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:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various mammalian cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini. Although the first Glu in the common motif is replaced by Gln (Q207EEVLIP) in rat T lymphocyte alloantigens RT6.1 and RT6.2, the two RT6s appear to have ADP-ribosyltransferase activity. To investigate the significance of the Glu-rich motif in the enzyme activity, we produced a mutant RT6.1, in which Gln207 was replaced by Glu (Q207E), together with wild-type RT6s, in Escherichia coli. The recombinant RT6.1 and RT6.2 displayed extremely low auto-ADP-ribosylation, though the latter modification was somewhat higher than the former one. In contrast, much higher the auto-modification was observed for Q207E mutant. Moreover, the mutant could effectively ADP-ribosylate agmatine as a substrate. Thus, the single amino acid mutation of RT6.1 caused remarkable increase in its ADP-ribosyltransferase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the enzyme activity.
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PMID:Molecular characterization of rat T lymphocyte alloantigen RT6.1 as an ADP-ribosyltransferase. 919 52

Several proteins with NAD+:arginine ADP-ribosyltransferase (ART) activity are expressed in T cells and affect their function. Rat T cells that express the ART designated RT6 are determinants of the expression of autoimmune diabetes. In the mouse, a 35-kDa ecto-ART modulates the proliferation and functional activity of CTL. Here we report on mouse ARTs designated Rt6-1 and Rt6-2 in BALB/c and C57BL/6 mice. mRNAs for Rt6-1 and Rt6-2 were found in spleen, thymus, and intestinal tissue of both strains, but Rt6-1 mRNA in C57BL/6 mice was detected only at low levels. Rt6-1 and Rt6-2 cDNAs from both strains were cloned and sequenced. Predicted amino acid sequences of Rt6-2 were identical in both strains, but there was an in-frame stop codon in the sequence of Rt6-1 in C57BL/6 mice not present in BALB/c mice. Recombinant C57BL/6 Rt6-2 and BALB/c Rt6-1 proteins expressed in COS1 cells exhibited ART activity and were documented to be glycosylphosphatidylinositol-linked membrane proteins. COS-1 cells transfected with a C57BL/6 Rt6-1 cDNA construct expressed a truncated protein consistent in size with that predicted by the presence of the stop codon. This approximately 21-kDa protein appeared not to be glycosylphosphatidylinositol linked to the cell surface and lacked ART activity. C57BL/6 Rt6-1 therefore appears to be a naturally occurring ART knockout. The expression of Rt6-1 and Rt6-2 mRNAs in lymphoid tissues suggests that these ARTs may regulate immune system functions. Expression of Rt6-2 or another redundant ART may compensate for the lack of enzymatically active Rt6-1 in C57BL/6 mice.
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PMID:Expression in BALB/c and C57BL/6 mice of Rt6-1 and Rt6-2 ADP-ribosyltransferases that differ in enzymatic activity: C57BL/6 Rt6-1 is a natural transferase knockout. 930 Jun 95

RT6 is a rat lymphocyte glycosylphosphatidylinositol (GPI)-anchored alloantigen with nicotinamide adenine dinucleotide (NAD) glycohydrolase (NADase) and auto-ADP-ribosyltransferase activities. RT6 may have immunoregulatory properties based in part on the observation that injection of diabetes-resistant (DR)-BB rats with depleting doses of anti-RT6.1 mAb induced autoimmune diabetes and thyroiditis. We now report that injection of DR-BB rats with anti-RT6.1 mAb increased plasma NADase activity, which localized, by fluid phase liquid chromatography fractionation, to the high density lipoprotein (HDL) fraction. Following ultracentrifugation in high salt, however, RT6 was found in the nonlipoprotein fraction, where it existed, under nondenaturing conditions, as a 200-kDa complex and, by SDS-PAGE, as a 30- to 36-kDa species. Thy-1, another GPI-linked protein, and proteins that reacted with anti-GPI-oligosaccharide Abs also translocated from HDL to the nonlipoprotein fraction under similar conditions. Injection of anti-RT6.1 mAb into thymectomized DR and diabetes-prone-BB rats increased soluble RT6 to levels comparable to those observed in euthymic DR-BB rats, suggesting that HDL-bound RT6 is not derived from peripheral lymphocytes. In agreement, NADase activity in the plasma of eviscerated DR-BB rats did not increase following injection of anti-RT6 mAb. These data suggest that HDL is a carrier of plasma RT6 and other GPI-linked proteins, with equilibrium between the lipoprotein and nonlipoprotein fractions being salt dependent. Since GPI-linked proteins in HDL can transfer to cells in a functionally active form, the presence of RT6 in HDL is consistent with it having a role in signaling in nonlymphoid cells.
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PMID:Characterization of high density lipoprotein-bound and soluble RT6 released following administration of anti-RT6.1 monoclonal antibody. 968 81

