The long-term objective of this proposal is to increase our understanding of the role of complement in human disease and its interaction with other components of the immune system. The serum complement system in man is important in both the afferent and efferent immune response. The central component or C3 participates as both a cofactor in formation of convertase in the cascade and interacts directly with both humoral and cellular components of the immune system. A critical step in this intersection is covalent attachment of C3 to antibody-antigen complexes on activation of the alternative pathway. Following activation, split products of C3 serve as ligands for specific receptors on blood cells, e.g. CD35, CD21 & CD11b,c/CD18, which are important in such diverse roles as cellular adhesion and B-cell signaling. As a first step towards the long-term objective, this proposal will examine the biochemical interaction between C3 and the antibody molecule. The hypothesis that C3 binds to a specific site on the IgG heavy chain will be tested. The first specific aim is to identify the site/sites of covalent attachment of human C3 onto human IgG1 heavy chain. Preliminary results have shown that the major site of attachment is within the CH1 domain.
This aim will use a biochemical approach (amino acid sequencing and mass spectrometry) to identify the site/sites of attachment. The second specific aim is to identify specific residues for C3 attachment within this region of the heavy chain for both human and murine isotypes by using a site-directed mutagenesis approach. The approach in general is to synthesize recombinant antibody bearing neutral substitutions (e.g., Ala) at potential acceptor sites and test their activity for activation of the alternative pathway. Understanding the role of complement in its interaction with the immune system in host protection is to significant importance. Genetic deficiencies in complement often lead to autoimmune disorders and increased susceptibility to disease. By increasing our understanding of its role, a therapeutic approach can be realized.
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