycoprotein, fetuin (lane 2) and asialofetuin (lane 3) were included as positive and negative controls respectively (C). HEK293T cells were transfected with control pcDNA, or plasmids encoding TLR4-YFP alone or with MD2 and CD14 expression plasmids, and proteins from cell lysates were immunoprecipitated with ant-GFP antibody and probed on immunoblot with either anti-GFP antibody (top) or SNA (bottom) (D). Proteins from the medium of MD2-transfected cells were immunoprecipitated with anti-FLAG antibody and probed on immunoblot with anti-FLAG antibody (left) or SNA (right) (E). The expected molecular weights of MD2 and TLR4 are indicated by arrows. Results shown are representative of data from at least 2 independent experiments, each with similar results.</p

Effect of P714H, N35A/N173A, and N35A/N173A/N205A mutations on the ability of TLR4 to mediate LPS-induced activation.

<p>HEK293T cells were co-transfected with expression vectors encoding wild type TLR4 (WT) or P714H, N35A/N173A, and N35A/N173A/N205A mutants along with pEFBOS-MD2, pCDNA3-CD14, pELAM-luc, and pTK-<i>Renilla</i>-luc. Transfected cells were stimulated with LPS for 6 h, and firefly vs. renilla luciferase activities were measured in cell lysates. Data were processed using Student t-test. *p<0.005; **p<0.05 (vs. WT).</p

Optimal LPS-induced signaling when both TLR4/CD14 and MD2 treated with neuraminidase.

<p>HEK293T cells that were transfected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032359#pone-0032359-g002" target="_blank">Figure 2</a> and TLR4/CD14-transfected cells were supplemented with NA-treated SNT from MD2 transfected cells (MD2*), heated-inactivated NA-treated SNT (MD2**), or SNT from empty vector transfected cells (pcDNA) before LPS stimulation (5 ng/ml). Cell lysates were harvested after 16 h for reporter assay (A). TLR4/CD14-transfected HEK293T cells were treated with NA, heat-inactivated NA (ΔNA), or were untreated (-) for one hour at 37°C, stimulated with LPS (5 ng/ml) for 16 h in the presence of SNT from MD2 transfected cells (sMD2), which had been pre-treated with NA agarose (NA), heated-inactivated NA agarose (ΔNA), or were untreated, and luciferase activities were determined in cell lysates (B). TLR4/MD2-transfected HEK293T cells were supplemented with NA treated recombinant human CD14 (rhCD14[NA]), untreated CD14 (rhCD14), or medium (none) prior to LPS stimulation (1–5 ng/ml). Cell lysates were harvested after 16 h for reporter assay (C). Results shown are representative of data from at least 3 independent experiments, each with similar results.</p