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1.  Nucleotide Pyrophosphatase/Phosphodiesterase 1 Exerts a Negative Effect on Starch Accumulation and Growth in Rice Seedlings under High Temperature and CO2 Concentration Conditions 
Plant and Cell Physiology  2013;55(2):320-332.
Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes (NPP1–NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5′-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28°C/23°C (12 h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33°C/28°C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions.
PMCID: PMC3913438  PMID: 24092883
ADP-glucose; CO2; NPP; Oryza sativa; Plastid; Starch
2.  Autotaxin (ATX): a multi-functional and multi-modular protein possessing enzymatic lysoPLD activity and matricellular properties 
Biochimica et biophysica acta  2008;1781(9):525-530.
Recent studies have established that autotaxin (ATX), also known as phosphodiesterase Iα/autotaxin (PD-Iα/ATX) or (ecto)nucleotide pyrophosphatase/phosphodiesterase 2 [(E)NPP2], represents a multi-functional and multi-modular protein. ATX was initially thought to function exclusively as a phosphodiesterase/pyrophosphatase. However, it has become apparent that this enzymatically active site, which is ultimately responsible for ATX’s originally discovered property of tumor cell motility stimulation, mediates the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). In addition, a separate functionally active domain, here referred to as the Modulator of Oligodendrocyte Remodeling and Focal adhesion Organization (MORFO) domain, was discovered in studies analyzing the role of ATX during the differentiation of myelinating cells of the central nervous system (CNS), namely oligodendrocytes. This novel domain was found to mediate anti-adhesive, i.e. matricellular, properties and to promote morphological maturation of oligodendrocytes. In this review, we summarize our current understanding of ATX’s structure-function domains and discuss their contribution to the presently known main functional roles of ATX.
PMCID: PMC2564869  PMID: 18485925
autotaxin; lysoPLD; phosphodiesterase-Iα; matricellular; tumor progression; vascular development; oligodendrocyte development
3.  Benzyl and Naphthalene-Methyl Phosphonic Acid Inhibitors of Autotaxin with Anti-invasive and Anti-metastatic Actions 
ChemMedChem  2011;6(5):922-935.
Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) via a lysophospholipase D activity that leads to the generation of the growth factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Here we report the synthesis and pharmacological characterization of inhibitors of ATX based on the 4-tetradecanoylaminobenzyl phosphonic acid scaffold that was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzyl phosphonic acid and 6-substituted naphthalen-2-yl-methyl phosphonic acid analogs blocked ATX with Ki values in the low-micromolar-nanomolar range against FS-3, LPC, and nucleotide substrates through a mixed-mode mechanism of inhibition. None of the compounds tested inhibited the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor subtypes. Analogs 22 and 30b, the two most potent ATX inhibitors, dose-dependently inhibited the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro. The average terminal half-life for compound 22 was 10h ± 5.4h and it caused a long-lasting reduction plasma LPA levels. Compounds 22 and 30b significantly reduced lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The described 4-substituted benzyl phosphonic acids and 6-substituted naphthalen-2-yl-methyl phosphonic acids represent new lead compounds that effectively inhibit the ATX-LPA-LPA receptor axis both in vitro and in vivo.
PMCID: PMC3517046  PMID: 21465666
ATX inhibitors; LPA receptors; 4-substituted benzyl phosphonic acids; 6-substituted naphthalen-2-yl-methyl phosphonic acids; structure-activity relationships
4.  Promiscuous sulfatase activity and thio-effects in a phosphodiesterase of the alkaline phosphatase superfamily† 
Biochemistry  2008;47(48):12853-12859.