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

Recent evidence suggests that a new member of the mono-ADP-ribosyltransferase/NAD glycohydrolase family, RT6, may be important in immune regulation. RT6 is expressed in two allelic forms and is present on post-thymic T cells in the rat. RT6-expressing T cells in the rat may have a regulatory role, a conclusion based on their ability to prevent autoimmune diabetes in the BB rat model of insulin-dependent diabetes mellitus. This observation led to investigation of RT6 at a molecular and biochemical level resulting in the determination that RT6 protein exists as both glycosylated and non-glycosylated glycosylphosphatidylinositol (GPI)-linked cell surface molecules. RT6, like many GPI-linked proteins, can mediate cell signal transduction events associated with T cell activation, and is also present in a soluble form in the circulation. The discovery that RT6 is an NAD glycohydrolase and auto-ADP-ribosyltransferase led to the ongoing investigations into the role that enzymatic activity may have in the immunoregulatory function of rat RT6+ T cells. A homologue of rat RT6, termed Rt6, has been identified in the mouse. Rt6 is predominately an ADP-ribosyltransferase enzyme as determined using simple guanidino compounds (e.g. arginine) as ribose acceptors. Abnormalities in mouse Rt6 mRNA are associated with the expression of autoimmunity. In the present manuscript, we review recent data on RT6/Rt6, and discuss the potential mechanisms by which RT6-expressing cells, and perhaps RT6 protein itself, may mediate immune regulation.
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PMID:The RT6 (Art2) family of ADP-ribosyltransferases in rat and mouse. 1033 39

ADP-ribosylation of membrane proteins on mouse T cells by ecto-ADP-ribosyltransferase(s) (ARTs) can down-regulate proliferation and function. The lack of mAbs against mouse ARTs has heretofore prevented analysis of ART expression on T cell subsets. Using gene gun technology, we immunized a Wistar rat with an Art2b expression vector and produced a novel mAb, Nika102, specific for ART2.2, the Art2b gene product. We show that ART2.2 is expressed as a GPI-anchored protein on the surface of mature T cells. Inbred strain-dependent differences in ART2.2 expression levels were observed. C57BL/6J and C57BLKS/J express the Ag at high level, with up to 70% of CD4+ and up to 95% of CD8+ peripheral T cells expressing ART2.2. CBA/J and DBA/2J represent strains with lowest expression levels. T cell-deficient mice and NZW/LacJ mice with a defective structural gene for this enzyme were ART2.2 negative. In the thymus, ART2.2 expression is restricted to subpopulations of mature cells. During postnatal ontogeny, increasing percentages of T cells express ART2.2, reaching a peak at 6-8 wk of age. Interestingly, ART2.2 and CD25 are reciprocally expressed: activation-induced up-regulation of CD25 is accompanied by loss of ART2.2 from the cell surface. Nika102 thus defines a new differentiation/activation marker of thymic and postthymic T cells in the mouse and should be useful for further elucidating the function of the ART2.2 cell surface enzyme.
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PMID:A new monoclonal antibody detects a developmentally regulated mouse ecto-ADP-ribosyltransferase on T cells: subset distribution, inbred strain variation, and modulation upon T cell activation. 1057 Feb 89

