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 RT6 alloantigen of the rat is expressed on most peripheral T cells but not on thymocytes and thus represents a marker for postthymic T lymphocyte maturation in this species. Diabetes-prone (DP) BB rats exhibit a genetically determined T cell lymphopenia associated with a deficiency of RT6+ T cells. In this study we have analyzed the expression of RT6 on lymph node (LN) cells and intestinal intraepithelial lymphocytes (IEL) in two DP BB strains (BB/OK and BB/Mol) and two control strains (non-lymphopenic BB/PhiK and LEW) by flow cytometry. In the DP BB rats the number of LN T cells was substantially reduced (less than 25% TcR2+ cells) and completely lacked RT6 expression. The IEL population was also reduced in number and in marked contrast to normal rats consisted predominantly of CD4+ cells. The majority of IEL, however clearly expressed RT6. Treatment with a phosphatidylinositol (PI)-specific phospholipase C markedly reduced the RT6 density showing that PI-mediated anchoring of RT6 in the cell membrane also applies to IEL of DP BB rats. The results demonstrate that the DP BB strains possess a functional RT6 gene and are also able to generate the PI anchor. The defect in RT6 expression is thus unlikely to be the primary cause of the T cell lymphopenia.
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PMID:Diabetes-prone BB rats express the RT6 alloantigen on intestinal intraepithelial lymphocytes. 171 8

Lymphocytes bearing the T-lymphocyte differentiation antigen RT6 play an important immunoregulatory role in the development of autoimmune diabetes in BB rats. Immunofluorescence studies suggest that diabetes-prone (DP)- but not diabetes-resistant (DR)-BB rat lymphocytes fail to express RT6 antigen during ontogeny. Two alloantigenic forms of the molecule exist, i.e., RT6.1 and RT6.2; both are linked to cell membranes by a phosphatidylinositol (PI) linkage. In these studies, PI-phospholipase C (PLC) treatment of lymphocytes from BB and normal rats followed by immunoabsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of released proteins with anti-RT6 allotype-specific monoclonal antibodies was performed. RT6.1 in several nondiabetic rat strains was found to consist of a family of nonglycosylated and variably glycosylated molecules: an N-Glycanase-resistant 24,000- to 26,000-Mr peptide and four N-Glycanase-sensitive peptides of 29,000, 31,000, 33,000, and 34,000 Mr. In contrast, RT6.2 was found to be a 24,000- to 26,000-Mr nonglycosylated polypeptide. The electrophoretic pattern of RT6.1 was observed to be the same when the antigen was extracted from W3/25+ (CD4+) versus W3/25- T lymphocytes or from resting versus mitogen-activated cells. A pattern of bands characteristic of the RT6.1 antigen found in normal rat strains was detected after PLC treatment or detergent solubilization of lymphocytes obtained from DR rats. In contrast, no evidence of either RT6 species was found after PLC or detergent treatment of comparable numbers of T lymphocytes from DP-BB rats. Interestingly, T lymphocytes from Wistar-Furth (RT6.2+) x DP (RT6-) F1 crosses were observed to coexpress both RT6.2 and RT6.1 molecules, with the electrophoretic pattern of RT6.1 being similar to that obtained in DR and other rat strains. This study provides biochemical evidence that DP rats may have an intact RT6a structural gene.
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PMID:Biochemical studies of RT6 alloantigens in BB/Wor and normal rats. Evidence for intact unexpressed RT6a structural gene in diabetes-prone BB rats. 221 79

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.
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PMID:Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei. 243 27

The rat T cell alloantigen RT6.2 shows a slow rate of synthesis in isolated T cells which hampers studies on the metabolism of RT6.2 in these cells (1). In order to facilitate further molecular and functional characterization of this molecule we have established a T-T hybridoma cell line which stably expresses RT6.2. Only 1 of 102 T cell hybridomas obtained upon fusion of the rat thymoma C58NT with DA rat lymph node cells expressed this antigen. This clone--EpD3--initially showed a relatively slow rate of cell division and a highly unstable pattern of expression of RT6.2. Thus, the relative number of RT6.2 bearing cells consistently decreased during cultivation of EpD3 and of EpD3-derived subclones. Cell separation studies suggest that the switch in RT6.2 phenotype of EpD3 cultures was due to the appearance of variant cells of RT6.2- phenotype with a higher proliferative capacity and was not induced by factors in the culture medium. By repeated panning and subcloning procedures to select for RT6.2 expressing cells, a subline of EpD3--EpD3/87--was obtained which stably expresses high levels of RT6.2. Metabolic labeling studies of RT6.2 in EpD3 show that it is synthesized very efficiently in these cells and support our previous suggestion that RT6.2 does not bear any classic oligosaccharide side chains. Moreover, RT6.2 can be released almost completely from EpD3 cells by phosphatidylinositol-specific phospholipase C (PI-PLC).
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PMID:Production of a rat T cell hybridoma that stably expresses the T cell differentiation marker RT6.2. 326 67

