Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol-containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.
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PMID:Release of C8 binding protein (C8bp) from the cell membrane by phosphatidylinositol-specific phospholipase C. 341 71

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic disorder characterized by complement-mediated hemolytic anemia, pancytopenia, and venous thrombosis. These clinical manifestations arise from an underlying molecular defect of bone marrow stem cells. Specifically, somatic mutations in the phosphatidylinositol glycan class A gene result in the ability of blood cells to anchor complement-regulatory proteins (CD59 and DAF) to the cell surface via glycosyl phosphatidylinositol (GPI). In an attempt to circumvent the functional defect in PNH cells, a recombinant transmembrane form of CD59 (CD59-TM) was analyzed for the ability to regulate complement activity. Balb/3T3 stable transfectants expressing similar levels of either CD59-TM or native CD59 (CD59-GPI) were equally protected against human complement-mediated membrane damage. Treatment of these cells with phosphatidylinositol-specific phospholipase C failed to release CD59-TM from the cell surface. Retroviral transduction of GPI-anchoring deficient mouse L cells with CD59-TM resulted in surface expression of the protein and rendered these cells resistant to human complement-mediated membrane damage. Conversely, L cells transduced with CD59-GPI failed to express this protein on the cell surface. A GPI-anchoring deficient complement-sensitive B-cell line derived from a PNH patient was successfully transduced with CD59-TM, resulting in surface expression of the protein. The PNH B cells expressing CD59-TM were protected against classical complement-mediated membrane damage by human serum. Taken together, these data establish that a functional recombinant transmembrane form of CD59 can be expressed on the surface of GPI-anchoring deficient PNH cells and suggest that retroviral gene therapy with this molecule could provide a treatment for PNH patients.
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PMID:Expression of recombinant transmembrane CD59 in paroxysmal nocturnal hemoglobinuria B cells confers resistance to human complement. 752 35

In human monocytes, superoxide (O2-) generation accompanies phagocytosis and is important for bactericidal activity. It also contributes to tissue damage in inflammation. In the present study we investigated, whether lipopolysaccharide (LPS) directly stimulates monocyte O2- production with kinetics known for other LPS effects and, if so, by which mechanism. LPS caused a time- and dose-dependent O2- release in nonadherent purified monocytes. The effect appeared after 5 min, peaked at 30 min, and disappeared after 2 h. It was maximal with 10 ng/ml lipid A (+148 +/- 22%, P < .001), 1 ng/ml LPS Escherichia coli Re (+226 +/- 68%, P < .001), and 100 ng/ml LPS Salmonella abortus equi sm (+272 +/- 52%, P < .001), respectively. The effect was not observed in buffer, even when using 10 micrograms/ml LPS. It was dependent on the presence of heat-inactivated AB serum, with a maximal effect at > or = 0.5%. Serum could be replaced by LPS-binding protein (LBP). Polymyxin B and anti-LBP antiserum, respectively, blocked the LPS effect. LPS-induced O2- generation was also completely blocked by anti-CD14 antibodies (3C10 and 63D3) and by their corresponding F(ab')2 fragments. Monocytes treated with phosphoinositol-specific phospholipase C and monocytes from patients with paroxysmal nocturnal hemoglobinuria, lacking the phosphatidylinositol-anchored CD14, did not respond to LPS stimulation with O2- production. Similarly to LPS, E. coli caused stronger O2- production with heat-inactivated serum than without, and this effect was blocked by anti-CD14 antibodies. In conclusion, these data indicate that LPS directly stimulates O2- production in human monocytes via CD14 depending on LBP.
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PMID:LPS directly induces oxygen radical production in human monocytes via LPS binding protein and CD14. 753 19

