Purification of an active opioid-binding protein from bovine striatum

We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors.     

Evidence for the presence of disulfide bridges in opioid receptors essential for ligand binding. Possible role in receptor activation

The mobility of purified mu opioid binding protein in SDS-polyacrylamide gek electrophoresis is sensitive to the presence of reducing agents. In the presence of increasing concentrations of DTT the apparent molecular weight increases in a stepwise fashion from 53 kDa to 65 kDa. This reduction in mobility is attributed to the successive breakage of disulfide bridges, resulting in an increasingly asymmetric molecule. Treatment of cell membranes from various brain areas with reducing agents, such as DTT, produced a concentration-dependent inhibition of opioid binding. Sensitivity to DTT inhibition varied between receptor types, mu greater than delta much greater than kappa. For mu receptors, agonist binding was considerably more sensitive to DTT than antagonist binding. Inhibition by DTT is readily reversible and is unaffected by Na+ and/or Mg2+ ions. Reversibility may be partially prevented by the inclusion of a low concentration of a reducing reagent such as glutathione which does not inhibit binding but blocks reformation of disulfide bonds. Scatchard analysis of saturation data shows that DTT causes a pronounced decrease in binding affinity with little effect on receptor number. It is suggested that disulfide bonds are essential for ligand binding and that cleavage of one or more of these bonds may play a role in opioid receptor activation by agonists.     

Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.     

Specific high affinity interactions of monomeric endotoxin.protein complexes with Toll-like receptor 4 ectodomain

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin.protein complexes of high specific radioactivity. The binding of endotoxin.MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD+/-MD-2. These interactions are specific, of high affinity (KD<300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr approximately 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin.MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin.protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.     

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.     

The carboxyl-terminal domain of closely related endotoxin-binding proteins determines the target of protein-lipopolysaccharide complexes.

The bactericidal/permeability increasing (BPI) and lipopolysaccharide (LPS)-binding (LBP) proteins are closely related two-domain proteins in which LPS binding is mediated by the NH(2)-terminal domain. To further define the role of the COOH-terminal domain of these proteins in delivery of LPS to specific host acceptors, we have compared interactions of LBP, BPI, LBP(N)-BPI(C) (NH(2)-terminal domain of LBP, COOH-terminal domain of BPI), and BPI(N)-LBP(C) with purified (3)H-LPS and, subsequently, with purified leukocytes and soluble (s)CD14. The COOH-terminal domain of LBP promotes delivery of LPS to CD14 on both polymorphonuclear leukocytes and monocytes resulting in cell activation. In the presence of Ca(2+) and Mg(2+), LBP and BPI each promote aggregation of LPS to protein-LPS aggregates of increased size (apparent M(r) > 20 x 10(6) Da), but only LPS associated with LBP and BPI(N)-LBP(C) is disaggregated in the presence of CD14. BPI and LBP(N)-BPI(C) promote apparently CD14-independent LPS association to monocytes without cell activation. These findings demonstrate that the carboxyl-terminal domain of these closely related endotoxin-binding proteins dictates the route and host responses to complexes they form with endotoxin.     

Endotoxin{middle dot}albumin complexes transfer endotoxin monomers to MD-2 resulting in activation of TLR4

Response to Gram-negative bacteria (GNB) is partially mediated by the recognition of GNB-derived endotoxin by host cells. Potent host response to endotoxin depends on the sequential interaction of endotoxin with lipopolysaccharide binding protein (LBP), CD14, MD-2 and TLR4. While CD14 facilitates the efficient transfer of endotoxin monomers to MD-2 and MD-2·TLR4, activation of MD-2·TLR4 can occur in the absence of CD14 through an unknown mechanism. Here, we show that incubation of purified endotoxin (E) aggregates (E(agg), M ( r )?≥?20 million) in PBS with?≥?0.1% albumin in the absence of divalent cations Ca(2+) and Mg(2+), yields E·albumin complexes (M ( r ) ～70,000). E·albumin transfers E monomers to sMD-2 or sMD-2·TLR4 ectodomain (TLR4(ecd)) with a 'K (d)' of ～4?nM and induces MD-2·TLR4-dependent, CD14-independent cell activation with a potency only 10-fold less than that of monomeric E·CD14 complexes. Our findings demonstrate, for the first time, a mechanistic basis for delivery of endotoxin monomers to MD-2 and for activation of TLR4 that is independent of CD14.     