Rat T lymphocyte alloantigen 6.1 (RT6.1), which was synthesized as the fusion protein with a maltose-binding protein in Escherichia coli, displayed NAD(+)-dependent auto-ADP-ribosylation in addition to an enzyme activity of NAD+ glycohydrolase. Such ADP-ribosylation of RT6.1 was also observed in lymphocytes isolated from rat tissues as follows. When intact rat lymphocytes expressing RT6.1 mRNA were incubated with [alpha-32P]NAD+, its radioactivity was incorporated into a cell surface protein with the M(r) of 31,000. The radiolabeled 31-kDa protein was released from the cell surface by treatment of the cells with phosphatidylinositol-specific phospholipase C and immunoprecipitated with anti-RT6.1 antiserum. The radioactivity incorporated into the 31-kDa protein was recovered as 5'-[32P]AMP upon incubation with snake venom phosphodiesterase and also removed by NH2OH treatment. These results suggested that the NAD(+)-dependent modification of the 31-kDa protein was due to ADP-ribosylation of glycosylphosphatidylinositol-anchored RT6.1 at an arginine residue. When intact lymphocytes, in which RT6.1 had been once modified by [32P]ADP-ribosylation, were further incubated in the absence of NAD+, there was reduction of the radioactivity in the [32P]ADP-ribosylated RT6.1. The reduced radioactivity was recovered from the incubation medium as [32P]ADP-ribose. This reduction was effectively inhibited by the addition of ADP-ribose to the reaction mixture. Moreover, readdition of NAD+ caused the ADP-ribosylation of RT6.1 again. Thus, the ADP-ribosylation of RT6.1 appeared to proceed reversibly in intact rat lymphocytes.
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PMID:NAD(+)-dependent ADP-ribosylation of T lymphocyte alloantigen RT6.1 reversibly proceeding in intact rat lymphocytes. 755

RT6.2 is a 26-kDa alloantigen expressed only on post-thymic T cells and attached to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. It has been reported that expression of RT6.2 in animal models may correlate with lymphopenia and genetically-induced insulin-dependent diabetes mellitus. Its physiological function is unclear. Since RT6.2 has significant amino acid identity with a GPI-anchored rabbit muscle NAD:arginine ADP-ribosyltransferase, RT6.2 was expressed in rat mammary adenocarcinoma cells and the ability of the expressed protein to catalyze ADP-ribose transfer reactions was examined. Cells transformed with the RT6.2 gene expressed NAD glycohydrolase activity that was released from intact cells by phosphatidylinositol-specific phospholipase C, consistent with its presence on the cell surface. A similar activity was not detected with vector-transformed cells. RT6.2 did not ADP-ribosylate simple guanidino compounds. The molecular weight of the phosphatidylinositol-specific phospholipase C-released NAD glycohydrolase, determined by SDS-polyacrylamide gel electrophoresis, was 22,000-24,000, in good agreement with that of native RT6.2. These results strongly suggest that the rat T cell alloantigen RT6.2 is a GPI-anchored NAD glycohydrolase.
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PMID:Expression of NAD glycohydrolase activity by rat mammary adenocarcinoma cells transformed with rat T cell alloantigen RT6.2. 814 25

Mouse T-cell antigens Rt6.1 and Rt6.2 are glycosylphosphatidylinositol-anchored arginine-specific adenosine diphosphate (ADP)-ribosyltransferases. In the present study, we obtained evidence that an arginine-specific ADP-ribosyltransferase activity liberated from BALB/c mouse splenocytes by phosphatidylinositol-specific phospholipase C increased fivefold in the presence of dithiothreitol and that the activity was immunoprecipitated by polyclonal antibodies generated against recombinant rat RT6.1. When mouse Rt6.1 was expressed as a recombinant protein, the transferase activity of Rt6.1 was stimulated by dithiothreitol, and inhibited by N-ethylmaleimide, while activities of recombinant mouse Rt6.2 and the Glu-207 mutant of rat RT6.1 [Hara, N., Tsuchiya, M., and Shimoyama, M. (1996) J. Biol. Chem. 271, 29552-29555] were unaffected by either agent. In addition to four cysteine residues conserved among mouse Rt6 and rat RT6 antigens, Rt6.1 has two extra cysteine residues at positions 80 and 201. To investigate a contribution of these extra cysteines in mouse Rt6.1 to thiol dependency of Rt6.1 transferase activity, Cys-80 and Cys-201 of Rt6.1 were replaced with serine and phenylalanine, respectively, the corresponding residues of mouse Rt6. 2 and rat RT6.1. Transferase activity of the Phe-201 mutant of Rt6.1 lost thiol dependency while that of the Ser-80 mutant remained thiol-dependent. Thus, we conclude that mouse Rt6.1 is a thiol-dependent arginine-specific ADP-ribosyltransferase, and that Cys-201 confers thiol dependency on Rt6.1 transferase. Our study indicates that arginine-specific ADP-ribosyltransferase activity detected on BALB/c mouse splenocytes is attributed to Rt6.1 and that Rt6.1 differs from Rt6.2 in enzymatic property of the transferase and perhaps in immunoregulatory functions.
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PMID:Mouse Rt6.1 is a thiol-dependent arginine-specific ADP-ribosyltransferase. 991 5

