Reconstitution of C5 convertase of the alternative complement pathway with isolated C3b dimer and factors B and D.

C5 convertase of the alternative complement pathway is a trimolecular complex consisting of two molecules of C3b and one molecule of Bb. We previously proposed a model of the alternative pathway C5 convertase in which the second C3b molecule binds covalently to the first C3b molecule bearing Bb, and the C5 molecule binds to each C3b molecule of the covalently linked C3b dimer, resulting in its appropriate presentation to the catalytic site on Bb. In the present study, we purified the covalently linked C3b dimer and reconstituted the C5 convertase with the C3b dimer and factors B and D to obtain evidence in support of this model. An insoluble glucan, OMZ-176, was incubated with human serum to activate the alternative pathway and to allow formation of the alternative C5 convertase on the surface of the glucan, and the glucan bearing the C5 convertase was then solubilized by incubation with glucosidases. In this way, the covalently linked C3b dimer was obtained in solution without using a detergent. The C3b dimer was then separated from enzymes, C3b monomer, C3b oligomer, and other materials by chromatographies. SDS-PAGE analysis demonstrated that the purified C3b dimer had intact alpha'-chains. Alternative pathway C5 convertase was reconstituted when the isolated C3b dimer was incubated with factors B and D. The presence of P enhanced C5 convertase formation threefold. These results support the notions that the formation of the covalently linked C3b dimer is a general phenomenon associated with activation of the alternative pathway and that the C3b dimer acts as a part of the C5 convertase.

[A factor from the venom of the Central Asiatic cobra Naja naja oxiana, which inactivates component C4 of human complement]. / Faktor iada sredneaziatskoi kobry Naja naja oxiana inaktiviruiushchii komponent C4 komplementa cheloveka.

An acid glycoprotein (mol. m. 60 kDa) containing 6 sialic acid residues and N-terminal Thr was isolated from the venom of the central asian cobra Naja naja oxiana. The protein has an anticomplementary activity selectively inactivating of the C4 component of the human complement. This factor (CFA-Ib) binds C4 with Ki = 0.27 +/- 0.13 microM and then irreversible inactivates it with a rate constant k = 0.75 +/- 0.25 min-1. Membrane bound C4b restores its ability of CFA-Ib binding. This binding hinders component C2 sorption on C4b and C3 convertase formation.

C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme.

C5 convertase of the alternative C pathway is a complex enzyme consisting of three C fragments--one molecule of a major fragment of factor B (Bb) and two molecules of a major fragment of C3 (C3b). Within this C3bBbC3b complex, the first C3b binds covalently to the target surface, and Bb, which bears a catalytic site, binds noncovalently to the first C3b. In the present investigation, we studied the nature of the convertase that is assembled on E surfaces and obtained evidence that the second C3b binds directly to the alpha'-chain of the first through an ester bond rather than to the target surface. Thus, the alternative pathway C5 convertase could be described as a trimolecular complex in which Bb binds noncovalently to a covalently linked C3b dimer. We also obtained evidence that not only the second C3b but also the first C3b participates in binding C5, that is, covalently-linked C3b dimer acts as a substrate-binding site. Because of this two-site binding, the convertase has a much higher affinity for C5 than the surrounding monomeric C3b molecules. Based on this evidence, a new model of the alternative pathway C5 convertase is proposed. Covalent association of two subunits and the bivalent binding of the substrate are then common properties of the alternative and classical pathway C5 convertases.

A simple isolation procedure for functionally pure components of the bovine alternative complement pathway (ACP) C3 convertase and bovine conglutinin (K).

A simple multicomponent isolation procedure for bovine C3, factor B, factor D and conglutinin (K) from a single serum sample is described. The components of the alternative pathway C3 convertase were isolated in milligram quantities from 800 ml bovine serum and were found to be functionally pure with respect to each other and to factors H and I.

Purification and characterization of the C3 convertase of the classical pathway of human complement system by size exclusion high-performance liquid chromatography.

The C3 convertase of the classical pathway of the complement system is a liable complex, C4b,2a, and is activated by limited proteolysis of two components, C4 and C2, by C1s. By utilizing iodine-treated C2 and size exclusion high-performance liquid chromatography (HPLC), we have succeeded in isolating for the first time the classical pathway C3 convertase. Size exclusion HPLC demonstrated that the apparent molecular mass of the C3 convertase was 280K daltons. The C3 convertase decay-dissociates spontaneously into C4b and C2a. The decay-dissociation is a temperature-dependent reaction and the half-lives of the C3 convertase at 24, 30, and 37 degrees C were estimated to be 400, 180, and 60 min, respectively. The decay-dissociation was also dependent on pH and was accelerated by increasing pH. In addition, the decay-dissociation of the C3 convertase was accelerated by C2b. This result suggests that C2b acts as a feedback inhibitor on the activation of the classical pathway of complement system.

Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in the complement regulatory protein, decay accelerating factor.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired defect of bone marrow stem cells in which the affected clones produce erythrocytes (also granulocytes and platelets) with membranes that are abnormally sensitive to complement-mediated lysis. Abnormal erythrocytes (E) from patients with PNH (PNH-E) are 3-5 times more sensitive (type II PNH-E) or 15-25 times more sensitive (type III PNH-E) to lysis in vitro by human complement than normal E from unaffected individuals and the functionally normal E that arise from unaffected clones and the functionally normal E that arise from unaffected clones in PNH patients (type I PNH-E). After complement activation by either the classical or alternative pathway, abnormal amounts of C3b are deposited on the membranes of PNH-E compared with normal E, suggesting that the PNH-E membrane cannot regulate the events responsible for C3b deposition. Two proteins that decrease the stability of the classical and alternative pathway C3 convertases on target cells have been isolated from normal human E stroma: the 70,000 Mr decay accelerating factor of stroma (DAF) and the 250,000 Mr C3b receptor (C3bR). Specific immune precipitates of solubilized membranes from 125I-surface-labeled normal E demonstrate both proteins. In contrast, specific immune precipitates of PNH-E from three patients show C3bR but are deficient in DAF; type II PNH-E are relatively deficient and type III PNH-E are totally deficient in DAF. Antibody that neutralizes the activity of isolated DAF is adsorbed by intact normal E under conditions in which it is weakly adsorbed by type II PNH-E and not adsorbed by type III PNH-E. The deficiency of DAF antigen in PNH-E, as assessed by lack of immunoprecipitation and antibody adsorption, could explain the abnormal sensitivity of PNH-E to complement-mediated lysis and suggests that DAF may protect the membranes of normal E from damage resulting from autologous complement activation.