Complement component H is a member of a superfamily of proteins that are comprised in part or in whole, of 60-aa short consensus repeats (SCRs) that display a characteristic framework of highly conserved residues. The long- term objectives of this study are to better understand the biosynthesis and regulation of expression of H and the correlation of its structural framework of SCRs with its function. Further knowledge of this molecule will enable its comparison with other members of this family, which include the interleukin-2 receptor and the C3d/Epstein-Barr virus receptor. To approach these objectives, the following specific aims are proposed: (1) examination of the 5' region of the murine H gene and its tissue-specific regulation, (2) determination of the functional regions in H necessary for C3 binding and I cofactor activity and (3) investigation of the H-related transcripts observed in murine liver. Various tissues and cell-lines will be examined for expression of H mRNA and protein using NOrthern blot analysis, RNase protection assays and metabolic labeling experiments. The effect of various immunomodulatory agents on H mRNA expression and biosynthesis will be assessed. 5' genomic DNA sequence will be cloned into a plasmid containing the luciferase gene as a reporter gene to determine which regions are important for regulation of the H gene. The functional domains of H will be determined by a combination of deletion and site- directed mutagenesis of H cDNA sequence. These mutants will be constructed in a eucaryotic expression vector, transfected into COS cells and the resultant proteins assessed for their ability to bind C3b and to act as a cofactor in the I-mediated cleavage of C3b. The H-related transcripts, cDNA clones and cosmid clones that have been observed will be further characterized. The tissue-specific expression of these transcripts will be determined by Northern blot analysis and RNase protection assays. The protein encoded by these cDNAs will be expressed in COS cells and assessed for functional activity. The loci in the genome for these sequences will be more precisely mapped using pulse-field gel electrophoresis.
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| Munoz-Canoves, P; Vik, D P; Tack, B F (1990) Mapping of a retinoic acid-responsive element in the promoter region of the complement factor H gene. J Biol Chem 265:20065-8 |