We performed a flow cytometric analysis using monoclonal antibodies to decay accelerating factor (DAF) and CD59/membrane attack complex inhibitory factor (CD59/MACIF) in order to investigate the leukemic cells and erythrocytes from a patient with paroxysmal nocturnal hemoglobinuria (PNH) who developed acute myelocytic leukemia. In May 1990, the leukemic cells comprised 70% of the mononuclear cells in the bone marrow and 76% of those in the peripheral blood. They consisted of a mixture of positive and negative populations, including single DAF-positive cells. In August 1990, almost 100% of the peripheral mononuclear cells were leukemic blasts, and these consisted of a single population with reduced DAF expression. Single-color flow cytometric analysis showed that the leukemic cells lacked CD59/MACIF, while control leukemic cells (n = 3) expressed both DAF and CD59/MACIF. Leukemic blasts from this patient and six control patients expressed lymphocyte function-associated antigen 3 and FcIII receptors (CD 16) both before and after treatment with phosphatidylinositol-specific phospholipase C. The patient's erythrocytes lacking DAF and CD59/MACIF expression corresponded to the proportion of complement-sensitive cells at the onset of acute leukemia. These DAF- and CD59/MACIF-deficient erythrocytes disappeared almost completely with progression of the leukemia. In conclusion, it appears that the expression of glycosylphosphatidylinositol-linked membrane proteins by leukemic cells was heterogeneous and discordant in our patient, and that the leukemic cells were derived from the PNH clone because of their deficiency of CD59/MACIF. It is also suggested that DAF could compete more effectively than CD59/MACIF for a limited number of anchor molecules available on the proliferating leukemic cells.
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PMID:Discordant and heterogeneous expression of GPI-anchored membrane proteins on leukemic cells in a patient with paroxysmal nocturnal hemoglobinuria. 768 3

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inositol-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.
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PMID:GPI phospholipase C from Trypanosoma brucei causes a GPI-negative phenotype in Leishmania major: I. Implications for GPI-negative mammalian cells; II. Compartmentalization of two GPI biosynthetic pathways. 808 Dec 27

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.
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PMID:A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways. 813 15

Hemolytic anemia is a major feature of paroxysmal nocturnal hemoglobinuria (PNH). Intravascular red blood cell (RBC) destruction is caused by increased sensitivity of the abnormal erythrocyte to complement-mediated lysis, due to the GPI absence of a membrane-bound glycosylphosphatidylinositol (GPI)-linked protein, which functions as an inhibitor of reactive lysis (CD59). Both in vivo and in vitro models have suggested the feasibility of cell-to-cell transfer of GPI proteins, and patients with hemolysis could potentially benefit from transfer of CD59 to their deficient erythrocytes. We studied the ability of RBC components prepared from outdated packed RBC collections, as well as high-density lipoprotein (HDL) preparations, rich in CD55 and CD59, to promote protein transfer, as assessed by flow cytometry, immunoblotting, and susceptibility to complement-mediated lysis. By flow cytometry, CD55 and CD59 were present on RBC-derived microvesicles that stained with an antiglycophorin antibody Ab; in addition, soluble CD59 and CD55 were detected by immunoblot in soluble fractions eluated from RBC units stored for more than 35 days, but not in fresh blood. Both commercial HDL preparations and those prepared in our laboratory contained CD55 and CD59, as assayed by immunoblot. When RBC that were deficient (GPI)-anchored protein, obtained from five patients, with PNH were incubated with HDL preparations for 2 to 4 hours, there was significant transfer of both proteins to the cell surface, as demonstrated by flow cytometry. Washed RBC microvesicles, prepared by ultrasonification, also mediated transfer of GPI-linked proteins to deficient RBC. Pretreatment of microvesicles, RBC eluate preparations, and HDL with phosphatidylinositol-specific, phospholipase C, abrogated protein transfer to deficient cells, indicating that increased cell-associated CD55 and CD59 levels were related to insertion of the intact GPI moiety, rather than to simple adhesion. PNH RBC that were exposed to HDL, RBC eluate preparations, or microvesicles demonstrated decreased in vitro complement-mediated hemolysis in the Ham test. Transfer of GPI-linked proteins from soluble preparations containing CD55 and CD59 to PNH erythrocytes is feasible and may have clinical utility.
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PMID:Correction of the PNH defect by GPI-anchored protein transfer. 1074 88

