Over the past 10 years, recombinant DNA cloning and sequencing studies have elucidated the primary structures of the complement protein family that is composed of components C3, C4, and C5. This application proposes to use this sequence information, together with protein expression and mutagenesis methods, and a peptide inhibition strategy to probe the structural basis for the most important functional property shared by these 3 proteins: activation by proteolytic cleavage. Despite having very similar sequences at their activation sites, C3, C4, and C5 are specifically cleaved by distinct proteases. The proposed studies are aimed at identifying the structural features of C3 and C5 that are important for recognition by their specific proteases. The mutational strategy to be used focuses on regions marked by length polymorphisms (indels) in this protein family. This strategy was chosen because indels are usually associated with loops on the protein surface, and surface loops or turns are, in general, likely to be involved in a variety of intermolecular recognition events. Indels may play an important role in the evolution of distinct functions among members of a protein family. In providing a rationale for identifying the protease recognition sites on C3 and C5, this strategy suggests a comprehensive view of the structures of these proteins which combines what we know of their primary structures, the structures of their genes (indels are often found at intron-exon junctions), and the functional properties of the native proteins. Hence, the proposed studies also provide a test of this view. The long-term goal of this research program is to identify, for each member of this family, the structural features that together form their unique biochemical and functional properties. This information will provide detailed molecular insights into how these proteins function, and into complement function in general, as these proteins interact directly with most other complement proteins, and are the focus of complement activation, regulation, and complement receptor-mediated cellular interactions. This insight into complement function may aid in the design of intervention strategies such as the use of synthetic peptide inhibitors in situations where complement activation is undesirable, such as in chronic inflammation and in the hyperacute rejection associated with xenogeneic tissue transplantation.
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