The nucleotide phosphodiesterase/pyrophosphatase from Xanthomonas axonopodis (NPP) is a structural and evolutionary relative of alkaline phosphatase that preferentially hydrolyzes phosphate diesters. With the goal of understanding how these two enzymes with nearly identical Zn2+ bimetallo sites achieve high selectivity for hydrolysis of either phosphate monoesters or diesters, we have measured a promiscuous sulfatase activity in NPP. Sulfate esters are nearly isosteric with phosphate esters but carry less charge, offering a probe of electrostatic contributions to selectivity. NPP exhibits sulfatase activity with kcat/KM value of 2 × 10−5 M−1s−1, similar to the R166S mutant of alkaline phosphatase. We further report the effects of thio-substitution on phosphate monoester and diester reactions. Reactivities with these non-cognate substrates illustrate a reduced dependence of NPP reactivity on the charge of the nonbridging oxygen situated between the Zn2+ ions relative to that in alkaline phosphatase. This reduced charge dependence can explain about 102 of the 107-fold differential catalytic proficiency for the most similar monoester and diester substrates in the two enzymes. The results further suggest that active site contacts to substrate oxygen atoms that do not contact the Zn2+ ions may play an important role in defining the selectivity of the enzymes.
PMCID: PMC2662379  PMID: 18975918
5.  Pho5p and Newly Identified Nucleotide Pyrophosphatases/ Phosphodiesterases Regulate Extracellular Nucleotide Phosphate Metabolism in Saccharomyces cerevisiae 
Eukaryotic Cell  2005;4(11):1892-1901.
Extracellular nucleotides play many biological roles, including intercellular communication and modulation of nucleotide receptor signaling, and are dependent on the phosphorylation state of the nucleotide. Regulation of nucleotide phosphorylation is necessary, and a specialized class of enzymes, nucleotide pyrophosphatases/phosphodiesterases (E-NPPs), has been identified in mammals to perform this function. Although the E-NPP class is conserved among complex eukaryotes, this system has not yet been identified in Saccharomyces cerevisiae. Using genetic and biochemical experiments, we show that two orthologs of the E-NPP family, referred to as Npp1p and Npp2p, exist in budding yeast and can perform nucleotide phosphate hydrolysis. This activity is enhanced during phosphate starvation, where hydrolyzed phosphates can be imported from extracellular sources and utilized to overcome phosphate starvation through the activity of the Pho5p acid phosphatase. The added compensatory effect by Pho5p is also a newly established role for Pho5p. This study demonstrates that extracellular nucleotide phosphate metabolism appears to be controlled by at least two independent regulatory mechanisms, uniting phosphate starvation with extracellular nucleotide regulation.
PMCID: PMC1287867  PMID: 16278456
6.  Physiologic and pathologic functions of the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP1 in calcification 
Purinergic Signalling  2006;2(2):371-377.
The catabolism of ATP and other nucleotides participates partly in the important function of nucleotide salvage by activated cells and also in removal or de novo generation of compounds including ATP, ADP, and adenosine that stimulate purinergic signaling. Seven nucleotide pyrophosphatase/phosphodiesterase NPP family members have been identified to date. These isoenzymes, related by up conservation of catalytic domains and certain other modular domains, exert generally non-redundant functions via distinctions in substrates and/or cellular localization. But they share the capacity to hydrolyze phosphodiester or pyrophosphate bonds, though generally acting on distinct substrates that include nucleoside triphosphates, lysophospholipids and choline phosphate esters. PPi generation from nucleoside triphosphates, catalyzed by NPP1 in tissues including cartilage, bone, and artery media smooth muscle cells, supports normal tissue extracellular PPi levels. Balance in PPi generation relative to PPi degradation by pyrophosphatases holds extracellular PPi levels in check. Moreover, physiologic levels of extracellular PPi suppress hydroxyapatite crystal growth, but concurrently providing a reservoir for generation of pro-mineralizing Pi. Extracellular PPi levels must be supported by cells in mineralization-competent tissues to prevent pathologic calcification. This support mechanism becomes dysregulated in aging cartilage, where extracellular PPi excess, mediated in part by upregulated NPP1 expression stimulates calcification. PPi generated by NPP1modulates not only hydroxyapatite crystal growth but also chondrogenesis and expression of the mineralization regulator osteopontin. This review pays particular attention to the role of NPP1-catalyzed PPi generation in the pathogenesis of certain disorders associated with pathologic calcification.
