Biochemical Characterization of CD39L4

Mulero, Julio J.; Yeung, George; Nelken, Sarah T.; Bright, Jessica M.; McGowan, Daniel W.; Ford, J. E.

doi:10.1021/bi000960y

Cited by 30 publications

(22 citation statements)

References 15 publications

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“…Although E-NTPDases are heavily glycosylated enzymes, the role of N-glycans in enzymatic activity has remained unclear. Some studies support an importance of glycosylation for enzymatic activity of E-NTPDases (33)(34)(35), while others report little or no effect (17,23).…”

Section: Discussionmentioning

confidence: 99%

“…A novel rat ecto-ATPase mRNA was identified that con-tains an additional 193-bp exon arising from alternative splicing in the 3Ј-end of the gene (22). Splice variants also were reported for the related ecto-nucleotide pyrophosphatase/phosphodiesterase family (E-NPP), where human phosphodiesterase-I␣ (PD-I␣) and autotaxin likely represent splice variants of the NPP2 gene (23)(24)(25)(26) In this study we delineate the genomic organization of the human ecto-ATPase gene and describe three splice variants of the human NTPDase2 gene encoding proteins of 495, 472, and 450 amino acids, respectively. Additionally, we demonstrate the importance of Cys 399 for correct processing and trafficking to plasma membrane of the human NTPDase2.…”

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confidence: 99%

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Requirement of Cys399 for Processing of the Human Ecto-ATPase (NTPDase2) and Its Implications for Determination of the Activities of Splice Variants of the Enzyme

Mateo

Kreda

Henry

et al. 2003

Journal of Biological Chemistry

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Ecto-ATPase (CD39L1) corresponds to the type 2 enzyme of the ecto-nucleoside triphosphate diphosphohydrolase family (E-NTPDase). We have isolated from human ECV304 cells three cDNAs with high homology with members of the E-NTPDase family that encode predicted proteins of 495, 472, and 450 amino acids. Sequencing of a genomic DNA clone confirmed that these three sequences correspond to splice variants of the human ecto-ATPase (NTPDase2␣, -2␤, and -2␥). Although all three enzyme forms were expressed heterologously to similar levels in Chinese hamster ovary cells clone K-1 (CHO-K1) cells, only the 495-amino acid protein (NTPDase2␣ exhibited ecto-ATPase activity. Immunolocalization studies demonstrated that NTPDase2␣ is fully processed and trafficked to the plasma membrane, whereas the NTPDase2␤ and -2␥ splice variants were retained in not fully glycosylated forms in the endoplasmic reticulum. The potential roles of two highly conserved residues, Cys 399 and Asn 443 , in the activity and cellular trafficking of the ecto-ATPase were examined. Mutation of Cys 399 , which is absent in NTPDase2␤ and -2␥, produced a protein completely devoid of nucleotidase activity, while mutation of Asn 443 to Asp resulted in substantial loss of activity. Neither the Cys 399 nor Asn 443 mutants were fully glycosylated, and both were retained in the endoplasmic reticulum. These results indicate that the lack of ecto-nucleotidase activity exhibited by NTPDase2␤ and -2␥ and the C399S mutant, as well as the large reduction of activity in the N443D mutant are due to alterations in the folding/maturation of these proteins.Extracellular nucleotides modulate many physiological responses through interaction with G protein-coupled metabotropic receptors (P2Y) and ligand-gated ionotropic receptors (P2X) (1-3). Termination of nucleotide-promoted signaling is accomplished by rapid degradation and/or interconversion of extracellular nucleotides by ecto-nucleotidases. Although the primary function of ecto-nucleotidases involves the hydrolysis of extracellular nucleotides, the complete physiological significance of these enzymes is not fully understood. For example, ecto-nucleotidases also have been implicated in physiological phenomena as diverse as cell adhesion (4), purine recycling (5), pain transmission (6), immune function (7), blood hemostasis (8), and others (9, 10).The ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) 1 are a family of ecto-nucleotidases, including ecto-ATPases and ecto-ATPDases (apyrases), that hydrolyze nucleoside 5Ј-tri-and diphosphates with isozyme-dependent nucleotide selectivities and biochemical properties. The E-NTPDase family includes membrane-associated enzymes (NTPDase1 to NTPDase4) and soluble enzymes (NTPDase5 and NTPDase6) (see Zimmermann (10) for a review). NTPDase2 (also known as ecto-ATPase or CD39L1) exhibits a strong preference for nucleoside triphosphates (NTPs) over diphosphates (nucleoside diphosphates), hydrolyzing nucleoside diphosphates at 3-5% of the rate of NTPs (10 -14). NTPDase2 contai...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Requirement of Cys399 for Processing of the Human Ecto-ATPase (NTPDase2) and Its Implications for Determination of the Activities of Splice Variants of the Enzyme

Mateo

Kreda

Henry

et al. 2003

Journal of Biological Chemistry

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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

854

922

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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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“…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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Biochemical Characterization of CD39L4

Cited by 30 publications

References 15 publications

Requirement of Cys399 for Processing of the Human Ecto-ATPase (NTPDase2) and Its Implications for Determination of the Activities of Splice Variants of the Enzyme

Requirement of Cys399 for Processing of the Human Ecto-ATPase (NTPDase2) and Its Implications for Determination of the Activities of Splice Variants of the Enzyme

Cellular function and molecular structure of ecto-nucleotidases

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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