Many cell surface proteins are anchored to a membrane via a glycosylphosphatidylinositol (GPI), which is attached to the C termini in the endoplasmic reticulum. The inositol ring of phosphatidylinositol is acylated during biosynthesis of GPI. In mammalian cells, the acyl chain is added to glucosaminyl phosphatidylinositol at the third step in the GPI biosynthetic pathway and then is usually removed soon after the attachment of GPIs to proteins. The mechanisms and roles of the inositol acylation and deacylation have not been well clarified. Herein, we report derivation of human and Chinese hamster mutant cells defective in inositol acylation and the gene responsible, PIG-W. The surface expressions of GPI-anchored proteins on these mutant cells were greatly diminished, indicating the critical role of inositol acylation. PIG-W encodes a 504-amino acid protein expressed in the endoplasmic reticulum. PIG-W is most likely inositol acyltransferase itself because the tagged PIG-W affinity purified from transfected human cells had inositol acyltransferase activity and because both mutant cells were complemented with PIG-W homologs of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The inositol acylation is not essential for the subsequent mannosylation, indicating that glucosaminyl phosphatidylinositol can flip from the cytoplasmic side to the luminal side of the endoplasmic reticulum.

The cloning of the PIG-A gene has facilitated the unraveling of the complex pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH). Of current major concern is the mechanism by which a PNH clone expands. Many reports have suggested that an immune mechanism operates to cause bone marrow failure in some patients with PNH, aplastic anemia, and myelodysplastic syndromes. Because blood cells of PNH phenotype are often found in patients with these marrow diseases, one hypothesis is that the PNH clone escapes immune attack, producing a survival advantage by immunoselection. To test this hypothesis, we examined the sensitivity of blood cells, with or without PIG-A mutations, to killing by natural killer (NK) cells, using 51 Cr-release assay in vitro. To both peripheral blood and cultured NK cells, PIG-A mutant cells prepared from myeloid and lymphoid leukemic cell lines were less susceptible than their control counterparts (reverted from the mutant cells by transfection with a PIG-A cDNA). NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was perforin-dependent. There were no differences in major histocompatibility (MHC) class I expression or sensitivity to either purified perforin or to interleukin-2-activated NK cells between PIG-A mutant and control cells. From these results, we infer that PIG-A mutant cells lack molecules needed for NK activation or to trigger perforin-mediated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the set- IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is an acquired, clonal stem cell disorder that manifests clinically as intravascular hemolysis, venous thrombosis, frequent episodes of infection, and rare leukemic conversion. 1,2 In addition, PNH is often a bone marrow (BM) failure syndrome, strongly related to aplastic anemia and the myelodysplastic syndromes (MDSs). 3,4 All 3 processes may occur in a single patient. 5,6 Among the clinical manifestations, the molecular events leading to hemolysis have been elucidated 7-9 and the responsible gene PIG-A has been cloned. 10 PNH cells with PIG-A mutation do not produce glycosylphosphatidylinositol (GPI) and lack cell surface expression of a large family of proteins that utilize GPI to localize in the plasma membrane. 10 Therefore, PNH cells, deficient in complement regulatory GPI-linked proteins such as decay-accelerating factor (DAF) and CD59, undergo complement-mediated hemolysis characteristic of PNH. 11 A mechanism of infection-associated precipitation of hemolysis also has been shown. 12 Proclivity to thrombosis is also attributable to PIG-A mutations, but a molecular mechanism has not been found. 13,14 Despite rapid progress in understanding PNH pathophysiology, the etiology and the mechanism by which a PNH clone expands are still unknown. 15 Murine pig-a knockout models have shown that clonal expansion of PNH cells does not follow on the presence of the ...