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)

In this study we describe the biochemical features of the Toxoplasma gondii tachyzoite surface glycoprotein, gp23, demonstrating that it is attached to the parasite membrane by a glycosyl-phosphatidyl inositol anchor. Gp23 was metabolically labeled with tritiated palmitate, myristate, ethanolamine, inositol, glucosamine, mannose and galactose, as expected for a GPI-anchor structure. Gp23 was released from the surface of living parasites after treatment with phosphatidylinositol-specific phospholipase C(PI-PLC) and the resulting water-soluble protein was immunoprecipitated with a monoclonal antibody specific for gp23. The GPI-core glycan was generated after aqueous-HF dephosphorylation followed by nitrous acid deamination and its carbohydrate structure was analyzed using selective exo- and endoglycosidase treatments. Finally, the phosphatidylinositol moiety of gp23 was characterized using PI-PLC and phospholipase A2 (PLA2) digestions. Our cumulative data suggest that gp23 of T gondii tachyzoites contains a modified GPI-backbone similar to the mammalian Thy-1 anchor, consisting of a conserved core structure (ethanolamine-PO4-6-Man alpha1-2-Man alpha1-6-Man alpha1-4-GlcN alpha1-6-PI) bearing beta-linked N-acetylgalactosamine residue(s).
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PMID:Structural analysis of glycosyl-phosphatidylinositol membrane anchor of the Toxoplasma gondii tachyzoite surface glycoprotein gp23. 824 58

Many cell surface proteins are anchored into the cell membrane by glycosylphosphatidylinositol (GPI), among those a recently discovered arginine-specific mono-ADP-ribosyltransferase on cytotoxic T cells (CTL). This enzyme transfers ADP-ribose to cell surface proteins resulting in inhibition of cytotoxic and proliferative activity. Here we report that ADP-ribosyltransferase is released in active forms by crosslinking CD3, exposure to Il-2 or PMA stimulation. Release of transferase is specific, as another GPI-anchored protein, Thy-1 is not released. Transferase molecules released by cell activation are indistinguishable in size from molecules released by phospholipase C, suggesting that the release mechanism acts close to or within the GPI anchor. Protease inhibitors fail to inhibit transferase release with exception of 1,10-phenanthroline and its 4,7-diphenyl derivative. This suggests that the release mechanism acts on the cell surface but does not discriminate between action of a metalloprotease or phospholipase D. Release of transferase is shown to be rapid, it is not suppressed by monensin or brefeldin A and independent of serum phospholipase D, consistent with a mechanism acting on the cell surface. Transferase expression is shown to be dependent on the cell activation stage. In CTL clones, the transferase is demonstrable as a phospholipase C releasable molecule at early but not later stages of Ag specific activation.
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PMID:Release of a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase from cytotoxic T cells upon activation. 859 99

Thy-1 glycoprotein is expressed in rat glomerular mesangial cells, and anti-Thy-1 nephritis induced by anti-Thy-1 antibodies is a model of human renal diseases. In this study, we examined Thy-1-mediated biological reactions in cultured rat glomerular mesangial cells utilizing two anti-Thy-1 monoclonal antibodies (mAbs), 1-22-3 and OX-7. Incubation of the cells with these mAbs resulted in increased inositol trisphosphate (IP3) levels. The rise in IP3 produced by mAb 1-22-3 was greater than that produced by mAb OX-7 at the same dose. Incubation of mesangial cells with these mAbs resulted in an increase in the intracellular free calcium concentration ([Ca2+]i). mAb 1-22-3 induced a sustained increase in [Ca2+]i, while that induced by mAb OX-7 lasted 1-2 min, then decreased to the basal level. An transient increase in [Ca2+]i was also observed in Ca(2+)-free medium, indicating that these [Ca2+]i increases are due to release of Ca2+ from internal stores by IP3 without calcium flux across cell membrane. When cells were pretreated with protein tyrosine kinase (PTK) inhibitors (herbimycin A or genistein), Thy-1-mediated increases in [Ca2+]i were inhibited. These data suggest that Thy-1 induces the production of IP3 (including inositol 1,4,5-triphosphate, an intracellular Ca(2+)-releasing factor) and that PTKs may contribute to the Thy-1-mediated elevation of [Ca2+]i which presumably results from phospholipase C activation following Thy-1-mediated signaling in rat mesangial cells.
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PMID:Thy-1-mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell. 881 25

