Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase

Murphy-Piedmonte, Deirdre M.; Crawford, Patrick A.; Kirley, Terence L.

doi:10.1016/j.bbapap.2004.11.017

Cited by 34 publications

(31 citation statements)

References 27 publications

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“…Glycosylation does not appear to be required for enzymatic activity of NTPDase5 [147,148] and NTPDase6 [79,146]. This is shown by bacterial expression of the non-glycosylated proteins or by heterologous expression in the presence of tunicamycin (NTPDase5, [147]).…”

Section: Glycosylationmentioning

confidence: 99%

Cellular function and molecular structure of ecto-nucleotidases

Zimmermann

Zebisch

Sträter

2012

Purinergic Signalling

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Ecto-nucleotidases play a pivotal role in purinergic signal transmission. They hydrolyze extracellular nucleotides and thus can control their availability at purinergic P2 receptors. They generate extracellular nucleosides for cellular reuptake and salvage via nucleoside transporters of the plasma membrane. The extracellular adenosine formed acts as an agonist of purinergic P1 receptors. They also can produce and hydrolyze extracellular inorganic pyrophosphate that is of major relevance in the control of bone mineralization. This review discusses and compares four major groups of ectonucleotidases: the ecto-nucleoside triphosphate diphosphohydrolases, ecto-5′-nucleotidase, ecto-nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Only recently and based on crystal structures, detailed information regarding the spatial structures and catalytic mechanisms has become available for members of these four ecto-nucleotidase families. This permits detailed predictions of their catalytic mechanisms and a comparison between the individual enzyme groups. The review focuses on the principal biochemical, cell biological, catalytic, and structural properties of the enzymes and provides brief reference to tissue distribution, and physiological and pathophysiological functions.

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Section: Glycosylationmentioning

confidence: 99%

Cellular function and molecular structure of ecto-nucleotidases

Zimmermann

Zebisch

Sträter

2012

Purinergic Signalling

890

922

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“…The NTPDase1 enzyme acts via the same mechanism as the SCAN enzymes used in the present study -inhibition of platelet aggregation and clotting by hydrolysis of the platelet aggregation agonist, ADP, thereby preventing activation of the platelet purinergic receptors, which normally lead to platelet activation, shape change, aggregation, and clotting. Our laboratory has experience with the bacterial expression, refolding, purification and characterization of the two naturally occurring soluble NTPDases -NTPDase5 [32] and NTPDase6 [33]. In comparison to SCAN, both of these enzymes are less efficiently refolded after bacterial expression, as well as less stable proteins that are more prone to aggregation.…”

Section: Discussionmentioning

confidence: 99%

“…In comparison to SCAN, both of these enzymes are less efficiently refolded after bacterial expression, as well as less stable proteins that are more prone to aggregation. NTPDase5 is especially unstable and prone to aggregation [32], making it particularly unsuitable as a potential therapeutic protein. Thus, the modified human SCAN enzymes are preferable to the naturally soluble human NTPDases for development as potential injectable anti-coagulant and anti-thrombotic therapeutic agents.…”

