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)

The cellular urokinase-type plasminogen-activator (uPA) receptor (uPAR) is a glycolipid-anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand-binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol-specific phospholipase C (PLC) or endoproteinase Asp-N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem-cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA-binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH-affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR-specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns-specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C-terminus close to the proposed glycolipid-attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as-yet-unidentified transamidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.
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PMID:A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria. 132 6

We have previously shown that 3F8, a murine IgG3, monoclonal antibody (MoAb) specific for the ganglioside GD2, mediates tumor cell kill in vitro and in vivo. We now describe receptor requirements of polymorphonuclear leukocytes (PMN) in 3F8-mediated cytotoxicity (ADCC) of human GD2 (+) melanoma and neuroblastoma cell lines. PMN from a child with leukocyte adhesion deficiency (LAD) were devoid of CD11/CD18 adhesion molecules and mounted no detectable ADCC. MoAb to CD11b, CD11c, and CD18 each efficiently blocked ADCC by normal PMN. In contrast, a panel of different MoAbs to CD11a had no significant inhibitory effect on ADCC, a finding consistent with the low-to-absent expression of the CD11a ligand, intercellular adhesion molecule-1, on the target cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the expression of CD11b, CD11c, and CD18 on normal PMN, decreased the expression of Fc receptors (FcR), and enhanced ADCC by normal but not by LAD PMN. MoAbs to FcRII and FcRIII each efficiently blocked ADCC; anti-FcRI MoAb had no effect. Flow cytometry using anti-FcRII MoAb versus anti-FcRIII MoAb did not show cross competition, suggesting that inhibition of ADCC was not a steric effect resulting from FcRII proximity to FcRIII. PMN deficient in FcRIII (obtained from patients with paroxysmal nocturnal hemoglobinuria) and PMN depleted of FcRIII by treatment with elastase or phosphatidylinositol (PI)-specific phospholipase C produced low ADCC, supporting a role for the PI-liked FcRIII. Thus, optimal ADCC using human PMN, human solid tumor cells, and a clinically active MoAb (conditions that contrast with the heterologous antibodies and nonhuman or nonneoplastic targets used in most models of PMN ADCC) required CD11b, CD11c, FcRII, and the PI-linked FcRIII. Furthermore, in this clinically relevant system, GM-CSF enhancement of antitumor PMN ADCC correlated with increased expression of CD11/CD18 molecules.
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PMID:Absolute requirement of CD11/CD18 adhesion molecules, FcRII and the phosphatidylinositol-linked FcRIII for monoclonal antibody-mediated neutrophil antihuman tumor cytotoxicity. 134 7

JMH is a high-frequency human erythrocyte blood group antigen. Previous work has shown that JMH is absent from complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH); such cells have a broad defect in expression of phosphatidylinositol (PI)-linked proteins. Using both human JMH antisera and a JMH-like murine monoclonal antibody, we have identified a 76-Kd membrane protein present in JMH-positive but not JMH-negative erythrocytes. A similar 76-Kd JMH protein was also identified on a human lymphoid T-cell line, HSB-2. Using PI-specific phospholipase C, a small amount of JMH antigen could be cleaved from intact erythrocytes and immunoprecipitated from the supernate of treated erythrocytes, thus confirming that the protein bearing the JMH antigen is anchored by a PI-linkage to the erythrocyte membrane. This protein was further shown not to be identical to decay accelerating factor (70 Kd), a previously identified PI-anchored protein of somewhat similar molecular weight.
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PMID:Isolation of the JMH antigen on a novel phosphatidylinositol-linked human membrane protein. 137 90