Identification of distinct binding site subunits of mu and delta opioid receptors

Iodinated human beta-endorphin was affinity-cross-linked to opioid receptors present in membrane preparations from bovine frontal cortex, bovine striatum, guinea pig whole brain, and rat thalamus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed covalently labeled peptides of 65, 53, 41, and 38 kilodaltons (kDa). The 65- and 38-kDa peptides were present in all four tissues. The 41-kDa peptide was seen only in bovine caudate and guinea pig whole brain while the 53-kDa peptide was absent in rat thalamus. All four labeled peptides were constituents of opioid receptors since their labeling was fully suppressed by the presence of excess opiates, such as bremazocine, during binding. The distribution and levels of the labeled species in the brain tissues examined and, in earlier work, in the neuroblastoma X glioma NG 108-15 cell line suggested that the 65-kDa peptide is a binding component of mu receptors while the 53-kDa peptide is a binding subunit of delta receptors. This result was strongly supported by the finding that the labeling of the 65-kDa peptide is selectively reduced by the presence of the highly mu-selective ligand Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (DAMGE) during binding, while while the labeling of the 53-kDa peptide is selectively reduced or eliminated by the highly mu-selective ligand [D-Pen2, D-Pen5]enkephalin (DPDPE). The labeling of the 41- and 38-kDa bands was reduced by either DAMGE or DPDPE. The relationship of these lower molecular weight opioid-binding peptides to mu and delta receptors is not understood. Several possible explanations are presented.     

Affinity chromatography of solubilized opioid binding sites using CH-Sepharose modified with a new naltrexone derivative

An affinity gel containing a newly synthesized derivative of naltrexone, beta-naltrexyl-6-ethylenediamine (NED), was used to purify opioid binding sites by 300-450-fold. Binding sites solubilized from brains of toad, rat and cow using digitonin in the presence of 0.5 M NaCl are retained by the gel and can be eluted with naloxone (1-3 microM) in yields of 20-25%. The biospecificity of the interaction of opioid binding sites with modified beads is supported by the following: 1) unmodified beads do not retain the binding sites, 2) binding sites prebound with an opioid are not retained, 3) dilution of NED gels with unmodified Sepharose progressively reduced efficiency of retention and 4) specific receptor ligands elute binding sites retained on the NED gels.     

An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation

Experiments utilizing endotoxin aggregates, lipooligosaccharides (LOS) isolated from metabolically labeled Neisseria meningitidis serotype group B, demonstrate that albumin is an essential component of lipopolysaccharide binding protein- (LBP) and sCD14-dependent 1) disaggregation of LOS and 2) LOS activation of human umbilical vein endothelial cells (HUVEC). Aggregates of LOS (LOS(agg)) with an apparent M(r) >or= 2 x 10(7) were isolated by gel sieving on Sephacryl HR S500 in buffered balanced salts solution plus albumin. Incubation of LOS(agg) with LBP and sCD14 promoted LOS(agg) disaggregation in an albumin-dependent fashion to complexes that contain LOS and sCD14, but no LBP, with an apparent M(r) approximately 60,000 (LOS:sCD14) as determined by Sephacryl S200 chromatography. Isolation by gel filtration of LOS(agg):protein aggregates formed by the interaction of LOS(agg) with either LBP or sCD14 alone revealed that the sequence of LOS-protein interactions as well as the step(s) at which albumin is necessary for the production of bioactive LOS:sCD14 were specific. Efficient generation of LOS:sCD14 required 1) interaction of LOS(agg) with LBP before interaction with CD14 and 2) the presence of albumin during the interaction of LBP with LOS(agg). Activation of HUVEC by LOS(agg), as measured by IL-8 production, required both LBP and sCD14 and was thirty times more potent in the presence of albumin. In contrast, LOS:sCD14 did not require additional LBP, sCD14, or albumin to activate HUVEC but depended on the presence of albumin for optimal solubility/stability once formed. The albumin effect is apparently specific, because neither ovalbumin nor gelatin substituted for albumin in facilitating LBP:sCD14-dependent disaggregation of LOS(agg) or activation of endothelial cells. These results indicate that albumin is an essential facilitator of LBP/sCD14-induced LOS disaggregation that is required for activation of endothelial cells by LOS(agg).