Section: Discussionmentioning

confidence: 99%

Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo

Yang

Kirley

2008

Thrombosis Research

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Human soluble calcium-activated nucleotidase (human SCAN) is a homologue of the salivary anticoagulant apyrases injected by insects into their hosts to allow blood feeding. However, the human enzyme, unlike its insect counterparts, does not efficiently hydrolyze the platelet agonist, ADP. By site-directed mutagenesis, two mutant human SCANs were constructed and expressed in bacteria. Following refolding from inclusion bodies and purification, these enzymes were assessed for anticoagulant and anti-thrombotic efficacy. These engineered proteins include both active site mutations and a dimer interface mutation to increase the stability and ADPase activity of the modified human nucleotidase. The ADPase activity of these mutants increased more than ten fold. The E130Y/ K201M/E216M SCAN mutant efficiently inhibited platelet aggregation in vitro. In addition, the E130Y/K201M/T206K/T207E/E216M mutant inhibited jugular vein thrombosis in the murine ferric chloride-induced model of thrombosis, as assessed by laser Doppler blood flow measurements. The bed bug insect homologue of human SCAN was also expressed and purified, and used in these in vivo experiments as a benchmark to assess the therapeutic potential of the engineered human enzymes. The most active modified human enzyme was able to completely inhibit the thrombosis induced by ferric chloride at roughly double the protein dose used for the bed bug enzyme. Thus, for the first time, we show that an engineered form of this human protein is efficacious in an in vivo model of thrombosis, demonstrating that suitably modified human SCAN enzymes have therapeutic potential as anti-coagulant and anti-thrombotic therapeutic agents. This suggests their utility in future treatment strategies for thrombotic cardiovascular diseases, including myocardial infarctions and ischemic strokes. Keywordscalcium-activated nucleotidase; ferric chloride-induced thrombosis; aggregometry; platelets; anticoagulant proteins; laser Doppler blood flow Cardiovascular and cerebrovascular diseases continue to be the leading causes of death in the developed world, with more than 450,000 patients in the United States afflicted by stroke annually [1]. The platelet is a major contributor to occlusive thrombus formation in acute Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest StatementThere are no conflicts of interest to declare. The sole funding for this study was obtained via NIH grant HL72882 to T.L.K. Mammals express a protein homologous to the nucleotidases found in the saliva of bloodsucking insects. In insects, these soluble pro...

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“…NTPDase1-4, 7, and 8 are always membrane-bound, having two membrane-spanning domains, one each at the C-and N-termini of the proteins [3]. NTPDase5 and 6 have a unique topology in that they have just one membrane-spanning domain and, after cleavage of these respective N-terminal signal peptides, they can be released into the extracellular space as soluble enzymes [4][5][6][7][8][9]. NTPDases that are found at the cell surface modulate signaling mediated by cell-surface purinergic receptors, which are known to control many physiological processes, including blood clotting, pain perception, and smooth muscle contraction [1,10].…”

Section: Introductionmentioning

confidence: 99%

“…For example, NTPDase1 (aka ecto-apyrase, CD39) hydrolyzes di-and triphosphates almost equally well (ATPase:ADPase ratio≈ 1), while NTPDase2 (ecto-ATPase) preferentially hydrolyzes triphosphates (NTPase:NDPase ratios greater than 25:1). NTPDase5 and 6 greatly prefer diphosphates over triphosphates as substrates [4][5][6][7][8][9]. These differential nucleotide hydrolysis profiles are important for distinguishing the functions of these various NTPDases [10].…”

Section: Introductionmentioning

confidence: 99%

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

Crawford¹,

Gaddie²,

Smith³

et al. 2007

Archives of Biochemistry and Biophysics

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Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) is a cell surface, membrane-bound enzyme that hydrolyzes extracellular nucleotides, thereby modulating purinergic signaling. An alternatively spliced variant of NTPDase3 was obtained and analyzed. This alternatively spliced variant, termed "NTPDase3β", is produced through the use of an alternative terminal exon (exon 11) in place of the terminal exon (exon 12) in the full-length NTPDase3, now termed "NTPDase3α". This results in an expressed protein lacking the C-terminal cytoplasmic sequence, the C-terminal transmembrane helix, and apyrase conserved region 5. The cDNA encoding this truncated splice variant was detected in a human lung library by PCR. Like the full-length NTPDase3α, the alternatively spliced NTPDase3β was expressed in COS cells after transfection, but only the fulllength NTPDase3α is enzymatically active and properly trafficked to the plasma membrane. However, when the truncated NTPDase3β was co-transfected with full-length NTPDase3α, there was a significant reduction in the amount of NTPDase3α that was properly processed and trafficked to the plasma membrane as active enzyme, indicating that the truncated form interferes with normal biosynthetic processing of the full-length enzyme. This suggests a role for the NTPDase3β variant in the regulation of NTPDase3 nucleotidase activity, and therefore the control of purinergic signaling, in those cells and tissues expressing both NTPDase3α and NTPDase3β.

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Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase

Cited by 34 publications

References 27 publications

Cellular function and molecular structure of ecto-nucleotidases

Cellular function and molecular structure of ecto-nucleotidases

Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo

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

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