The overall objective of this research program is to contribute to current understanding of the formation, regulation and mechanism of action of the two key enzymes of complement activation, the classical and alternative pathway C3-convertases. To fulfill this goal we will continue collecting detailed information at the molecular level on the proteins participating in the assembly of these bimolecular enzymes. The emphasis will be on defining the structure of binding sites on C2 and B and on describing structural correlates of function of complement protein D. We propose to approach our objectives by pursuing three specific aims: 1) The topology and chemical nature of the C4b-binding sites on C2b and C2a will be described by using monoclonal antibodies of predetermined specificity and site-directed mutagenesis. Mutant C2's will be the products of the full- length C2 cDNA C2HL5-3, altered at key codons and expressed transiently in COS cells or permanently in CHO (dhfr-) cells by using the p91023 expression vector. 2) Structural correlates of function of complement protein B will be defined by following a similar methodology. The full- length cDNA clone for B, lambda BHL4-1 will be initially characterized and expressed as above. C3b and other binding sites of B will be described by using mutants and/or monoclonals. 3) Mechanisms regulating the enzymatic activity of D will be investigated. We will initially isolate, characterize and express a full-length cDNA for D. Investigation of structural correlates of function will then proceed in parallel with collaborative studies aimed at describing the crystal structure of the enzyme. We believe that the acquisition of detailed information at the molecular level on the complex protein-protein interactions leading to C3 cleavage represents and obligatory step towards the important goal of pharmacologic manipulation of complement activation during human diseases.
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