C4b-binding protein, a multimeric structure composed of seven alpha-chains (C4BPa), 1 beta-chain (C4BPb) and a protein S component, functions as a regulatory component of the complement system. The initial goal of this application is to elucidate structure/function relationships, biosynthesis and regulation of expression for murine C4BP-alpha. Longer-term goals include extending these studies to humans where the potential role of C4BP in normal and autoimmune conditions will be investigated, with emphasis on further insight into pathogenesis and on developing novel methods of therapy.
Specific aims to be addressed during the proposed funding period include (1) an assessment of the effects of immunomodulatory agents (IL-1, IL-6, TNF and dexamethasone) on C4BP-alpha gene expression, (2) the identification of functional regulatory elements in the 5' promoter region of the C4BP-alpha gene and associated transcription factors, (3) the determination of the functional domains in C4BP-alpha necessary for C4b binding and protein I-mediated cleavage of C4b to C4c and C4d and (4) identification and characterization of the murine C4BP-beta cDNA and genomic DNA. Recent studies in the investigator's laboratory have implicated C4BP as an acute phase reactant. In this regard, the steroid hormone dexamethasone has been observed to markedly elevate the level of C4BP-alpha mRNA in mouse liver cells. The effects of additional inflammatory mediators on C4BP-alpha mRNA levels will be assessed by Northern analysis, whereas changes in C4BP synthesis will be studied by biosynthetic labeling experiments. Sequence from the 5' region of the C4BP-alpha gene will be subcloned upstream of the luciferase reporter gene. Deletion constructs will be made to determine which regions have enhancer or regulatory activity. Protein factors involved in transcription of the C4BP-alpha gene will be identified and regions that serve as protein binding sites mapped by performing DNA """"""""footprint"""""""" studies with nuclear protein extracts from appropriate cells. The domains of C4BP-alpha that interact with C4b and protein I will be determined by a combination of deletion and site-directed mutagenesis of C4BP-alpha cDNA. These mutants will be constructed in a eukaryotic expression vector, transfected into COS cells and the resultant proteins assessed for their ability to bind C4b and to act as a cofactor in the I-mediated cleavage of C4b to C4c and C4d. Sequence from human C4BP-beta cDNA will be used to probe lambda clones from the 5' end of the murine C4BP-alpha gene to determine whether the murine alpha and beta genes are tightly linked as they are in humans and rats. Once C4BP-beta specific cDNA and genomic clones are obtained and their structures characterized, regulation of the C4BP-beta gene will be investigated and compared with C4BP-alpha. Expression/mutagenesis studies with murine C4BP-beta cDNA will be undertaken to identify sites in the beta-chain that interact with the protein S component and C4BP-alpha.
| Moffat, G J; Tack, B F (1992) Regulation of C4b-binding protein gene expression by the acute-phase mediators tumor necrosis factor-alpha, interleukin-6, and interleukin-1. Biochemistry 31:12376-84 |
