Sucharit Bhakdi

Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes

The action of the 107 kDa hemolysin from Escherichia coli on planar lipid membranes was investigated. We report that a single toxin molecule can form a cation-selective, ion-permeable channel of large conductance in a planar phospholipid bilayer membrane. The conductance of the pore is proportional to that of the bulk solution, indicating that the channel is filled with water. A pore diameter of about 2 nm can be evaluated. The pore formation mechanism is voltage-dependent and essentially resembles that of pore-forming colicins; this implies that opening of the channel is dependent on transfer of an electrical charge through the membrane. We propose that the physiological effects of E. coli hemolysin result from its ability to form ion channels in the membrane of attacked cells, and show that there is quantitative agreement between the effects of this toxin on model membranes and its hemolytic properties.

Interaction of complement S-protein with thrombin-antithrombin complexes: A role for the S-protein in haemostasis☆

Complement S-protein is known to function as an inhibitor of the terminal complement sequence (Bhakdi, S. and Tranum-Jensen, J., Biochim. Biophys. Acta 737, 343, 1983). We here report that the S-protein may also play a functional role in haemostasis. Electro-immunoassays performed with the use of poly- and monoclonal antibodies to the protein revealed that it binds to thrombin-anti-thrombin III (T X AT-III) to form stable S X T X AT-III complexes. These complexes form naturally during blood coagulation. They have been identified in human serum and have also been generated in vitro with the respective purified proteins. Formation of the complexes is paralleled by a net protection of thrombin towards the inactivating effects of AT-III, demonstrable in functional assays using a synthetic polypeptide or fibrinogen as substrates for the protease. S-protein may thus exert a promoting effect on blood coagulation and could emerge as a novel component of the blood coagulation system in the future.

Sensitive ELISA for quantitating the terminal membrane C5b-9 and fluid-phase SC5b-9 complex of human complement

Activation of the complement system to completion results either in the generation of a pore-forming, cytolytic C5b-9(m) complex, or of a cytolytically inactive, fluid-phase SC5b-9 complex. In this paper, we describe a sensitive and reliable, sandwich ELISA for C5b-9(m) and SC5b-9, which is based on the use of a monoclonal antibody to a neoantigen of C5b-9 in combination with affinity-purified, polyclonal rabbit antibodies. The ELISA has been calibrated with purified C5b-9(m) and SC5b-9, and can detect 3 ng/ml C5b-9(m) and 20 ng/ml SC5b-9. We show that maximal conversion of C5-C9 in pooled human serum by insulin or zymosan activation generates 220 +/- 40 micrograms/ml SC5b-9. 65 of 100 normal human EDTA plasma samples analyzed in this study contained 100-600 ng/ml SC5b-9, corresponding to 0.04-0.24% of maximal conversion. Levels of circulating SC5b-9 in other donors were below the limit of detection. Incubation of serum at 37 degrees C always led to spontaneous generation of SC5b-9; concentrations ranged from 490-4725 ng/ml after 60 min, 37 degrees C, with a mean of 1848 +/- 1031 (SD) ng/ml amongst 25 donors studied. The terminal complement complex present in EDTA plasma was partially purified by PEG precipitation, DEAE-ion exchange chromatography and sucrose density gradient centrifugation, and was found to contain C8, C9 and S-protein as demonstrable by SDS-PAGE immunoblotting. Thus, the material most probably represented genuine SC5b-9. No significant age- or sex-dependent variations in SC5b-9 levels were noted. The present data call for a critical re-appraisal of several previously published methods for the determination of SC5b-9 levels in human plasma and serum.

Functions and relevance of the terminal complement sequence

The terminal complement sequence is initiated upon cleavage of C5 with liberation of C5a anaphylatoxin, and involves the assembly of macromolecular C5b-9 complexes either on cell surfaces or in plasma. Cell-bound C5b-9 complexes generate transmembrane pores that can cause cell death, or they can elicit secondary cellular reactions triggered, for example, by passive flux of calcium ions into the cells. In vivo functions of the fluid-phase SC5b-9 complex have not yet been defined, but the identity of S-protein with vitronectin (serum spreading factor) provokes the anticipation that significant biological functions of this complex do exist. The terminal complement sequence may fulfill protective functions when it is triggered on alien cells that are marked for destruction. Dysregulation in the complement sequence may, however, result in detrimental attack by C5b-9 on autologous cells. Examples include not only autoimmune disease states, but also the activation of complement on dead or dying cells, and bystander attack on blood cells during cardiopulmonary bypass. Methods for detecting and quantifying C5b-9 are outlined, and the potential usefulness of such assays in clinical research is discussed.

