Phylogeny of the third component of complement, C3: Analysis of the conservation of human CR1, CR2, H, and B binding sites, concanavalin A binding sites, and thiolester bond in the C3 from different species

Abstract

The third component of complement, C3, binds to several other complement proteins. To study the diverse reactivities of C3, we analyzed the conservation of structural and functional features in the C3 from different species. First, we developed a method to purify swine (Po), rabbit (Rb), mouse (Mo), cobra (Co), Xenopus (Xe), axolotl (Ax), and trout (Tr) C3 from plasma. This involved protein precipitation by polyethylene glycol, followed by anion-exchange, gel filtration, and cation exchange chromatography. All C3's tested were comprised of two chains (α/β-chain) and contain a thiolester bond within the α-chain. The two N-linked high-mannose carbohydrates found in human C3 were only conserved (as detected by ConA binding) in Rb C3. In contrast, Xe, Ax, and Tr C3 have this moiety only in the β-chain and Po and Mo C3 only in the α-chain. Co C3, in contrast to cobra venom factor (CVF), lacks ConA binding carbohydrates in both chains. N-terminal amino acid sequence analysis of the α-, α′-, and β-chains showed varying degrees of similarity within the different C3's. The N-termini of the Xe and Ax C3 β-chains were found to be blocked. The conservation of binding sites in the different C3's for human complement receptors type one (CR1) and two (CR2) and for factors H and B was investigated due to the structural and functional similarities of these molecules and to the ability of some of them to bind to the same domain(s) in human C3. By using microtiter plate-fixed iC3's (ELISA), microsphere-bound iC3's (FACS analysis), and/or zymosan-C3b/iC3b's, it was found that Hu CR1 and CR2, but not Hu H, bind to Xe iC3, and that Hu H, but not Hu CR1 and CR2, interacts with Tr iC3. This suggests that although human H, CR1, and CR2 recognize the same domains in human C3 their exact binding sites are different. Furthermore, zymosan-C3b/iC3b prepared using Xe and Tr serum bound exclusively human CR1 and H, respectively, but did not bind human B. These results indicate that although H, CR1, and B bind to the same domain in C3 their exact binding sites are distinct. In addition, they suggest that the ability of H and CR1 to compete with B for binding to C3b is due to an allosteric or steric effect and not to a competition for the same binding site(s).