Glycosylphosphatidylinositol (GPI)-deficient Jurkat T cells as a model to study functions of GPI-anchored proteins (original) (raw)
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Blood, 1999
Patients with paroxysmal nocturnal hemoglobinuria (PNH) have one or a few clones of mutant hematopoietic stem cells defective in glycosylphosphatidylinositol (GPI) synthesis as a result of somatic mutation in the X-linked gene PIG-A. The mutant stem cell clone dominates hematopoiesis by a mechanism that is unclear. To test whether a lack of multiple GPI-anchored proteins results in dysregulation and expansion of stem cells, we generated mice in which GPI-anchor negative cells are present only in the hematopoietic system. We transplanted lethally irradiated mice with female fetal liver cells bearing one allele of the Piga gene disrupted by conditional gene targeting. Because of the X-chromosome inactivation, a significant fraction of the hematopoietic stem cells in fetal livers was GPI-anchor negative. In the transplanted mice, cells of all hematopoietic lineages contained GPI-anchor negative cells. The percentage of GPI-anchor negative cells was much higher in T lymphocytes includin...
British Journal of Haematology, 2003
Paroxysmal nocturnal haemoglobinuria (PNH) has a dual pathogenesis. PIG-A mutations generate clones of haemopoietic stem cells (HSC) lacking glycosylphosphatidylinositol (GPI)-anchored proteins and, secondly, these clones expand because of a selective advantage related to bone marrow failure. The first aspect has been elucidated in detail, but the mechanisms leading to clonal expansion are not well understood. We have previously shown that apoptosis and Fas expression in HSC play a role in bone marrow failure during aplastic anaemia. We have now investigated apoptosis in PNH. Ten patients were studied. Apoptosis, measured by flow cytometry, was significantly higher among CD34 + cells from patients compared with healthy controls. Fas expression was also increased. Cells that were stained for CD34, CD59 and apoptosis showed a significantly lower apoptosis in CD34 + /CD59) compared with CD34 + /CD59 + cells from the same patient. In three patients, staining for CD34, CD59 and Fas revealed lower Fas expression on CD34 + /CD59) cells. Differential apoptosis of CD34 + /CD59) HSC may be sufficient in itself to explain the expansion of PNH clones in the context of aplastic anaemia. In addition to demonstrating a basic mechanism underlying PNH clonal expansion, these results suggest further hypotheses for the evolution of PNH, based on the direct or indirect resistance of GPI-negative HSC to pro-inflammatory cytokines.
Glycosyl phosphatidylinositol-linked blood group antigens and paroxysmal nocturnal hemoglobinuria
Transfusion Clinique et Biologique, 1995
Human erythrocyte cell surface molecules that are attached to the cell membrane by glycosyl-phosphatidylinositol (GPI) anchors include the complement regulatory proteins decay accelarating factor (DAF, CD55) and membrane inhibitor of reactive 1ysis (MIRL, CD59), as well as the proteins that bear the Cartwright, Dombrock, and JMH blood group antigens. The acquired hematopoietic stem cell disorder paroxysmal nocturnal hemoglobinuria {PNH) results from the absence or marked deficiency in expression of GPI-anchored proteins in affected hematopoietic cells. PNH usually if not always results from a somatic mutation of an X-linked gene called PIG-A; the product of the PIG-A gene is a glycosyl transferase necessary for construction * This review contains material extracted from previously published reviews by the author and updated with the addition of recently acquired information.
Proceedings of the National Academy of Sciences, 1993
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder arising in a multipotent hemopoietic stem cell. PNH manifests clinically with intravascular hemolysis resulting from an increased sensitivity of the red cells belonging to the PNH clone to complement-mediated lysis. Numerous studies have shown that surface proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor (including proteins protecting the cell from complement) are deficient on the cells of the PNH clone, leading to the notion that GPI-anchor biosynthesis may be abnormal in these cells. To investigate the biochemical defect underlying PNH we have used lymphoblastoid cell lines (LCLs) with the PNH phenotype obtained by Epstein-Barr virus immortalization of lymphocytes from nine patients with PNH. By labeling cells with myo-[3H]inositol we have found that PNH LCLs produce phosphatidylinositol normally. By contrast, PNH LCLs fail to incorporate [3H]mannose into GPI anchor precursors. When cel...
The Hematology Journal, 2000
The association of paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA) raises the yet unresolved questions as t o whether these two disorders are different forms of the same disease. We compared two groups of patients with respect t o cytogenetic features, glycosylphosphatidylinositol (GPII-linked protein expression, protein C/ protein Slthrombomodulinlantithrombin 111 activity, and PIG-A gene expression. The first group consisted of eight patients with PNH (defined as positive Ham and sucrose tests at diagnosis), and the second, 37 patients with AA. Twelve patients with AA later developed a PNH clone. Monoclonal antibodies used t o study GPI-linked protein expression (CD14 [on monocytesl, CD16 [on neutrophils], CD48 [on lymphocytes and monocytes], CD67 [on neutrophils and eosinophils], and, more recently, CD55, 0 5 8 , and CD59 [on erythrocytesl) were also tested on a cohort of 20 normal subjects and five patients with constitutional AA. Ham and sucrose tests were performed on the same day as flow-cytometric analysis. Six of 12 patients with A A , who secondarily developed a PNH clone, had clinical symptoms, while all eight patients with PNH had pancytopenia and/or thrombosis andlor hemolytic anemia. Cytogenetic features were normal in all but t w o patients. Proteins C and S, thrombomodulin, and antithrombin 111 levels were within the normal range in patients with PNH and in those with AA (with or without a PNH clone). In patients with PNH, CD16 and CD67 expression were deficient in 78% t o 98% of the cells and CD14 in 7696 t o 100Y0. By comparison, a GPI-linked defect was detected in 13 patients with AA, affecting a mean of 32% and 33% of CD16/CD67 and CD14 cell populations, re-CQUIRED APLASTIC ANEMIA (AA) is a heterogeneous disease, in which several pathophysiologic factors are involved.' In contrast to patients who undergo bone marrow transplantation (BMT), those who are successfully treated with immunosuppressive therapy (IST) are at risk for subsequently developing paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndromes, and acute myeloid l e~k e m i a .~.~ D e novo PNHh-' is an acquired clonal disorderY"" characterized by complement-mediated hemolysis and the expansion of affected cells of various hematopoietic lineages. The most typical manifestation of PNH is intravascular hemolysis due to abnormal sensitivity of red blood cells
Implication of PIGA genotype on erythrocytes phenotype in Paroxysmal Nocturnal Hemoglobinuria
Leukemia, 2021
Somatic mutations of the X-linked phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIGA) gene in hematopoietic stem cells are the key molecular events in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH) [1]. PIGA mutations lead to impaired biosynthesis of glycosylphosphatidylinositol (GPI)-anchor [2]. Resultant lack of GPI-anchored proteins (GPI-AP), primarily CD55 and CD59, generates clonal selection of PNH clone(s) and increases sensitivity of PNH red cells (RBCs) to complement-mediated hemolysis [3]. Based on immuno-fluorescent staining, the grade of GPI-APs deficiency on the RBCs can distinguish: PNH type-1 (TI) with normal expression; PNH type-2 (TII) with partial reduction; and PNH type-3 (TIII) with total absence of two common GPI-APs (CD55 and CD59) [4]. Fractions of these RBC types are believed to be a result of the molecular spectrum of PIGA mutations, ranging from missense in various amino acids positions to ✉