The dot-immunoassay has been adapted for rapid detection and partial characterization of glycosylphosphatidylinositol (GPI)-linked, transmembrane, and intracellular proteins in Triton X-100 (TX-100) extracts of lymphoma cells and intestinal tissue. The GPI-anchored proteins tend to concentrate into specialized plasma membrane domains enriched in glycosphingolipids. The dot-immunoassay has been successfully used to demonstrate the differential distribution of GPI-linked and transmembrane surface glycoproteins of T lymphocytes in sucrose density gradient fractions of TX-100 lysate. The type II transmembrane protein CD26 and the intracellular tyrosine kinase p56lck partially cofractionated with GPI-linked glycoproteins, and the extent to which they partition into GPI-rich plasma membrane domains could be evaluated. Preferential association of the acidic glycosphingolipid GM1 with these domains could be demonstrated by cholera toxin binding directly to the dot-blotted sucrose density gradient fractions. Treatment of whole cell TX-100 lysates or sucrose gradient fractions dotted onto nitrocellulose filter strips with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) proved to be an efficient method to assay for the presence of a GPI-anchor in Thy-1 and Ly6 surface glycoproteins. We have used three criteria, namely flotation to light density fractions in sucrose gradients, colocalization with GM1, and sensitivity to PI-PLC cleavage, to assess the presence of a GPI modification in a putative GPI-linked protein in intestinal tissue extract. It is envisaged that the techniques described in this report would find a wider application to rapidly assess the contents of GPI-rich plasma membrane domains in different cells and tissues.
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PMID:Evaluation by dot-immunoassay of the differential distribution of cell surface and intracellular proteins in glycosylphosphatidylinositol-rich plasma membrane domains. 885 May 46

Aerolysin is a channel-forming protein secreted by virulent Aeromonas spp. Some eucaryotic cells, including T-lymphocytes, are sensitive to very low concentrations of the toxin (<10(-9) M). Here we show that aerolysin binds selectively and with high affinity to the glycosylphosphatidylinositol (GPI)-anchored surface protein Thy-1, which is found on T-lymphocyte populations as well as in brain. Less than 1 ng of purified Thy-1 could be detected by probing Western blots with the toxin. Mutant T-cell lines that lack the ability to add GPI anchors to Thy-1 and other surface proteins were much less sensitive to aerolysin, as were wild-type cells that were pretreated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins. Phosphatidylcholine/cholesterol liposomes containing purified Thy-1 in their membranes were much more sensitive to aerolysin than protein-free liposomes.
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PMID:The glycosylphosphatidylinositol-anchored surface glycoprotein Thy-1 is a receptor for the channel-forming toxin aerolysin. 911 89

Antibody-mediated cross-linking of Thy-1 glycoprotein on the surface of rat mast cells and rat basophilic leukemia (RBL) cells initiates biochemical events which culminate in secretion of allergy mediators. Thy-1, like some other glycosylphosphatidylinositol (GPI)-anchored proteins, forms detergent-insoluble complexes containing protein tyrosine kinases (PTK) and some other molecules which are implicated in the signaling pathway. On the surface of a rat mast cell there are more than 10(6) Thy-1 molecules; however, it is not known which fraction of them is involved in transmembrane signaling, and what exactly is the heterogeneity of Thy-1 complexes. Using sucrose density gradient ultracentrifugation of detergent-lysed RBL cells we found that the density of Thy-1 complexes depended on the detergent used and the lysis conditions employed. Sepharose 4B gel chromatography fractionation followed by density gradient ultracentrifugation revealed both size and density heterogeneity of Thy-1 and Lyn PTK complexes. Cross-linking of surface Thy-1 caused significant changes in the density of these complexes, and an increase in Lyn kinase activity in low/medium-density fractions. Thy-1 in low-density fractions was relatively resistant to cleavage with phosphatidylinositol-specific phospholipase C (PI-PLC). Interestingly, removal of only a small fraction of surface Thy-1 by PI-PLC abolished the cell activation as determined by tyrosine phosphorylation of certain proteins. When Triton X-100 lysates were fractionated at 12000 x g, about 50 % of Thy-1 remained associated with the nuclear/cytoskeleton pellet; this fraction of Thy-1 exhibited an increased sensitivity to PI-PLC. Confocal laser scanning microscopy on fixed cells revealed that the total Thy-1 was relatively homogeneously distributed over the plasma membrane, whereas the PI-PLC-resistant Thy-1 was found mostly in small clusters. The combined data suggest that specialized membrane microdomains enriched in Thy-1 with increased sensitivity to PI-PLC are directly involved in coupling Thy-1 aggregation to transmembrane signaling.
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PMID:Functional heterogeneity of Thy-1 membrane microdomains in rat basophilic leukemia cells. 964 66