Mouse Rt6.1 and Rt6.2, homologues of rat T-cell RT6 antigens, catalyze arginine-specific ADP-ribosylation. Without an added ADP-ribose acceptor, Rt6.2 shows NAD glycohydrolase (NADase) activity. However, Rt6.1 has been reported to be primarily an ADP-ribosyltransferase, but not an NADase. In the present study, we obtained evidence that recombinant Rt6.1 catalyzes NAD glycohydrolysis but only in the presence of DTT. The NADase activity of Rt6.1 observed in the presence of DTT was completely inhibited by N-ethylmaleimide (NEM). Native Rt6.1 antigen, immunoprecipitated from BALB/c mouse splenocytes with polyclonal antibodies generated against recombinant RT6.1, also exhibited NADase activity in the presence of DTT. Compared with Rt6.2, Rt6.1 has two extra cysteine residues at positions 80 and 201. When Cys-80 and Cys-201 in Rt6.1 were replaced with the corresponding residues of Rt6.2, serine and phenylalanine, respectively, Rt6.1 catalyzed the NADase reaction even in the absence of DTT. Conversely, replacing Ser-80 and Phe-201 in Rt6.2 with cysteines, as in Rt6.1, converted the thiol-independent Rt6.2 NADase to a thiol-dependent enzyme. Kinetic study of the NADase reaction revealed that the affinity of Rt6.1 for NAD and the rate of catalysis increased in the presence of DTT. Moreover, the NADase activity of Rt6.1 expressed on COS-7 cells was stimulated by culture supernatant from activated mouse macrophages, even in the absence of DTT. From these observations, we conclude that t!he Rt6.1 antigen has thiol-dependent NADase activity, and that Cys-80 and Cys-201 confer thiol sensitivity to Rt6.1 NADase. Our results also suggest that upon the interaction of T-cells expressing Rt6.1 with activated macrophages, the NADase activity of the antigen will be stimulated.
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PMID:Mouse T-cell antigen rt6.1 has thiol-dependent NAD glycohydrolase activity. 1101 Nov 42

T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-alpha-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.
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PMID:Metalloprotease-mediated shedding of enzymatically active mouse ecto-ADP-ribosyltransferase ART2.2 upon T cell activation. 1103 85

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

Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in their target proteins. The purpose of this study was to determine the structure, chromosomal localization, and expression profile of the gene for mouse ecto-ADP-ribosyltransferase ART5. Southern blot analyses indicate that Art5 is a single copy gene which maps to mouse chromosome 7 at offset 49.6 cM in close proximity to the Art1, Art2a and Art2b genes. Northern blot and RT-PCR analyses demonstrate prominent expression of Art5 in testis, and lower levels in cardiac and skeletal muscle. Sequence analyses reveal that the Art5 gene encompasses six exons spanning 8 kb of genomic DNA. The 5' end of the Art5 gene overlaps with that of the Art1 gene. A single long exon encodes the predicted ART5 catalytic domain. Separate exons encode the N-terminal leader peptide and a hydrophilic C-terminal extension. Sequencing of RT-PCR products and ESTs identified six splice variants. The deduced amino acid sequence of ART5 shows 87% sequence identity to its orthologue from the human, and 37 and 32% identity to its murine paralogues ART1 and ART2. Unlike ART1 and ART2, ART5 lacks a glycosylphosphatidylinositol-anchor signal sequence and is predicted to be a secretory enzyme. This prediction was confirmed by transfecting an Art5 cDNA expression construct into Sf9 insect cells. The secreted epitope-tagged ART5 protein resembled rat ART2 in exhibiting potent NAD-glycohydrolase activity. This study provides important experimental tools to further elucidate the function of ART5.
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PMID:Structure, chromosomal localization, and expression of the gene for mouse ecto-mono(ADP-ribosyl)transferase ART5. 1158 54


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