To identify mannosyl (Man)-containing intermediates of the human glycoinositol phospholipid (GPI) anchor pathway and examine their expression in paroxysmal nocturnal hemoglobinuria (PNH), mannolipid products deriving from in vitro guanosine diphosphate [3H]Man labeling of HeLa cell microsomes were characterized. The defined GPI species were correlated with products deriving from in vivo [3H]Man labeling of normal and (GPI-anchor defective) affected leukocytes. In vitro analyses in HeLa cells showed dolichol-phosphoryl (Dol-P)-[3H]Man and a spectrum of [3H]Man lipids exhibiting TLC mobilities approximating those of Trypanosoma brucei (Tryp) GPI precursors. Iatrobead HPLC separations and partial characterizations of the major isolated [3H]Man species (designated H1-H8) showed that all but H1 (Dol-P-Man) were sensitive to HNO2 deamination and serum GPI-specific phospholipase D digestion but were resistant to phosphatidylinositol-specific phospholipase C digestion unless previously deacylated with mild alkali. [3H]Man label in H3, H4, and H6 but not in H5 or H7 was efficiently released into the aqueous phase by jack bean alpha-mannosidase digestion. BioGel P-4 and AX-5 sizing of the dephosphorylated core glycan fragments of H6 and H7 gave values that coincided precisely with the corresponding glycan fragments from the fully assembled Tryp anchor donor A' (P2). Affected leukocytes from four patients with PNH supported formation of GlcNAc- and GlcN-PI but all failed to express H6 and H7 as well as H8 and two showed complete absence of earlier Man-containing intermediates. These findings argue that human intracellular GPI mannolipids are built on acylated inositol phospholipids, that H6 and H7 contain differentially phosphoethanolamine-substituted Man3-GlcN-inositol cores, and that PNH cells are defective in conversion of GlcN-PI into these more mature mannolipid structures.
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PMID:Synthesis of mannosylglucosaminylinositol phospholipids in normal but not paroxysmal nocturnal hemoglobinuria cells. 137 20

Many proteins of eukaryotic cells are anchored to membranes by covalent linkage to glycosyl-phosphatidylinositol (GPI). These proteins lack a transmembrane domain, have no cytoplasmic tail, and are, therefore, located exclusively on the extracellular side of the plasma membrane. GPI-anchored proteins form a diverse family of molecules that includes membrane-associated enzymes, adhesion molecules, activation antigens, differentiation markers, protozoan coat components, and other miscellaneous glycoproteins. In the kidney, several GPI-anchored proteins have been identified, including uromodulin (Tamm-Horsfall glycoprotein), carbonic anhydrase type IV, alkaline phosphatase, Thy-1, BP-3, aminopeptidase P, and dipeptidylpeptidase. GPI-anchored proteins can be released from membranes with specific phospholipases and can be recovered from the detergent-insoluble pellet after Triton X-114 treatment of membranes. All GPI-anchored proteins are initially synthesized with a transmembrane anchor, but after translocation across the membrane of the endoplasmic reticulum, the ecto-domain of the protein is cleaved and covalently linked to a preformed GPI anchor by a specific transamidase enzyme. Although it remains obscure why so many proteins are endowed with a GPI anchor, the presence of a GPI anchor does confer some functional characteristics to proteins: (1) it is a strong apical targeting signal in polarized epithelial cells; (2) GPI-anchored proteins do not cluster into clathrin-coated pits but instead are concentrated into specialized lipid domains in the membrane, including so-called smooth pinocytotic vesicles, or caveoli; (3) GPI-anchored proteins can act as activation antigens in the immune system; (4) when the GPI anchor is cleaved by PI-phospholipase C or PI-phospholipase D, second messengers for signal transduction may be generated; (5) the GPI anchor can modulate antigen presentation by major histocompatibility complex molecules. Finally, at least one human disease, paroxysmal nocturnal hemoglobinuria, is a result of defective GPI anchor addition to plasma membrane proteins.
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PMID:Glycosyl-phosphatidylinositol-anchored membrane proteins. 145 Mar 66

The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol-specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.
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PMID:Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell-associated protein. 168 97

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.
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PMID:H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement. 168 19

