Functional epitope mapping of human interleukin-1 beta by surface plasmon resonance.

A panel of monoclonal antibodies to human IL-1 beta has been used to probe its conformational and functional characteristics. Real time antibody-protein interaction was assessed by surface plasmon resonance with a BIAcore apparatus, in order to determine the kinetic and thermodynamic parameters of the interaction and to map the recognition sites of the antibodies on the IL-1 beta surface. Topological analysis was thus compared to the inhibitory capacity of antibodies for IL-1 beta bioactivity and binding to the activating receptor IL-1RI. This functional mapping analysis allows the following hypothesis. At least two discrete areas of IL-1 beta, located within the sequences 133-147 and 177-186 (as defined by mAbs MhC1 and BRhD2, respectively), are apparently involved in IL-1RI-independent agonist activity, and thus possibly take part in the interaction with the receptor accessory protein IL-1RAcP. Another area in the 133-147 sequence (defined by mAb BRhC3) is involved in agonist binding to its receptor CDw121a (IL-1RI), whereas a site recognized by mAb BRhG5 within the sequence 218-243 is selectively responsible for non-agonist binding to the activating receptor. The loop between the 4th and the 5th beta-strand, at the open end of the IL-1 beta-barrel structure, may possibly take part in both non-agonist binding to IL-1RI and in the interaction with IL-1RAcP.

Multimer formation and ligand recognition by the long pentraxin PTX3. Similarities and differences with the short pentraxins C-reactive protein and serum amyloid P component.

PTX3 is a prototypic long pentraxin consisting of a C-terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. The present study was designed to characterize the structure and ligand binding properties of human PTX3, in comparison with the classical pentraxins C-reactive protein and serum amyloid P component. Sequencing of Chinese hamster ovary cell-expressed PTX3 revealed that the mature secreted protein starts at residue 18 (Glu). Lectin binding and treatment with N-glycosidase F showed that PTX3 is N-glycosylated, sugars accounting for 5 kDa of the monomer mass (45 kDa). Circular dichroism analysis indicated that the protein consists predominantly of beta-sheets with a minor alpha-helical component. While in gel filtration the protein is eluted with a molecular mass of congruent with900 kDa, gel electrophoresis using nondenaturing, nonreducing conditions revealed that PTX3 forms multimers predominantly of 440 kDa apparent molecular mass, corresponding to decamers, and that disulfide bonds are required for multimer formation. The ligand binding properties of PTX3 were then examined. As predicted based on modeling, inductive coupled plasma/atomic emission spectroscopy showed that PTX3 does not have coordinated Ca2+. Unlike the classical pentraxins CRP and SAP, PTX3 did not bind phosphoethanolamine, phosphocholine, or high pyruvate agarose. PTX3 in solution, bound to immobilized C1q, but not C1s, and, reciprocally, C1q bound to immobilized PTX3. Binding of PTX3 to C1q is specific and saturable with a Kd 7.4 x 10(-8) M as determined by solid phase binding assay. The Chinese hamster ovary cell-expressed pentraxin domain bound C1q when multimerized. Thus, as predicted on the basis of computer modeling, the prototypic long pentraxin PTX3 forms multimers, which differ from those formed by classical pentraxins in terms of protomer composition and requirement for disulfide bonds, and does not recognize CRP/SAP ligands. The capacity to bind C1q, mediated by the pentraxin domain, is consistent with the view that PTX3, produced in tissues by endothelial cells or macrophages in response to interleukin-1 and tumor necrosis factor, may act as a local regulator of innate immunity.

Expression and functional analysis of water channels in a stably AQP2-transfected human collecting duct cell line.

In this study, we describe the establishment of a stably transfected epithelial cell line with the cDNA for the rat aquaporin 2 (AQP2). To this end, we used a human cell line (HCD) derived from the cortical collecting duct and having characteristics of principal cells (Prié, D., Friedlander, G., Coureau, C., Vandewalle, A., Cassigena, R., and Ronco, P. M. (1995) Kidney Int. 47, 1310-1318). The HCD cells were first screened for the constitutive expression of AQPs. By Western blot analysis, we found a low expression of immunoreactive AQP2 and AQP4 proteins. In contrast, transfected cells (clone CD8) probed with AQP2 antiserum expressed an intense 29-kDa protein on immunoblot in addition to a broad band between 35-45 kDa corresponding to the glycosylated form of the protein, indicating that full maturity of the protein is attained in transfected cells. Immunofluorescence demonstrated that AQP2 was located in intracellular vesicles. After vasopressin stimulation, the staining redistributed from an intracellular site to the apical pole of the cells, an effect similar to that described on collecting duct principal cells in vivo (Sabolic, I., Katsura, T., Verbavatz, J. M., and Brown, D. (1995) J. Membr. Biol. 143, 165-175) and in perfused tubules (Nielsen, S., Chou, C. L., Marples, D., Christensen, E. I., Kishore, B. K., and Knepper, M. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1013-1017). The redistribution of AQP2 in CD8 cells was accompanied by an approximately 6-fold increase in osmotic water permeability coefficient (Pf), which was inhibited by 0.3 m HgCl2. These data indicate that functional vasopressin-sensitive water channels are expressed in transfected cells. The stably transfected cells represent a suitable model to unravel by direct experimental approach the intracellular signals involved in the translocation of AQP2 to the apical plasma membrane in the presence of vasopressin.

