Complement-induced solubilization of C-reactive protein-pneumococcal C-polysaccharide precipitates: evidence for covalent binding of complement proteins to C-reactive protein and to pneumococcal C-polysaccharide

Insoluble precipitates between C-reactive protein and pneumococcal C-polysaccharide were solubilized by fresh but not heat-inactivated human serum. Solubilization was dependent on the C-reactive protein to C-polysaccharide ratio and the serum concentration and proceeded optimally at 37 degrees C. On 12 to 30% sucrose density gradients solubilized complexes sedimented as a broad peak between the 7S region and the bottom of the tube. In C2-deficient human serum, solubilization was delayed by 30 min and then proceeded at a slow rate. Immunoprecipitation experiments indicated that C3, C4, C5, and to a lesser extent C1q were deposited on solubilized complexes. After dissociating solubilized complexes with EDTA, a considerable percentage of C-polysaccharide coprecipitated with C3, C4, and C5, whereas C-reactive protein coprecipitated with only C3. SDS-PAGE analysis of solubilized precipitates indicated the formation of covalent complexes between fragments of C3 and both C-polysaccharide and C-reactive protein.     

Human C-reactive protein protects mice from Streptococcus pneumoniae infection without binding to pneumococcal C-polysaccharide

Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection of mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcgamma receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.     

A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine.

Streptococcus mitis strain SK598, which represents a subgroup of biovar 1, possesses a unique variant of the C-polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast to the previously characterized forms of C-polysaccharide, which all contain one or two choline residues per repeat. The following structure of the repeating unit of the SK598 polysaccharide was established: where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This structure is identical to the double choline-substituted form of C-polysaccharide, except that it is substituted with ethanolamine instead of choline. This extends the number of recognized C-polysaccharide variants to four.     

Further evidence that eugenol does not bind to DNA in vivo

The naturally-occurring alkenylbenzene, eugenol, was examined for its ability to form DNA adducts in the livers of mice that had been treated with up to 10 mg of the compound. No adducts were detected by 32P-postlabelling with a limit of detection of 1 adduct in 10(9) nucleotides. Under these conditions adducts were readily detected in liver DNA from the structurally-related hepatocarcinogen safrole.     

Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.     

The pneumococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine.

The structure of the pneumococcal common antigen, C-polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C-polysaccharide is composed of a backbone of tetrasaccharide-ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a D-ribitol residue and position 6 of a beta-D-glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N-acetylated. O)-P-Cho | 6 6)-beta-D-Glcp-(1-->3)-alpha-AATp-(1-->4)-alpha-D-GalpNAc-(1-->3)- bet a-D-GalpNAc-(1-->1)-D-ribitol-5-P-(O--> where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N-acetylation, from those reported previously for pneumococcal C-polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry 19, 4712-4719; Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem. 71, 644-648]. The structures of the C-polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C-polysaccharides was identical and in all cases both galactosamine residues appeared to be N-acetylated.     

Inhibition of antibody responses to phosphocholine by C-reactive protein

C-reactive protein (CRP) is an acute phase serum protein in man that binds to the cell wall C-polysaccharide (PnC) of Streptococcus pneumoniae via phosphocholine (PC) determinants. We have previously shown that in mice CRP increases splenic clearance of PnC-coated autologous erythrocytes and S. pneumoniae, and increases survival after pneumococcal infection. Because CRP alters clearance of particulate PnC antigens, we tested its effect on immunization with pneumococci. Pretreatment of mice with 50 to 200 micrograms CRP 30 min before immunization with serotype 3 S. pneumoniae resulted in dose-dependent inhibition of the antibody response to PC. Both serum hemagglutinin and splenic PFC against PC were decreased in CRP-treated mice tested from 1 to 10 days after injection of antigen. CRP treatment had no effect on the antibody response to the serotype 3 capsular polysaccharide, another T-independent antigen. To determine whether CRP inhibition was related to altered processing of particulate antigen, mice were immunized with horse red blood cells (HRBC) conjugated with PC or PnC and the PFC responses to PC and HRBC were determined. CRP treatment resulted in specific inhibition of the PFC response to PC in both cases without affecting the response to HRBC. These results indicate that inhibition of the antibody response by CRP is not the result of altered antigen localization and processing, and that CRP may prevent immunization by masking determinants on bacterial or other surfaces.     

