Structural/Functional Analysis of Chicken and Lamprey C3

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C3 plays a critical role in complement activation due to its ability to bind to other complement proteins, and interact with several cell surface receptors. The elucidation of the molecular features related to these C3 associated functions requires further analysis of its structure. The goal of this project is to analyze the structure and functions of C3 from two phylogenetically key species, the chicken and the lamprey, and to compare and correlate this information with what is known about mammalian C3 in order to further our understanding of this molecule. At the protein level, the objectives will be to purify C3 from the plasma of chickens and lampreys and test their ability to react with human complement proteins CR1, CR2, CR3, C5, H, B, I, and P as well as with autologous complement receptors. Previously established purification procedures will be used. The binding of C3-binding proteins to C3 from the two species will be analyzed by direct binding ELISA, by binding to C3 fragment-coated erythrocytes, and by inhibition assays. Amino acid sequences for the different C3s will be obtained by sequencing proteolytic fragments of C3 . In order to obtain N-terminal amino acid sequences of fragments generated by autologous C3 convertases and factor I (in the presence of H), serum of the two species will be activated by zymosan, the zymosan bound C3 fragments will be eluted, separated by SDS-PAGE, and then blotted to PVDF membranes. The different C3 fragments will be sequenced directly from the PVDF membranes. The presence of carbohydrates and thiolester bonds will also be determined. Oligonucleotide probes constructed from the determined amino acid sequences, cDNA probes or affinity purified anti-C3 antibodies will be used to screen lambda gt11 cDNA liver libraries to obtain complete primary structure for the various C3s. (Lambda gt11 cDNA liver libraries from the two species have been prepared already, and four candidate clones for chicken C3 have been isolated thus far.) The obtained sequences will be compared and correlated to functional data. Of special interest are the C3 segments known to mediate the binding of C3-binding proteins. The interaction of the protein, "C3", with other plasma proteins and with several cell surface receptors make it a key participant in phagocytic and immunoregulatory processes involved in animal defense mechanisms. Preliminary experiments suggest that it is possible to identify the structural features of C3 that are important for its functions by comparing the C3 amino acid sequences between species, and correlating this information with the ability of these C3s to bind to different C3-binding proteins. The proposed studies, in addition to providing new information on the phylogenetic history of C3, will provide basic information on how the structural features of C3 relate to its multiple functions.