Reactivity of cysteine-67 of the human immunodeficiency virus-1 protease: studies on a peptide spanning residues 59 to 75.

The protease encoded by the human immunodeficiency virus-1 (HIV-1) is essential for the processing of viral polyproteins into mature viral proteins. The 99-residue protease from HIV-1 contains two generally conserved cysteine residues, one of which (Cys-67) is located on the solvent-exposed surface. It was shown previously that Cys-67 of the native enzyme reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) at pH 6.2, causing a reversible inactivation of the protease. However, there was no reaction when the protein was denatured in 6 M guanidine hydrochloride, implying that the native conformation rendered Cys-67 more reactive. To investigate the structural basis of the lowered pKa in the native protein, we synthesized a 17-residue peptide matching the sequence of residues 59-75. The reactivity of this synthetic peptide with DTNB mimicked that of the protease, being more reactive in the absence of 6 M guanidine hydrochloride than in its presence. It was possible that His-69 or Lys-70 could facilitate ionization of the SH group of Cys-67, which is required for reaction with DTNB. Apparently both residues are important, because increased reactivity of the native peptide was eliminated when either His-69 or Lys-70 were changed to Ala. Replacement of His-69 by Glu reversed the reactivity, so that the native peptide was less reactive than that denatured in guanidine hydrochloride. Thus, the reactivity of Cys-67 is modulated by the charges on residues 69 and 70 in the protein. The presence of His-69 and Lys-70 renders the native protease especially susceptible to oxidation and disulfide formation. The resulting reversible inactivation of the protease may be important in the normal regulation of the viral life cycle, a suggestion supported by the strong conservation of the residues in this region.

Use of ZetaPrep cartridge for the purification of human recombinant interleukin 1 beta.

Zetaprep mass ion-exchange media represent a rapid and efficient chromatographic tool in the separation of proteins, in place of the conventional agarose or cellulose-based gels. We adopted this method, combined with classical steps, to purify to homogeneity human recombinant interleukin 1 beta (IL-1 beta) produced from E. coli and from S. cerevisiae. An anion exchanger QAE-ZetaPrep was used to achieve a rapid partial purification of both proteins. The IL-1 beta purification was completed by gel permeation chromatography on Sephadex G-50. When the protein was produced from yeast, an intermediate chromatographic step on a hydroxylapatite column was also necessary. The isolated proteins proved to be homogeneous by electrophoresis and amino acid analysis. The biological activity of IL-1 beta produced by E. coli is comparable to that of the natural protein, while the protein produced by yeast showed very low specific activity.

Effectiveness of defibrotide for prophylaxis of deep venous thrombosis in gynecological surgery: a double-blind, placebo-controlled clinical trial.

Defibrotide, a new antithrombotic compound without anticoagulant activity, has been tested for prevention of deep venous thrombosis (DVT) in patients undergoing gynecological surgery (mainly hysterectomy). Eighty-nine women (mean age 48.5) were randomly allocated to defibrotide (44 patients) or placebo (45 patients). 800 mg defibrotide was given daily (200 mg intravenously 4 times a day), starting on the day before operation and then for the next 7 days. DVT were detected by the conventional 125I-fibrinogen test. The two groups were homogeneous for known risk factors (age, varicosities, obesity, neoplasia and previous thromboembolic episodes). The results showed a statistically significant reduction of DVT incidence in patients on defibrotide, as compared with those on placebo: 4/44 = 9% vs. 13/45 = 28.8% (p less than 0.05). There were no side effects, including hemorrhagic complications. The numbers of units transfused were comparable for the 2 groups. In conclusion, the trial shows that defibrotide is an effective and safe drug for the prevention of DVT in gynecological surgery.