RT6 proteins are glycosylphosphatidylinositol (GPI)-linked alloantigens that are localized to cytotoxic T lymphocytes and that have nicotinamide adenine dinucleotide glycohydrolase and adenosine diphosphate (ADP)-ribosyltransferase activities. In view of the importance of GPI-linked surface proteins in mediating interactions of cells with their milieu, and the varied functions of airway cells in inflammation, we undertook the present study to determine whether human homologues of the RT6 superfamily of ADP-ribosyltransferases (ART) are expressed in pulmonary epithelial cells. We hypothesized that these surface proteins or related family members may be present in cells that interact with inflammatory cells, and that they may thereby be involved in intercellular signaling. Using in situ analysis and Northern blot analysis, we identified ART1 messenger RNA (mRNA) in airway epithelial cells. As expected for GPI-anchored proteins, the localization of ART1 at the apical surface of ciliated epithelial cells was demonstrated by staining with polyclonal anti-ART1 antibody, and was confirmed by loss of this immunoreactivity after treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), which selectively cleaves GPI anchors and releases proteins from the plasma membrane. Using in situ hybridization with specific ART3 and ART4 oligonucleotides, we also identified two additional members of the RT6 superfamily in epithelial cells. In accord with these findings, we identified ART3 and ART4 mRNAs through reverse transcription- polymerase chain reaction of polyadenine-positive RNA from human trachea. Interestingly, these proteins appeared to be preferentially localized to the airway epithelium. The localized expression of these members of the RT6 superfamily in human pulmonary epithelial cells may reflect a role for them in cell-cell signaling during immune responses within the airway.
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PMID:Selective expression of RT6 superfamily in human bronchial epithelial cells. 1046 Jul 51

Lymphocytes express a number of NAD-metabolizing ectoenzymes, including mono(ADP-ribosyl)transferases (ART) and ADP ribosylcyclases. These enzymes may regulate lymphocyte functions following the release of NAD in injured or inflammatory tissues We report here that extracellular NAD induces apoptosis in BALB/c splenic T cells with an IC(50) of 3-5 microM. Annexin V staining of cells was observed already 10 min after treatment with NAD in the absence of any additional signal. Removal of GPI-anchored cell surface proteins by phosphatidylinositol-specific phospholipase C treatment rendered cells resistant to NAD-mediated apoptosis. RT-PCR analyses revealed that resting BALB/c T cells expressed the genes for GPI-anchored ART2.1 and ART2.2 but not ART1. ART2-specific antisera blocked radiolabeling of cell surface proteins with both [(32)P]NAD and NAD-mediated apoptosis. Further analyses revealed that natural knockout mice for Art2.a (C57BL/6) or Art2.b (NZW) were resistant to NAD-mediated apoptosis. Labeling with [(32)P]NAD revealed strong cell surface ART activity on T cells of C57BL/6 and little if any activity on cells of NZW mice. T cells of (C57BL/6 x NZW)F(1) animals showed strong cell surface ART activity and were very sensitive to NAD-induced apoptosis. As in BALB/c T cells, ART2-specific antisera blocked cell surface ART activity and apoptosis in (C57BL/6 x NZW)F(1) T cells. The fact that T cells of F(1) animals are sensitive to rapid NAD-induced apoptosis suggests that this effect requires the complementation of (at least) two genetic components. We propose that one of these is cell surface ART2.2 activity (defective in the NZW parent), the other a downstream effector of ADP-ribosylation (defective in the C57BL/6 parent).
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PMID:Rapid induction of naive T cell apoptosis by ecto-nicotinamide adenine dinucleotide: requirement for mono(ADP-ribosyl)transferase 2 and a downstream effector. 1141 49

The presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2- T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.
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PMID:Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activities. 1148 87


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