The complement decay accelerating factor (DAF) functions intrinsically in the membranes of blood cells to prevent autologous complement activation on their surfaces. Its deficiency in affected cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) is causally related to the inability of these cells to control autologous C3b uptake, a characteristic PNH abnormality important in disease pathogenesis. DAF, as well as a number of other surface proteins also deficient in affected cells, are anchored by posttranslationally-added glycophospholipid (GPL) structures, suggesting that the cell lesion in PNH resides in the biosynthetic pathway for GPL-anchor assembly or protein attachment. In previous work, cDNA cloning has shown that DAF mRNA, like mRNAs of other GPL-anchored proteins, encodes C-terminal hydrophobic amino acids appropriate for a conventional polypeptide membrane spanning domain. Transfection analyses have shown that the signals for GPL-anchor processing of nascent DAF polypeptides reside in this region. Analyses of the DAF gene have demonstrated that it spans 35 kb of DNA (on chromosome 1) while investigations of DAF mRNA in different cells have revealed transcripts of 3.1, 2.7, and 2.0 kb. Comparative studies in PNH have shown no apparent DAF DNA or mRNA alterations in affected cells. Consistent with these findings, studies of DAF biosynthesis in affected leukocytes have disclosed aberrant (precursor and mature) DAF peptides. The experiments proposed in this application are directed at 1) further characterization of the DAF gene including its promoter region and RNA processing sites, 2) identification of the signals within the 3' end of DAF mRNA which regulate GPL-anchor processing, 3) investigations of the cellular site of control of DAF GPL-anchor structural modifications and of biological implications of GPL- anchoring in cells, 4) further characterization of abnormal DAF peptides synthesized in PNH cells through transfectional approaches and attempts at reconstitution of DAF expression with normal DNA and 5) further characterization of DAF mRNA and protein species in different cell types and application of the information to analyses of cellular mechanisms underlying unexplained observations concerning PNH progenitors and type I cells of patients.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI023598-07
Application #
3135918
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1985-09-30
Project End
1994-01-31
Budget Start
1992-02-01
Budget End
1993-01-31
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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