[24] Preparation and isolation of specific antibodies to complement components

Publisher Summary Complement activation through either the classical or alternative pathway generally leads to the deposition of complement components on the target cell surface. Immunoassays for C3and C4 and their molecular derivatives have contributed to the study of the role of the complement system in diverse immunopathological processes. This chapter discusses the preparation and isolation of specific antibodies to complement components. Complement activation can occur in two physicochemical environments—i.e., in solution (e.g., serum) or on a target cell membrane. In the former case, a noncytolytic “SC5b-9” complex consisting of noncovalently associated C5b-C9 in a molar ratio of 1 : 1 : 1 : 1 : 2-3 plus an additional “S” protein from serum is created. This complex may be isolated from inulin-activated human serum and can serve as material for immunization for raising antibodies to the C5b-9 complex. Studies have shown that antisera to SC5b-9 do not differ qualitatively from those raised against the membrane-derived complex. However, SC5b-9 preparations still contain several serum protein contaminants. Therefore, immunization with the membrane C5b-9 complex is recommend, which is easy to purify in adequate amounts from target cell membranes.

A simple method for quantitative measurement of C3d in human plasma

We describe a simple and reliable method for quantitating C3d in human plasma. The method rests on the finding that native C3 and its activation/inactivation product C3c bind to Concanavalin A, whereas C3d does not. Rocket affinoimmunoelectrophoresis with Con-A Sepharose incorporated into an intermediate gel permits quantitation of free C3d in 2-20 microliters aliquots of EDTA-plasma without any manipulation prior to sample application. Using this method, we found that the level of circulating C3d in plasma of 30 healthy donors was usually well below 3% of maximally convertable C3d.

Membrane damage by channel-forming proteins

Abstract Certain hydrophilic proteins self-associate on target cell membranes to form amphiphilic, channel-structured protein complexes whose embedment within the bilayer perturbs the membrane permeability barrier.

Plasma C3d/C3 quotient as a parameter for in vivo complement activation.

Plasma levels of C3 and C3d in 30 healthy donors and 58 patients with systemic lupus erythematosus were determined by rocket immunoelectrophoresis and Con-A affinoimmunoelectrophoresis. Significantly decreased levels of C3 were found in approximately 50% of the SLE sera. Slight increases of free C3d were found in 55%, and marked increases in 9% of the SLE sera; the remaining SLE sera did not show increased C3d levels. When results were expressed as quotients of plasma C3d/C3, a significant pattern emerged and 70% of the SLE sera clearly fell outside the normal range. 10% of the SLE sera had normal C3d/C3 indices but had markedly depressed C3 levels. We conclude that in cases where total serum C3 is not markedly reduced (less than 60% of normal), the C3d/C3 quotient may be a more sensitive parameter for assessing in vivo complement activation than C3 or C3d determinations alone.

Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolyticaIly inactive, fluid-phase C5b-9 complexes, Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3BI, 3Dg, and 2F3), sheep (clones 3Dg and 2F3) and guinea-pig (clone 3D8) neoantigens were identified . Three of four tested clones (3D8, 2F3, IA12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.

Electroimmunoassay-Immunoblotting (EIA-IB) for the utilization of monoclonal antibodies in quantitative immunoelectropheresis: the method and its applications

Immunoprecipitates formed in conventional electroimmunoassays were solubilized by brief immersion of the agarose gels in sodium dodecylsulfate. The proteins were then transferred to nitrocellulose sheets by electroblotting. The blotted proteins were readily amenable to analyses by non-precipitating monoclonal antibodies and the immunoblots were developed with second antibody/biotin-streptavidin-peroxidase staining. The electroimmunoassay-immunoblot (EIA-IB) method is of value in (1) specificity assays of monoclonal antibodies in crossed immunoelectrophoresis; (2) analysis of specific molecular interactions between proteins; (3) rapid screening and simple identification of monoclonal antibodies by line immunoelectrophoresis-immunoblotting.