The third component of complement is an important molecule in host defense and inflammation. It is a multi-functional protein playing a pivotal role in the complement cascade. We propose to study the structure-function relationship of this molecule. Our approach will take advantage of cobra venom factor (CVF), an analog of C3 occurring in cobra venom. We will perform a comparative analysis of structural properties with the functions of the two molecules in order to identify functionally important regions of the C3 molecule. Two methodological approaches are proposed in this grant application. First, we wish to study the structure and function of the carbohydrate moieties of CVF and human C3. This work will include the chemical characterization of the carbohydrate side chains, their location within the molecules, and their importance for known functions of the two molecules. In order to assess the possible biological significance of the carbohydrate moieties in the infant experiments are proposed to study the expression of the CRl receptor on neutrophils of differing degrees of maturation and their interaction with native and deglycosylated C3. The other experimental approach will be the molecular cloning of CVF and cobra C3 to obtain the nucleotide and amino acid sequences of the two molecules. Comparisons will be done with the known primary structures of murine and human C3 to obtain information on the evolution of the C3 molecule and to locate functionally important regions of the C3 molecule. The work proposed on this application might provide for information allowing to construct a human C3 derivative with CVF-like functions for coupling to monoclonal antibodies as complement-dependent cytotoxic immunoconjugates of low immunogenicity. If CVF and C3 are coded for by a single gene in the cobra genome, our work will also provide for an interesting model to study differential gene expression, RNA splicing, and protein processing in different organs of the same species.
