COMPLEMENT C5 DEFICIENCY--MOLECULAR ANALYSIS

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The fifth component of complement (C5) is a serum glycoprotein that mediates important inflammatory and cytolytic processes. Sera from C5-deficient individuals lack bactericidal activity and have severely impaired ability to induce chemotaxis. The recent isola- tion and characterization of a full-length cDNA for mouse C5, have thus far allowed us to demonstrate: (a) C5D mice synthesize small amounts (10-20% of normal) but do not secrete single chain intracellular C5 protein; (b) C5D mRNA was quantitatively (lO-fold less) and qualitatively (6.0 and 6.5 kb species in cytoplasm) different from sufficient (C5S) mRNA; (c) restriction fragment length polymorphisms (Hind III and Pvu II) between the C5S and C5D genes correlated with the protein deficiency. The experiments outlined in this proposal will extend these preliminary findings in the mouse and will initiate a parallel study regarding C5 deficiency in humans. We will define the structural abnormalities in the protein synthesized by the C5D cells that interfere with the secretion of the protein. cDNA libraries will be prepared from C5S and C5D mRNA and the C5 specific cDNA will be isolated and characterized to analyze the mRNA sequence abnormality(ies) responsible for production of the abnormal C5 protein. In addition, the full-length C5D cDNAs will be employed in in vitro and in vivo translational systems to determine which C5D mRNA is translated into the non-secreted C5D protein. Genomic libraries (cosmid or YAC) will be prepared from C5S and C5D DNA and C5 specific clones will be isolated and characterized to analyze the structural abnormalities in the C5D gene responsible for the abnormalities in the C5D mRNA. The C5S and C5D genomic clones will be transfected into mouse L-cells and the expression of the genes in these cells will be studied to examine the potential role of the C5D cells in producing the abnormalities in protein secretion. Finally, the defects in the human C5 gene which cause the C5 protein deficiency will be determined from restriction fragment length polymorphisms which correlate with the disease. Also, C5S and C5D genes will be isolated and characterized for structural defects which are ultimately responsible for the protein deficiency.