IDENTIFICATION OF 3 PHYSICALLY AND FUNCTIONALLY DISTINCT BINDING-SITES FOR C3B IN HUMAN-COMPLEMENT FACTOR-H BY DELETION MUTAGENESIS

Human complement factor H controls spontaneous activation of complemen t in plasma and appears to play a role in distinguishing host cells fr om activators of the alternative pathway of complement, In both mice a nd humans, the protein is composed of 20 homologous short consensus re peat (SCR) domains, The size of the protein suggests that portions of the structure outside the known C3b binding site (SCR 1-4) possess a s ignificant biological role. We have expressed the full-length cDNA of factor H in the baculovirus system and have shown the recombinant prot ein to be fully active, Mutants of this full-length protein have now b een prepared, purified, and examined for cofactor activity and binding to C3b and heparin, The results demonstrate (i) that factor H has at least three sites that bind C3b, (ii) that one of these sites is locat ed in SCR domains 1-4, as has been shown by others, (iii) that a secon d site exists in the domain 6-10 region, (iv) that a third site reside s in the SCR 16-20 region, and (v) that two heparin binding sites exis t in factor H, one near SCR 13 and another in the SCR 6-10 region, Fun ctional assays demonstrated that only the first C3b site located in SC R 1-4 expresses factor I cofactor activity, Mutant proteins lacking an y one of the three C3b binding sites exhibited 6- to 8-fold reductions in affinity for C3b on sheep erythrocytes, indicating that all three sites contribute to the control of complement activation on erythrocyt es. The identification of multiple functionally distinct sites on fact or H clarifies many of the heretofore unexplainable behaviors of this protein, including the heterogeneous binding of factor H to surface-bo und C3b, the effects of trypsin cleavage, and the differential control of complement activation on activators and nonactivators of the alter native pathway of complement.