Decay-accelerating factor (DAF) functions intrinsically in the plasma membranes of host cells to prevent autologous complement activation on their surfaces. The protein comprised of four -61 amino acid long short consensus repeats (SCRs) linked through a membrane proximal stock to a glycoinositol phospholipid (GPI) membrane anchor is freely mobile on cell surfaces and acts by destabilizing the amplification convertases of the cascade wherever autologous deposited C4b or C3b elicit their assembly. DAF's activity is essential physiologically as its deficient expression in paroxysmal nocturnal hemoglobinuria (PNH) underlies the heightened uptake of autologous C3b fragments that characterizes affected cells and contributes to disease pathogenesis. Considerable data have shown that DAF's activity is also important in a wide range of other clinical settings including neoplasms where it can inhibit immune attack and infectious and inflammatory states where augmented expression levels are needed to protect effector cells and nearby tissues from local inflammation. In previous work, we 1) isolated the DAF promoter region and mapped the locations of enhancer and response elements within it, 2) developed methods for producing large quantities of recombinant GPI-anchored variant DAF and other C4b/C3b regulatory proteins and for sensitively analyzing their functional properties, 3) demonstrated that the DAF deficit in PNH cells resides in defective GPI anchor biosynthesis and showed that PNH cells harbor a common defect in assembly of N-acetyl-D- glucosamine (G1cNAc)-inositol phospholipid, the first intermediate of the intracellular GPI anchor synthetic sequence, 4) analyzed subsequent intermediates of the GPI anchor synthetic pathway and prepared human K562 cell mutants with defined lesions in a number of different steps, and 5) characterized PNH marrow and formally demonstrated that affected DAF and unaffected DAF populations of CD34+, CD38 self-renewing progenitors are present. The experiments proposed in this application are directed at 1) characterization of cis sequence elements and transacting nuclear factors that participate in regulation of DAF gene transcription, 2) identification of the structural domains within the DAF molecule which mediate its critical regulatory activity on complement as well as its newly defined activity on certain types of cell-mediated cytotoxicity, 3) analysis of the genetic alteration(s) responsible for defective synthesis of G1cNAc-inositol phospholipid in different PNH patients, 4) isolation and characterization of the genes encoding other enzymes in the GPI anchor biosynthetic pathway, and 5) development of protocols for selectively depleting PNH patients of affected progenitors and returning their unaffected self-renewing CD34+, CD38 stem cells to them via autologous marrow transplants.
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