A transferrin-like protein that does not bind iron is induced by iron deficiency in the alga Dunaliella salina

Iron deficiency induces two major transferrin-like proteins in the plasma membrane (Pm) of the halotolerant alga Dunaliella salina. TTf, a 150-kDa protein, previously identified as a salt-induced triplicated transferrin, having iron-binding characteristics resembling animal transferrins, and a 100-kDa protein designated idi-100 (for iron-deficiency-induced 100 kDa protein). According to the predicted amino acid sequence of idi-100, it is only 30% identical to TTf and differs from it in having two, rather than three, homologous internal repeats and in a lower conservation of canonical iron/bicarbonate binding residues. Both are localized in the outer surface of the membrane; however, TTf can be dissociated from the membrane by treatment with EDTA, whereas release of idi-100 requires detergents. The accumulation of idi-100 under iron deficiency lags behind that of TTf and in contrast to TTf, it is not induced by high salinity, suggesting that induction of idi-100 requires lower Fe threshold levels than that of TTf. In contrast to TTf, idi-100 does not bind Fe; however, there are indications for interactions with bicarbonate ions. These results suggest that despite their common resemblance to transferrins, their similar subcellular localization and their induction by iron deficiency, idi-100 and TTf fulfill different functions.     

Evidence that tetrahydrofolate does not bind to serine hydroxymethyltransferase with positive homotropic cooperativity

Previous experiments suggesting that tetrahydrofolate binds to serine hydroxymethyltransferase with positive homotropic cooperativity have been reinvestigated. Our results show that the sigmoid-shaped tetrahydrofolate saturation curve, previously obtained by several other investigators, is due to the instability of tetrahydrofolate in the assay solution. Using a different assay method, we have shown that tetrahydrofolate gives a hyperbolic saturation curve with serine hydroxymethyltransferase. We could find no evidence, as suggested by other investigators, that heating the enzyme during purification destroyed its allosteric properties or that NADH binds to the enzyme as an allosteric effector. Evidence is presented that the loss of tetrahydrofolate during the assay period is due to oxidation by dissolved molecular oxygen.     

Evidence for an antagonist specific receptor that does not bind mineralocorticoid agonists

Kinetics of association--dissociation, competition and chromatography on two different resins, all revealed the presence of a new binding site which: specifically accepts 7-alpha-propyl spirolactone (3H-RU-26752), has little affinity for aldosterone, is present only in the target tissue (rat kidney), and is wanting in a non-target organ (liver). The presence of such sites could explain syndromes of mineralocorticoid excess where even trace amounts of an unusual aldosterone analogue, with little affinity for the classical mineralocorticoid receptor, can nevertheless produce hypertension through the intervention of an entirely new and abundant receptor system. This new molecule thus forms a novel tool to understand the nature and function of the soluble mineralocorticoid receptor in target organs.     

Tubulin does not bind glycerol irreversibly

Tubulin incorporates radioactivity when incubated with preparations of [¹⁴C]-glycerol, but the material that binds to tubulin is a contaminant rather than glycerol itself. The apparent binding of glycerol by tubulin was not stoichiometric, nor was it significantly affected by dilution of the radioactive glycerol preparation with unlabelled glycerol. Radioactive glycerol purified by prior exposure to protein was an order of magnitude less effective in binding to tubulin than unpurified glycerol. Cytochrome-C and bovine plasma albumin, as well as tubulin, incorporated radioactivity from isotopically labelled glycerol. The amount of radioactivity incorporated into tubulin was unaffected by assembly-disassembly.     

Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.     

An avidin-like domain that does not bind biotin is adopted for oligomerization by the extracellular mosaic protein fibropellin

The protein avidin found in egg white seems optimized for binding the small vitamin biotin as a stable homotetramer. Indeed, along with its streptavidin ortholog in the bacterium Streptomyces avidinii, this protein shows the strongest known noncovalent bond of a protein with a small ligand. A third known member of the avidin family, as similar to avidin as is streptavidin, is found at the C-terminal ends of the multidomain fibropellin proteins found in sea urchin. The fibropellins form a layer known as the apical lamina that surrounds the sea urchin embryo throughout development. Based upon the structure of avidin, we deduced a structural model for the avidin-like domain of the fibropellins and found that computational modeling predicts a lack of biotin binding and the preservation of tetramerization. To test this prediction we expressed and purified the fibropellin avidin-like domain and found it indeed to be a homotetramer incapable of binding biotin. Several lines of evidence suggest that the avidin-like domain causes the entire fibropellin protein to tetramerize. We suggest that the presence of the avidin-like domain serves a structural (tetrameric form) rather than functional (biotin-binding) role and may therefore be a molecular instance of exaptation-the modification of an existing function toward a new function. Finally, based upon the oligomerization of the avidin-like domain, we propose a model for the overall structure of the apical lamina.     

