Decay accelerating factor (DAF) is a surface glycoprotein which protects host cells from attack by autologous complement. Complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH) lack DAF. The affected cells, however, also lack acetylcholinesterase (AChE) as well as other membrane factors. Recent studies in our laboratory have revealed that DAF is anchored to cells by a C-terminal glycolipid structure which closely resembles that in AChE. This unconventional anchor is similar to C-terminal structures of Thy- 1 antigen and of trypanosome variant surface glycoproteins (VSGs) which are thought to be added to these surface proteins during a post-translational modification. We have found that soluble DAF molecules which resemble hydrophilic forms of these other proteins are present in numerous bodily fluids. The proposed experiments are directed at 1) analysis of the distribution of DAF in tissues, further structural characterization of DAF forms, and investigation of the mechanism of formation of the extracellular DAF species, 2) identification of biosynthetic precursors to study glycolipid anchor assembly/attachment in human cells and exploitation of the probes to characterize the steps involved in DAF anchor incorporation, 3) isolation of DAF cDNA and use of the cDNA to establish whether a C-terminal extension peptide absent from DAF protein is predicted as in VSG and Thy-1 cDNA and to determine whether DAF genomic DNA and DAF message are normal in PNH, and 4) investigation of DAF biosynthesis with specific attention to glycolipid anchor attachment in affected lymphocytes of PNH patients and characterization of any abnormalities. The information gained from the proposed studies of DAF could not only have relevance for PNH and molecular mechanisms accounting for the natural ability of host cells to resist injury from autologous effector systems, but could also provide insights about the expression of other glycolipid anchor- associated proteins.
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