A human E membrane protein that inhibits lysis by the purified human C5b-9 proteins was isolated and characterized. After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 inhibitory activity of the purified protein resided in the 18- to 20-kDa band. Phosphatidylinositol-specific phospholipase C digestion of the purified protein abolished 50% of its C5b-9 inhibitory activity, and removed approximately 15% of the protein from human E. Western blots of normal and paroxysmal nocturnal hemoglobinuria E revealed an absence of the 18- to 20-kDa protein in the paroxysmal nocturnal hemoglobinuria E cells. The identity of this E protein with leukocyte Ag CD59 (P18, HRF20) was confirmed immunochemically and by N-terminal amino acid sequence analysis. A blocking antibody raised against the purified protein reacted with a single 18- to 20-kDa band on Western blots of human erythrocyte membranes. Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). By contrast, CD59 did not inhibit either C5b67 deposition onto the cell surface, or, binding of 125I-C8 to preassembled membrane C5b67. Taken together, these data suggest that CD59 exerts its C-inhibitory activity by limiting incorporation of multiple C9 into the membrane C5b-9 complex.
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PMID:The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9. 169 60

The low affinity IgG Fc receptor, Fc gamma RIII, expressed on circulating neutrophils, natural killer (NK) cells, and tissue macrophages, is involved in effector functions such as cytotoxicity and immune complex clearance by these cells. While Fc gamma RIII is reported to be a phosphatidylinositol (PI)-linked, rather than peptide-linked, protein on neutrophils and NK cells, its membrane linkage in macrophages has not been studied. We examined the sensitivity of Fc gamma RIII to cleavage by PI-specific phospholipase C (PI-PLC) in cultured monocytes and alveolar tissue macrophages and report that this receptor is not PI-linked on these cells. We also observed normal levels of Fc gamma RIII on cultured monocytes of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which PI-linked proteins are deficient. The results suggest that Fc gamma RIII occurs solely in a transmembrane form in cells of the monocyte/macrophage lineage. In addition, we studied Fc gamma RIII on a cloned NK cell line and found it to be resistant to the effects of PI-PLC under conditions that cleaved Fc gamma RIII on neutrophils. Taken together, our results provide evidence for a distinct form of Fc gamma RIII that differs from the neutrophil receptor in its structure and, possibly, in its function.
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PMID:Monocyte/macrophage Fc gamma RIII, unlike Fc gamma RIII on neutrophils, is not a phosphatidylinositol-linked protein. 169 31

The Leu-8 molecule, the human homologue of the murine MEL-14 peripheral lymph node homing receptor, is expressed on neutrophils in both species and may be important in localization of cells to sites of inflammation. Most circulating human neutrophils express the Leu-8 molecule, and activation of neutrophils with phorbol myristate acetate causes a rapid decline in Leu-8 membrane fluorescence staining within 15 minutes. Northern blot analysis of total cellular RNA from neutrophils demonstrated two species of Leu-8 messenger RNA, a major one of 2.4 kb and a minor one of 1.9 kb. Because two different Leu-8 cDNA clones were obtained from human lymphocytes that were predicted to encode both transmembrane and phosphatidylinositol (PI)-anchored forms of the molecule, experiments were conducted to determine whether Leu-8 is anchored to neutrophils by a PI-anchor. There was a slight decrease in expression of Leu-8 on neutrophils when they were treated with PI-specific phospholipase C (PI-PLC). However, Leu-8 was abundant on neutrophils obtained from a patient with paroxysmal nocturnal hemoglobinuria. To determine the fate of the Leu-8 molecule during cell activation, neutrophils were labeled with 125I-anti-Leu-8. During activation antibody was rapidly lost from the cell surface and was not internalized, suggesting that Leu-8 is released from the cell membrane during cell activation. When cell extracts of neutrophils were compared with extracts of lymphoid cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, the Leu-8 species expressed on neutrophils had a significantly higher and more variable relative mobility (70 to 120 Kd for neutrophils v 70 Kd for Jurkat T cells). In addition, Leu-8 molecules were detected in the supernatants of activated neutrophils. These results indicate that human neutrophils express a high-molecular-weight form of the Leu-8 molecule that has a conventional transmembrane anchor and is rapidly released from the membrane during activation. The loss of the Leu-8 membrane glycoprotein during activation may be a mechanism for rapid alteration of neutrophil adhesion characteristics.
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PMID:Human neutrophils release the Leu-8 lymph node homing receptor during cell activation. 170 70


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