The cDNA cloning of human placental ecto-ATP diphosphohydrolases I and II.
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The cDNA clones of two isoforms (enzymes I and II) of human placental ecto-ATP diphosphohydrolases have been isolated based on the N-terminal amino acid (aa) sequence of the immunopurified 82 kDa protein and characterized. The cDNA clone encoding enzyme I consists of 2081 nucleotides and the predicted enzyme I consists of 517 aa residues. Enzyme I has a 5'-UTR and an N-terminal 11 aa sequence that differ from CD39, but the rest of the sequence is the same as CD39. The hydropathy plot indicated that enzyme I has two hydrophobic regions near the N- and C-termini of the molecule. In contrast, enzyme II consists of 1814 nucleotides and the predicted protein consists of 306 aa residues. The sequence of 1-1018 nucleotides of enzyme II is identical to that of enzyme I, but the 1019-1814 nucleotide sequence is different from both enzyme I and CD39. The hydropathy plot indicated that enzyme II has one hydrophobic region near the N-terminus, suggesting that enzyme II is also anchored to the cell membrane. It is, however, likely that some of enzyme II exists as a soluble form in plasma, possibly after proteolytic processing. (+info)
YND1, a homologue of GDA1, encodes membrane-bound apyrase required for Golgi N- and O-glycosylation in Saccharomyces cerevisiae.
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The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1 gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in the gda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and approximately 50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Deltaynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Deltagda1 disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Deltaynd1Deltagda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells. (+info)
Osmotic cell swelling-induced ATP release mediates the activation of extracellular signal-regulated protein kinase (Erk)-1/2 but not the activation of osmo-sensitive anion channels.
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Human intestine 407 cells respond to hypo-osmotic stress by the rapid release of ATP into the extracellular medium. A difference in the time course of activation as well as in the sensitivity to cytochalasin B treatment and BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester] loading suggests that ATP leaves the cell through a pathway distinct from volume-regulated anion channels. To evaluate a putative role for nucleotides as autocrinic/paracrinic factors in osmotic signalling, the effects of extracellular ATP on the regulation of volume-sensitive anion channels as well as on the hypotonicity-induced activation of extracellular signal-regulated protein kinases (Erk-1/2) were investigated. Micromolar concentrations of ATP were unable to elicit an isotope efflux from (125)I(-)-loaded cells by itself, but strongly potentiated the hypotonicity-provoked anion efflux through a Ca(2+)-dependent mechanism. The order of potency of nucleotides (ATP = UTP = ATP[S] > ADP = AMP >> adenosine = cAMP) indicated the involvement of P2Y(2) receptors. In contrast, millimolar concentrations of ATP markedly inhibited both the osmotically induced isotope efflux and whole-cell Cl(-) currents. Inhibition of whole-cell Cl(-) currents, not only by millimolar ATP but also by the purinoceptor antagonists suramin and reactive blue, was observed most prominently at depolarizing holding potentials, suggesting a direct interaction with volume-sensitive Cl(-) channels rather than interaction with purinoceptors. Both ATP and UTP, at submicromolar levels, were found to act as potent activators of Erk-1/2 in intestine 407 cells. Addition of the ATP hydrolase apyrase to the bath greatly reduced the hypotonicity-induced Erk-1/2 activation, but did not affect the swelling-induced isotope efflux or whole-cell Cl(-) currents. Furthermore, pre-treatment with suramin or reactive blue almost completely prevented the hypo-osmotic activation of Erk-1/2. The results indicate that extracellularly released ATP functions as an autocrinic/paracrinic factor that mediates hypotonicity-induced Erk-1/2 activation but does not serve as an activator of volume-sensitive compensatory Cl(-) currents. (+info)
A yeast Golgi E-type ATPase with an unusual membrane topology.
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E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+). The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized. (+info)
Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.
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A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. (+info)
Palmitoylation targets CD39/endothelial ATP diphosphohydrolase to caveolae.
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Ectonucleotidases influence purinergic receptor function by the hydrolysis of extracellular nucleotides. CD39 is an integral membrane protein that is a prototype member of the nucleoside 5'-triphosphate diphosphohydrolase family. The native CD39 protein has two intracytoplasmic and two transmembrane domains. There is a large extracellular domain that undergoes extensive glycosylation and can be post-translationally modified by limited proteolysis. We have identified a potential thioester linkage site for S-acylation within the N-terminal region of CD39 and demonstrate that this region undergoes palmitoylation in a constitutive manner. The covalent lipid modification of this region of the protein appears to be important both in plasma membrane association and in targeting CD39 to caveolae. These specialized plasmalemmal domains are enriched in G protein-coupled receptors and appear to integrate cellular activation events. We suggest that palmitoylation could modulate the function of CD39 in regulating cellular signal transduction pathways. (+info)
Modulation of nucleoside [correction of nucleotide] triphosphate diphosphohydrolase-1 (NTPDase-1)cd39 in xenograft rejection.
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BACKGROUND: There is increasing evidence showing that extracellular nucleosides [corrected] may be important mediators of vascular inflammation. Nucleoside [corrected] triphosphate diphosphohydrolase-1 (NTPDase-1, identical to CD39), the major vascular endothelial ectonucleotidase, is responsible for the hydrolysis of both extracellular ATP and ADP in the blood plasma to AMP. Studies were therefore conducted to evaluate the role of vascular NTPDase-1/cd39 in modulating platelet activation and vascular injury in cardiac xenografts. MATERIALS AND METHODS: Cardiac xenografts from both wild-type and cd39 knockout mice (C57BL/6 x 129 Svj) were transplanted into Lewis rats. Alterations in cd39 mRNA transcripts and NTPDase activity expression were evaluated in wild-type grafts in untreated rats and then following complement depletion and immunosuppression. Rejection responses were studied with both mutant and wild-type grafts in the following models: presensitization with or without complement depletion, complement depletion alone, and with chronic immunosuppression to induce long-term graft survival. RESULTS: NTPDase biochemical activity in wild-type xenografts rapidly decreased after transplantation but soon rebounded with graft survival. Elevated levels of cd39 mRNA with associated increases in NTPDase activity were observed in all long-term surviving wild-type grafts. Hyperacute xenograft rejection times were comparable in wild-type and mutant grafts but cd39-deficient grafts were subject to more rapid rejection and exhibited pronounced vascular injury in complement-depleted, presensitized rats. The cd39-deficient grafts in immunosuppressed recipients were subject to increased intravascular platelet sequestration and fibrin deposition; this resulted in focal myocardial infarction in long-term surviving mutant xenografts. CONCLUSIONS: Augmentation of NTPDase-1 activity may be an important adaptive response for graft survival. Our results suggest that NTPDase-1/cd39 influences pathways of vascular injury in cardiac xenografts. (+info)
H(2)O(2) activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation.
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Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H(2)O(2) concentrations on platelet adhesion to immobilized fibrinogen and on pp72(syk) and pp125(FAK) tyrosine phosphorylation. Our results demonstrate that: (1) H(2)O(2) does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H(2)O(2) does not affect pp72(syk) phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H(2)O(2) reduces, in a dose-dependent fashion, pp125(FAK) phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H(2)O(2) near to physiological values (10-12 microM) are able to strengthen the subthreshold activation of pp125(FAK) induced by epinephrine in apyrase-treated platelets; (5) H(2)O(2) doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H(2)O(2) to modulate pp125(FAK) phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation. (+info)