The decay accelerating factor (DAF or CD55) is a 70 kDa protein that functions intrinsically in the plasma membranes of self cells to prevent activation of autologous complement on their surfaces. The molecule, comprised of four approximately 60 amino acid long complement control protein repeats (CCPs) positioned above an O-glycosylated cushion, is linked to the cell surface membrane by a post-translationally-added glycosylphosphatidylinositol (GPI) anchor. It acts to rapidly dissociate C4b2a and C3bBb, the central amplification convertases of the cascade, wherever deposited autologous C4b or C3b elicits their assembly. Its absence in affected cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) leads to increased C3b uptake that contributes to disease pathogenesis. Its deficiency in this condition is due to failed GPI anchor assembly in a bone marrow stem cell resulting from mutation of an X-linked gene termed phosphatidylinositol glycan (PIG)-A. The proposed research is designed to provide new insights into several unresolved questions concerning DAF's function, PNH pathogenesis and mutation of the PIG-A gene.
Aim 1 focuses on the molecular mechanisms of DAF's action. Our previous studies have shown that regulation of C4b2a and C3bBb resides in different sets of CCPs and have yielded a molecular model of the protein. Experiments are designed to identify the critical residues involved in these interactions.
Aim 2 addresses DAF's functional importance in vivo. Longstanding studies have shown that DAF is expressed in high levels not only intra but extra-vascularly. Studies with DAF knock out mice will investigate its functional importance in both sites in different states of complement activation.
Aim 3 focuses on affected PNH stem cells and mutation of the PIG-A gene. Recent studies by ourselves and others indicate that PIG-A mutation does not confer a growth advantage. Comparisons of PIG-A and PIG- A+CD34+ cells are designed to uncover the mechanism(s) underling the difference in their growth properties. As an adjunct to these studies, investigations of cancer cells from individuals with Mutator (Mut) phenotypes are designed to determine if GPI-anchored protein deficiency resulting from PIG-A mutation can be exploited to identify individuals at increased cancer risk. The research overall will not only contribute to our fundamental understanding of complement regulation but should be clinically relevant to PNH as well as neoplasia.
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