9319111 Lambris The complement system is a complex group of proteins and glycoproteins which together play a very important role in immune surveillance and immune response pathways. The elucidation of the molecular features related to the functions of the different complement proteins require further analysis of their structure. Preliminary experiments strongly suggest that it is possible to identify the structural features of complement proteins that are important for their functions by comparing the primary structure of these proteins from different species, "natural analogs", and correlating this information with the ability of these proteins to cross react with proteins from other species. Except for the third complement component, C3, very little is known about the presence and nature of other complement proteins in species other than mammals. This laboratory has cloned trout (Tr), Xenopus (Xe), and chicken (Ch) C3 and provided preliminary data that trout and Xenopus have proteins with factor I, B, H, and D like activities. The goal of this project is to analyze the structure and functions of the alternative forms of C3 from trout and Xenopus and characterize the trout alternative pathway complement proteins, factor H and B. At the protein level, the objectives will be to purify the different forms of C3 from Xenopus and trout plasma and test their ability to react with human complement proteins CR1, CR2, H, B, I, C5 and P as well as with autologous H and B. Factor H and B will also be isolated from trout plasma and their function tested in the alternative complement pathway. A previously established purification procedure for mammalian complement proteins including PEG precipitation, Mono Q anion exchange, Mono S and gel filtration chromatography will be used to purify Xe, and Tr C3 and trout factors H and B. The binding of C3-binding proteins to trout C3 will be analyzed by direct binding ELISA, by binding inhibition assays. The functions of factor H will be analyzed by testing its ability to inhibit the C3bBb convertase, the binding of Factor B to C3b, and to exert cofactor activity in the cleavage of C3b by factor I. The function(s) of factor B will be investigated by testing its ability to bind C3b and cobra venom factor (CVF) and to form the C3bBb/CVFBb convertase. Amino acid sequence information for factors H and B will be obtained by sequencing their proteolytic fragments (HPLC purified or electroblotted to PVDF membranes). Oligonucleotide probes constructed from the obtained amino acid sequences, cDNA probes or affinity purified anti-C3 antibodies will be used to screen cDNA liver libraries to obtain complete primary structure for the alternative forms of C3 and that of factor H and B. cDNA liver libraries from both species have been prepared and partial cDNA clones for the alternative forms of Xe and Tr C3 have been isolated. The obtained sequences will be compared and correlated to functional data. Of special interest are the protein segments known to mediate the binding with other complement proteins. The proposed studies, in addition to the phylogenetic data on the alternative pathway of the complement system will provide basic information on the structural features of C3 and its binding proteins as they relate to their functions. %%% The complement system is a complex group of proteins found in the blood of vertebrate animals. These group of proteins together play a very important role in immune surveillance and immune response pathways. Understanding of how the molecular features of these proteins relate to their functions requires further analysis of their structure. One effective approach to identifying the structural features of complement proteins that are important for their functions is to compare the primary structure of these proteins from different species of animal and relating this information to the ability of these proteins to act in conjunction with proteins from other species. In add ition to contributing to the understanding of the relationship of complement proteins to their function, the results of this research will provide important new information on the immune defense systems of fish and amphibians. ***
