GENETIC ENGINEERING OF SHORT CONSENSUS REPEAT ELEMENTS

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The short consensus or complement repeat (SCR) is a modular protein structural unit that is usually encoded in distinct gene exons. SCRs have recently become the focus of substantial interest because they have been found in a wide range of apparently unrelated proteins including a component of the blood coagulation system, a vaccinia virus-encoded protein, a lymphokine receptor, 3 cellular adhesion receptors, and at least 10 complement proteins. SCRs range from 60-70 amino acids in length, they contain 4 or 6 conserved cysteines, and have a consensus sequence involving about 40% of the residues. They are likely to be independent structural units, similar in concept to immunoglobulin domains, consisting of a basic framework structure onto which sequence variations related to specific functions are superimposed. The function(s) of SCRs are in many cases unknown but their wide distribution suggests that they fulfill crucial structural and functional roles. They have been most extensively studied in the complement proteins. In most and perhaps all of these cases, their primary function is to bind to complement components C3 and C4, and perhaps secondarily to provide a linear, extended structure. Binding of SCR-containing proteins to both C3 and C4 are essential for activation and regulation of the two complement pathways. The binding of SCR-containing proteins to C3 and C4 is specific; with distinct SCRs binding to either C3 or C4. This grant proposes to use genetic engineering methods to dissect the interaction between SCRs and complement components C3 and C4. In particular we plan to identify amino acids within SCRs, and within C3 and C4 which are necessary for binding, and for binding specificity. As models of this interaction, we will examine the binding of murine C4 to murine C4-binding protein, and of murine C3 to murine factor H. The long-term goal of the program proposed is to understand at the amino acid sequence level the rules governing the binding specificities of SCRs and the structural features of their protein targets. This understanding should provide clues to the specificities and functions of these structural motifs in other (especially non- complement) proteins, and may provide a more unified understanding of the interactions and evolutionary relatedness of both soluble and membrane-bound proteins in the bloodstream. The ability to alter the specificities or reactivities of these proteins is likely to have implications for the eventual development of therapeutic agents.