Multimeric interactions between complement factor H and its C3d ligand provide new insight on complement regulation.

Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations. Surface plasmon resonance studies showed that native FH binds C3d in two stages. An equilibrium dissociation constant K(D)(1) of 2.6 microM in physiological buffer was determined for the first stage. Overlay experiments indicated that C3d formed multimeric complexes with FH. X-ray scattering showed that the maximum dimension of the C3d complexes with SCR-16/20 at 29 nm was not much longer than that of the unbound SCR-16/20 dimer. Molecular modelling suggested that the ultracentrifugation and scattering data are most simply explained in terms of associating dimers of each of SCR-16/20 and C3d. We conclude that the physiological interaction between FH and C3d is not a simple 1:1 binding stoichiometry between the two proteins that is often assumed. Because the multimers involve the C-terminus of FH, which is bound to host cell surfaces, our results provide new insight on FH regulation during excessive complement activation, both in the fluid phase and at host cell surfaces decorated by C3d.

Genetic fusion of three tandem copies of murine C3d sequences to diphtheria toxin fragment B elicits a decreased fragment B-specific antibody response.

Previous studies have demonstrated that the covalent modification of target protein and polysaccharide antigens with the activated complement product C3d results in dramatically enhanced immunogenicity of the target antigens. In this paper, we describe our attempts to enhance the immunogenicity of the non-toxic B fragment of diphtheria toxin (DT-B) by genetic fusion to polypeptides derived from the C3d coding sequence. Contrary to expectations, we found that the antibody responses elicited by immunizing mice with DT-B genetically linked to three tandem copies of C3d-derived sequences were markedly reduced relative to the antibody responses elicited by immunizing mice with DT-B alone. These results demonstrate levels of complexity in the immunomodulatory effects of the complement system that were not apparent in earlier reports on the adjuvant effects of C3d administered to mice as genetic fusions to target antigens, such as hen egg lysozyme, human immunodeficiency virus (HIV) gp120 or influenza virus hemaglutinnin. The data presented herein suggest that C3d may act as a negative regulator, in some immunological contexts, for antibody production in the mammalian host.

Structure at 1.44 A resolution of an N-terminally truncated form of the rat serum complement C3d fragment.

Complement component C3 plays a key role in the complement-mediated immune defence, and occupies a central position within the complement cascade system. One of its degradation products, C3dg, was purified from rat serum and crystallised in two different crystal forms as N-terminally truncated fragment. Despite the truncation and the lack of a significant portion of the N-terminus as compared to C3d, the structure of the fragment is highly similar to that of recombinant human C3d (Nagar et al., Science 280 (1998) 1277-1281). Structural details of the reactive site have been obtained, suggesting a possible mode of thioester bond formation between Cys-1010 and Gln-1013 and thioester bond cleavage in the transacylation reaction involving His-1126. The truncation at the N-terminus of C3d leads to the exposure of a surface of the molecule that favours dimerisation, so that in both crystal forms, the fragment is present as a dimer, with monomers related by a two-fold axis.

Ubiquitination and dimerization of complement receptor type 2 on sheep B cells.

Complement receptor type 2 (CR2) is a membrane-anchored glycoprotein that specifically binds C3d, as well as other ligands, and plays diverse roles in regulating immunity. Here we show that two distinct isoforms of CR2 are expressed on the surface of sheep B lymphocytes. One (CR2no 150 kDa) is structurally similar to known mammalian homologues while the other (CR2ub 190 kDa) has been modified by the covalent attachment of ubiquitin to the cytoplasmic domain and is identified for the first time. CR2no and CR2ub are expressed on the surface of sheep B cells as noncovalently associated dimers and the external topography of the two isoforms differs in some respect. The basis for these unusual higher-order structural properties may lie in the primary sequence of sheep CR2, since the transmembrane domain contains a region resembling a rare 7-amino acid dimerization motif, and two lysine residues in the cytoplasmic domain provide potential sites for posttranslational ubiquitination. The primary structures of sheep ubiquitin and C3d ligand are extensively conserved. In conjunction with the results of separate in vivo studies, these findings suggest that selective ubiquitination plays a role in modulating the higher-order structure and/or expression of CR2 during B cell development.

Isolation of the C3 complement component and its C3d subunit from IY-1 fraction of Cohn's fractionation of human plasma.

C3 complement component and its C3d subunit were isolated from the IY-1 Cohn's fraction, which is the waste of industrially produced albumin and immunoglobulins. The first step was the fractionation of precipitate IY-1 by polyethylene glycol (PEG) 4000 to a final concentration of 16% PEG. The precipitate formed was separated by centrifugation. The supernatant contained the C3d subunit of C3, and the redissolved 16% PEG precipitate contained the C3 component. Then the supernatant and the dissolved precipitate were subjected to anion-exchange chromatography on DEAE-Toyopearl 650 M. In the last step fractions containing C3 and C3d concentrated by ultrafiltration were chromatographed on Sephacryl S-200.

Isolation of the porcine complement activation fragments C3c and C3d and their use in the preparation of monospecific antibodies.

The hemolytic test to date has been the sole analytic technique applied to study the complement reaction in the swine. To improve the analytical possibilities for this species we have developed polyclonal antibody reagents with specificities for the C3c and C3d activation fragments of swine C3. Access to these reagents, by which activation products can be analysed in tissues and biological fluids, will offer new possibilities for a more precise analysis of the complement reaction.