Recombinant CD200 protein does not bind activating proteins closely related to CD200 receptor

CD200 (OX2) is a cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed mainly on myeloid cells and is involved in regulation of macrophage and mast cell function. In mouse there are up to five genes related to CD200R with conflicting data as to whether they bind CD200. We show that mouse CD200 binds the inhibitory receptor CD200R with a comparable affinity (Kd = 4 microM) to those found for the rat and human CD200 CD200R interactions. CD200 gave negligible binding to the activating receptors, CD200RLa, CD200RLb, and CD200RLc, by direct analysis at the protein level using recombinant monomeric and dimeric fusion proteins or to CD200RLa and CD200RLb when expressed at the cell surface. An additional potential activating gene, CD200RLe, found in only some mouse strains also did not bind CD200. Thus, the CD200 receptor family consists of both activatory and inhibitory members like several other paired ligand receptors, such as signal regulatory protein, killer cell Ig-like receptor/KAR, LY49, dendritic cell immunoreceptor/dendritic cell immunoactivating receptor, and paired Ig-like type 2 receptor. Although the ligand for the inhibitory product is a widely distributed host protein, the ligands of the activating forms remain to be identified, and one possibility is that they are pathogen components.     

Circulating hyaluronan, chondroitin sulphate and dextran sulphate bind to a liver receptor that does not recognize heparin

Chondroitin sulphate, injected intravenously into rats and given prior to intravenous ¹²⁵I-labelled hyaluronan with a mean Mw of about 400 kDa, was shown to inhibit the rapid receptor-mediated uptake of hyaluronan by the liver. The labelled hyaluronan that remained in the circulation was shown, by size exclusion chromatography of serum and urine, to be rapidly degraded down to fragments of lower Mw and filtered out into the urine and tissues. When the uptake of ¹²⁵I-hyaluronan was inhibited by unlabelled hyaluronan, only very low degradation and urinary excretion were found. Liver uptake could also be inhibited by dextran sulphate but not by heparin. Unlabelled hyaluronan could inhibit the liver uptake of labelled chondroitin sulphate but not labelled heparin. Unlabelled chondroitin sulphate and dextran sulphate inhibited cell association of labelled hyaluronan to liver endothelial cells in culture more effectively than unlabelled hyaluronan. Our data show that the liver hyaluronan receptors also recognize and effectively bind chondroitin sulphate and dextran sulphate but not heparin and that a hyaluronan-specific saturable degradative mechanism exists in the circulation. Such a mechanism could explain why hyaluronan in the general circulation has a much lower Mw than the hyaluronan in lymph. The results also indicate that increased hyaluronan levels in serum, and increased urinary excretion of hyaluronan, may be secondary to increased outflow of chondroitin sulphate from the tissues during some pathological conditions. This revised version was published online in November 2006 with corrections to the Cover Date.     

Human C-reactive protein does not protect against acute lipopolysaccharide challenge in mice

The physiological and pathophysiological functions of C-reactive protein (CRP), the classical acute-phase protein, are not well established, despite many reports of biological effects of CRP in vitro and in model systems in vivo. Limited, small scale experiments have suggested that rabbit and human CRP may both protect mice against lethal toxicity of Gram-negative bacterial LPS. However, in substantial well-controlled studies in C57BL/6 mice challenged with Escherichia coli O111:B4 LPS, we show in this work that significant protection against lethality was conferred neither by an autologous acute-phase response to sterile inflammatory stimuli given to wild-type mice 24 h before LPS challenge, nor by human CRP, whether passively administered or expressed transgenically. Male mice transgenic for human CRP, which mount a major acute-phase response of human CRP after LPS injection, were also not protected against the lethality of LPS from either E. coli O55:B5 or Salmonella typhimurium. Even when the acute-phase human CRP response was actively stimulated in transgenic mice before LPS challenge, no protection against LPS toxicity was observed. Indeed, male mice transgenic for human CRP that were pretreated with casein to stimulate an acute-phase response 24 h before LPS challenge suffered significantly greater mortality than unstimulated human CRP transgenic controls. Rather than being protective in this situation, human CRP may thus have pathogenic proinflammatory effects in vivo.