Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): A possible modulator of nucleotidase activity and purinergic signaling

Crawford, Patrick A.; Gaddie, Keith J.; Smith, Thomas M.; Kirley, Terence L.

doi:10.1016/j.abb.2006.10.013

Cited by 9 publications

(6 citation statements)

References 35 publications

(65 reference statements)

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“…For construction of ER-targeted NTPDase3, we deleted the C-terminal part of the cytosolic tail in the wild-type enzyme which is not necessary for normal processing or activity of NTPDase3 (31), and added the ER targeting signal from the wheat germ agglutinin binding protein 1 (WBP1, (16)) together with the hemagglutinin (HA) epitope and a spacer (Fig. 2 A ).…”

Section: Resultsmentioning

confidence: 99%

Trafficking and Intracellular ATPase Activity of Human Ecto-nucleotidase NTPDase3 and the Effect of ER-Targeted NTPDase3 on Protein Folding

2008

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Ecto-nucleoside triphosphate diphosphohydrolases, NTPDase1 (CD39) and NTPDase3, are integral plasma membrane proteins that hydrolyze extracellular nucleotides, thereby modulating the function of purinergic receptors. During processing in the secretory pathway, the active sites of ectonucleotidases are located in the lumen of vesicular compartments, thus raising the question whether the ecto-nucleotidases affect the ATP-dependent processes in these compartments, including protein folding in the endoplasmic reticulum (ER). It has been reported that CD39 is not active until it reaches the plasma membrane, suggesting that terminal glycosylation in Golgi is critical for its activity. To investigate the subcellular location and the mechanism of ecto-nucleotidase activation, we expressed human NTPDase3 in COS-1 cells and blocked the secretory transport with monensin or brefeldin A, or by targeting to ER with a signal peptide. Cell surface biotinylation, sensitivity to glycosidases, and fluorescence microscopy analyses suggest that, in contrast to the previous report on CD39, NTPDase3 becomes catalytically active in the ER or in the ER-Golgi intermediate compartment, and that terminal glycosylation in Golgi is not essential for activity. Moreover, ER-targeted NTPDase3, but not wild-type NTPDase3 or ER-targeted inactive G221A mutant, significantly diminished the folding efficiency and the transport to the plasma membrane of co-expressed CD39 used as a reporter protein. These data suggest that ER-targeted NTPDase3 significantly depletes ATP in ER, whereas wild-type NTPDase3 is likely to acquire ATPase activity in a post-ER, but pre-Golgi, compartment, thus avoiding unproductive ATP hydrolysis and interference with protein folding in the ER. ERtargeted NTPDase3 may be a useful experimental tool to study the effects of ER ATP depletion on ER function under normal and stress conditions.

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Section: Resultsmentioning

confidence: 99%

Trafficking and Intracellular ATPase Activity of Human Ecto-nucleotidase NTPDase3 and the Effect of ER-Targeted NTPDase3 on Protein Folding

2008

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“…This restriction prevents use of the common epitope mapping approach for making truncated proteins because truncation of anything more than the short C-terminal cytoplasmic tail portion (i.e. the A511Stop mutant) [54] results in NTPDase3 proteins that are misfolded and enzymatically inactive, and thus not recognized by the inhibitory monoclonal antibodies (data not shown). Therefore, we used two other approaches to define the monoclonal antibody epitope: (a) introduction of cysteine residues by mutagenesis followed by chemical cleavage of native NTPDase3 with NTCB at the introduced residues and (b) disruption of individual disulfide bonds by mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase‐3

Munkonda¹,

Pelletier²,

Ivanenkov³

et al. 2008

The FEBS Journal

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The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases), which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. In the present study, we report the first inhibitory monoclonal antibodies specific for an NTPDase, namely human NTPDase3 (EC 3.6.1.5), as assessed by ELISA, western blotting, flow cytometry, immunohistochemistry and inhibition assays. Antibody recognition of NTPDase3 is greatly attenuated by denaturation with SDS, and abolished by reducing agents, indicating the significance of the native conformation and the disulfide bonds for epitope recognition. Using site-directed chemical cleavage, the SDS-resistant parts of the epitope were located in two fragments of the C-terminal lobe of NTPDase3 (i.e. Leu220–Cys347 and Cys347–Pro485), which are both required for antibody binding. Additional site-directed mutagenesis revealed the importance of Ser297 and the fifth disulfide bond (Cys399–Cys422) for antibody binding, indicating that the discontinuous inhibitory epitope is located on the extracellular C-terminal lobe of NTPDase3. These antibodies inhibit recombinant NTPDase3 by 60–90%, depending on the conditions. More importantly, they also efficiently inhibit the NTPDase3 expressed in insulin secreting human pancreatic islet cells in situ. Because insulin secretion is modulated by extracellular ATP and purinergic receptors, this finding suggests the potential application of these inhibitory antibodies for the study and control of insulin secretion.

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“…NTPDase3β was expressed and targeted to the membrane; however, it had no measurable activity. When COS cells were co-transfected with the cDNAs of NTPDase3α and 3β, the amount of the active NTPDase3α delivered to the plasma membrane was reduced [90]. These results suggested that NTPDase3β may play a role in regulating NTPDase3α activity and purinergic signaling in tissues that express both proteins.…”

Section: Cr3 Acr3mentioning

confidence: 94%

The GDA1_CD39 superfamily: NTPDases with diverse functions

Knowles

2011

Purinergic Signalling

145

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The first comprehensive review of the ubiquitous "ecto-ATPases" by Plesner was published in 1995. A year later, a lymphoid cell activation antigen, CD39, that had been cloned previously, was shown to be an ecto-ATPase. A family of proteins, related to CD39 and a yeast GDPase, all containing the canonical apyrase conserved regions in their polypeptides, soon started to expand. They are now recognized as members of the GDA1_CD39 protein family. Because proteins in this family hydrolyze nucleoside triphosphates and diphosphates, a unifying nomenclature, nucleoside triphosphate diphopshohydrolases (NTPDases), was established in 2000. Membrane-bound NTPDases are either located on the cell surface or membranes of intracellular organelles. Soluble NTPDases exist in the cytosol and may be secreted. In the last 15 years, molecular cloning and functional expression have facilitated biochemical characterization of NTPDases of many organisms, culminating in the recent structural determination of the ecto-domain of a mammalian cell surface NTPDase and a bacterial NTPDase. The first goal of this review is to summarize the biochemical, mutagenesis, and structural studies of the NTPDases. Because of their ability in hydrolyzing extracellular nucleotides, the mammalian cell surface NTPDases (the ecto-NTPDases) which regulate purinergic signaling have received the most attention. Less appreciated are the functions of intracellular NTPDases and NTPDases of other organisms, e.g., bacteria, parasites, Drosophila, plants, etc. The second goal of this review is to summarize recent findings which demonstrate the involvement of the NTPDases in multiple and diverse physiological processes: pathogen-host interaction, plant growth, eukaryote cell protein and lipid glycosylation, eye development, and oncogenesis.

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Characterization of an alternative splice variant of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): A possible modulator of nucleotidase activity and purinergic signaling

Cited by 9 publications

References 35 publications

Trafficking and Intracellular ATPase Activity of Human Ecto-nucleotidase NTPDase3 and the Effect of ER-Targeted NTPDase3 on Protein Folding

Trafficking and Intracellular ATPase Activity of Human Ecto-nucleotidase NTPDase3 and the Effect of ER-Targeted NTPDase3 on Protein Folding

Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase‐3

The GDA1_CD39 superfamily: NTPDases with diverse functions

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