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called "raft." In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a kindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.
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PMID:Characterization of a novel rat brain glycosylphosphatidylinositol-anchored protein (Kilon), a member of the IgLON cell adhesion molecule family. 1007 27

Thy-1 protein, a member of the Ig superfamily, is bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. We demonstrate that following anchor cleavage by phospholipase C, the reactivity of the solubilized Thy-1 with several mAbs is lost, and its reactivity with polyclonal anti-Thy-1 Abs is markedly decreased. Hence, solubilized Thy-1 cannot be detected by a range of mAbs. In contrast, enzymatic cleavage of biotinylated Thy-1 yields an intact solubilized protein that can be detected by streptavidin. These results exclude a possible proteolytic degradation of solubilized Thy-1 and suggest that the marked decrease in Thy-1 immunoreactivity following delipidation is due to conformational changes in the Thy-1 protein. We further demonstrate that addition of phospholipase C to preformed Ab-Ag complexes causes dissociation and removal of Thy-1 from the complex, indicating that delipidation of Thy-1 induces a conformational change in Thy-1 that is sufficient to dissociate bound Ab. The possibility should therefore be considered that the GPI anchor affects the conformation of a protein to which it is linked.
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PMID:Cleavage of the glycosylphosphatidylinositol anchor affects the reactivity of thy-1 with antibodies. 1022 38

Aerolysin is a channel-forming toxin secreted by Aeromonas spp. that binds to glycosyl phosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on sensitive target cells. Receptor binding is followed first by oligomerization of the toxin and then by insertion of the oligomers into the membrane to form stable channels that disrupt the permeability barrier. Human immunodeficiency virus type 1 (HIV-1) produced from T cells is known to incorporate Thy-1 and other GPI-anchored proteins into its membrane. Here, we show that aerolysin is capable of neutralizing HIV-1 in a dose-dependent manner and that neutralization depends upon the presence of these proteins in the viral envelope. Pretreatment with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins greatly reduced HIV-1 sensitivity to the toxin, and virus originating from a mutant cell line that lacks GPI-anchored proteins was not neutralized. Aerolysin variants with single amino acid changes that prevent oligomerization or insertion of the toxin were unable to inactivate the virus, implying that channel formation is necessary for neutralization to occur. These findings represent the first evidence that a pathogenic human virus can be neutralized by a bacterial toxin.
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PMID:The channel-forming toxin aerolysin neutralizes human immunodeficiency virus type 1. 1041 55

Recent studies have demonstrated the existence of glycosyl-phosphatidylinositol (GPI)-anchored proteins in higher plants. In this study we tested whether GPI-addition signals from diverse evolutionary sources would function to link a GPI-anchor to a reporter protein in plant cells. Tobacco protoplasts were transiently transfected with a truncated form of the Clostridium thermocellum endoglucanase E reporter gene (celE') fused with a tobacco secretion signal (PR-1a) at the N-terminus and either a yeast (GAS1), mammalian (Thy-1) or putative plant (LeAGP-1) GPI-anchor addition signal at the C-terminus. The yeast and plant C-terminal signals were found to be capable of directing the addition of a GPI-anchor to the endoglucanase protein (EGE') as shown by the sensitivity of the lipid component of GPI to phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. In contrast, the mammalian signal was poorly processed for anchor addition. When EGE' was fused to a truncated form of the LeAGP-1 signal (missing three amino acids predicted to be critical to signal cleavage and anchor addition), a GPI-anchor was not linked to the EGE' protein indicating the necessity for the missing amino acids. Our results show the conservation of the properties of GPI-signals in plant cells and that there may be some similar preferences in GPI-addition signal sequences for yeast and plant cells.
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PMID:Glycosyl-phosphatidylinositol-anchor addition signals are processed in Nicotiana tabacum. 1065 49


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