Complement component C5 is an important participant in inflammatory and cell killing processes. The activation peptide (C5a) possesses potent spasmogenic and chemotactic activity. The macromolecular cleavage product (C5b) expresses a transient binding site for C6. The C5b,6 complex is the foundation upon which the membrane attack complex (C5b-9) is assembled. Earlier comparative studies of the primary structures of the human complement proteins pro-C3, pro-C4 and, more recently, C5 with the human proteinase binding proteins alpha 2- macroglobulin and pregnancy zone protein revealed up to eight regions of extended sequence similarity. It is presumed that these regions of each molecule comprise domains imparting an underlying common gross structure that is modified by other domains specific for each of these proteins. The goals of the proposed studies are to complete the protein structures of human and mouse C5 at the cDNA level, to further characterize C5- specific mRNA prepared from multiple tissues and cell lines with particular attention directed to the prospect of multiple C5- specific transcripts, to determine the molecular defect(s) inherent in the human C5-deficiency state utilizing DNA prepared from EBV-immortalized B-cells of affected individuals and C5-specific cDNA probes and to delineate and compare the gene structures of two members of the complement: proteinase binding protein family, C5 and alpha 2-macrogloblin. Existing well-characterized cDNAs for C5 and alpha 2-macroglobin will be used to screen human cosmid libraries. The genes for C5 and alpha 2- macroglobin will be isolated, restriction-mapped and sequenced by the Sanger dideoxy technique utilizing pre-selected, exon- containing templates produced from sheared DNA and subcloned in M13 and mp8. Attention will also be paid to sequences at the far 5' end of the gene thereby identifying common and perhaps unique control elements for expression of these genes. Correlation of the intron/exon arrangement of each gene with structural and/or functional domains of the protein products will be made. Information derived from the analysis of C5 and alpha 2-macroglobulin gene structures will also be compared to similar data for the human C3 and C4 genes. The collective data for this extended gene family will be used to analyze the pattern of protein structural domain segregation.
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