The long-term goal of the proposed program is to understand at the amino acid level how complement component C4, which plays a central role in both complement activation and regulation, carries out its numerous functions. In particular, we wish to exploit the tools of molecular biology to examine in depth how structural variations in C4 affect complement activity and the overall functioning of host defenses. An understanding of these relationships will be particularly valuable for interpreting the significance of polymorphic forms of C4 and it may also provide insights into the observed associations of C4 deficiency with immune complex disease and of the C4 gene with the major histocompatibility complex. Site-specific mutagenesis and recombinant DNA technology will be used to genetically engineer variants of C4. These variants will be obtained by placing recently isolated full-length C4 cDNAs into an eucaryotic cDNA expression vector, altering the native sequence by in vitro manipulation, and introducing the altered cDNA, carried by the expression vector, into a mammalian cell line capable of expressing the transfected cDNA as a mature secreted protein. Specific plans include construction and characterization of murine C4 variants designed (a) to pinpoint the structural defect(s) in a non-functional C4 isotype, sex-limited protein (Slp); (b) to assess the roles of glycosylation and sulfation in C4 function; (c) to identify the amino acids responsible for modulating the reactivity of the internal thiolester in C4; and (d) to identify the amino acid replacement(s) responsible for the low hemolytic activity of a natural polymorphic form of murine C4. Properties of the engineered proteins to be evaluated include: (a) hemolytic activity; (b) susceptibility to cleavage by C1s; (c) presence of an internal thiolester; (d) sensitivity to nucleophiles and chaotropes; (e) relative reactivity of the activated thiolester with hydroxyl and amino groups; (f) susceptibility to cleavage by Factor I in the presence of C4-binding protein (C4-bp); and (g) the ability to bind, after activation, to sensitized cells, to C2a, to C4-bp, and to complement receptor CR1.