Eosinophil-derived neurotoxin (EDN) is one of the four basic proteins stored in specific eosinophil granules. Here we demonstrate that EDN can also be detected at the surface of granulocytes. Reduction of EDN membrane expression after phosphatidylinositol-specific phospholipase C treatment suggests that a glycosylphosphatidylinositol (GPI) anchor is involved in the membrane association of EDN. The presence of a GPI anchor was confirmed by a lower expression of membrane EDN on granulocytes from patients with paroxysmal nocturnal hemoglobinuria which present cells lacking GPI anchor proteins. Furthermore, metabolic labeling with GPI anchor components supports biochemical evidence of GPI anchoring of EDN.
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PMID:Evidence for glycosylphosphatidylinositol (GPI)-anchored eosinophil-derived neurotoxin (EDN) on human granulocytes. 1260 41

Rituximab, an anti-CD20 monoclonal antibody used to treat B cell lymphoproliferative disorders and autoimmune diseases, kills cells through complement dependent cytotoxicity, antibody-dependent cellular toxicity and apoptosis. A mechanism of resistance to rituximab is upregulation of the complement regulatory proteins, CD59 and CD55. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic disorder caused by PIGA mutations that lead to a loss of all glycosylphospatidylinositol (GPI)-anchored proteins including, CD55 and CD59. We compared the cytotoxic activity of rituximab against a PNH B cell line, LD -, and the isogenic cell line LD - PIGA + in which GPI-anchor expression was restored by stable transfection of PIGA. The PNH cell line was more sensitive to rituximab-mediated killing than the LD - PIGA + cells. Biochemical disruption of GPI anchors with phosphatidylinositol specific phospholipase C (PIPLC), a phospholipase that cleaves GPI-anchored proteins, also increased rituximab-mediated killing. Thus, genetic and biochemical interruption of GPI anchor proteins augments sensitivity to rituximab.
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PMID:Enhanced cytotoxicity of rituximab following genetic and biochemical disruption of glycosylphosphatidylinositol anchored proteins. 1516 Sep 58

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by excessive activation of the alternative pathway of complement (APC). Atypical HUS is frequently a diagnosis of exclusion. Differentiating aHUS from other TMAs, especially thrombotic thrombocytopenic purpura (TTP), is difficult due to overlapping clinical manifestations. We sought to develop a novel assay to distinguish aHUS from other TMAs based on the hypothesis that paroxysmal nocturnal hemoglobinuria cells are more sensitive to APC-activated serum due to deficiency of glycosylphosphatidylinositol- anchored complement regulatory proteins (GPI-AP). Here, we demonstrate that phosphatidylinositol-specific phospholipase C-treated EA.hy926 cells and PIGA-mutant TF-1 cells are more susceptible to serum from aHUS patients than parental EA.hy926 and TF-1 cells. We next studied 31 samples from 25 patients with TMAs, including 9 with aHUS and 12 with TTP. Increased C5b-9 deposition was evident by confocal microscopy and flow cytometry on GPI-AP-deficient cells incubated with aHUS serum compared with heat-inactivated control, TTP, and normal serum. Differences in cell viability were observed in biochemically GPI-AP-deficient cells and were further increased in PIGA-deficient cells. Serum from patients with aHUS resulted in a significant increase of nonviable PIGA-deficient TF-1 cells compared with serum from healthy controls (P < .001) and other TMAs (P < .001). The cell viability assay showed high reproducibility, sensitivity, and specificity in detecting aHUS. In conclusion, we developed a simple, rapid, and serum-based assay that helps to differentiate aHUS from other TMAs.
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PMID:Modified Ham test for atypical hemolytic uremic syndrome. 2604 94


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