PMCID: PMC2254483  PMID: 18404477
ANK; ANKH; cartilage; inorganic phosphate; inorganic pyrophosphate; osteopontin
7.  QM/MM analysis suggests that Alkaline Phosphatase (AP) and Nucleotide pyrophosphatase/phosphodiesterase slightly tighten the transition state for phosphate diester hydrolysis relative to solution: implication for catalytic promiscuity in the AP superfamily 
Several members of the Alkaline Phosphatase (AP) superfamily exhibit a high level of catalytic proficiency and promiscuity in structurally similar active sites. A thorough characterization of the nature of transition state for different substrates in these enzymes is crucial for understanding the molecular mechanisms that govern those remarkable catalytic properties. In this work, we study the hydrolysis of a phosphate diester, MpNPP−, in solution, two experimentally well-characterized variants of AP (R166S AP, R166S/E322Y AP) and wild type Nucleotide pyrophosphatase/phosphodiesterase (NPP) by QM/MM calculations in which the QM method is an approximate density functional theory previously parameterized for phosphate hydrolysis (SCC-DFTBPR). The general agreements found between these calculations and available experimental data for both solution and enzymes support the use of SCC-DFTBPR/MM for a semi-quantitative analysis of the catalytic mechanism and nature of transition state in AP and NPP. Although phosphate diesters are cognate substrates for NPP but promiscuous substrates for AP, the calculations suggest that their hydrolysis reactions catalyzed by AP and NPP feature similar synchronous transition states that are slightly tighter in nature compared to that in solution, due in part to the geometry of the bimetallic zinc motif. Therefore, this study provides the first direct computational support to the hypothesis that enzymes in the AP superfamily catalyze cognate and promiscuous substrates via similar transition states to those in solution. Our calculations do not support the finding of recent QM/MM studies by López-Canut and coworkers, who suggested that the same diester substrate goes through a much looser transition state in NPP/AP than in solution, a result likely biased by the large structural distortion of the bimetallic zinc site in their simulations. Finally, our calculations for different phosphate diester orientations and phosphorothioate diesters highlight that the interpretation of thio-substitution experiments is not always straightforward.
PMCID: PMC3257412  PMID: 22097879
8.  Characterization of a Di-leucine–based Signal in the Cytoplasmic Tail of the Nucleotide-pyrophosphatase NPP1 That Mediates Basolateral Targeting but not Endocytosis 
Molecular Biology of the Cell  2001;12(10):3004-3015.
Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.
PMCID: PMC60151  PMID: 11598187
9.  Crystallization and preliminary X-ray crystallographic analysis of human autotaxin 
The α isoform of human autotaxin has been crystallized. Diffraction data were collected to 3.0 Å resolution using synchrotron radiation.
Autotaxin (ATX), which is also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2) or phosphodiesterase Iα (PD-Iα), is an extracellular lysophospholipase D which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). ATX stimulates tumour-cell migration, angiogenesis and metastasis and is an attractive target for cancer therapy. For crystallographic studies, the α isoform of human ATX was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.0 Å resolution from a monoclinic crystal form belonging to space group C2, with unit-cell parameters a = 311.4, b = 147.9, c = 176.9 Å, β = 122.6°.
PMCID: PMC3080147  PMID: 21505238
autotaxin; lysophosphatidic acid; lysophospholipase D; ectonucleotide pyrophosphatase/phosphodiesterase 2
10.  Anticancer activity of FTY720: phosphorylated FTY720 inhibits autotaxin, a metastasis-enhancing and angiogenic lysophospholipase D 
Cancer letters  2008;266(2):203-208.
FTY720 is an immunomodulator that is phosphorylated in vivo and inhibits lymphocyte mobilization by targeting sphingosine 1-phospate receptors. At doses higher than required for immunomodulation, FTY720 inhibits tumor progression through an unknown mechanism. Here we show that FTY720-phosphate is a competitive inhibitor (Ki ~0.2 µM) of autotaxin (ATX or NPP2), a nucleotide phosphodiesterase/pyrophosphatase (NPP) that enhances metastasis and angiogenesis and acts as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA). FTY720-phosphate did no affect the activity of NPP1, the closest relative of ATX. After oral administration in mice, FTY720 (3 mg/kg) significantly reduced plasma LPA levels. These results suggest that FTY720 may exert its anticancer effects, at least in part, by targeting the ATX-LPA axis.
PMCID: PMC2943588  PMID: 18378389
FTY720; autotaxin; lysophosphatidic acid; tumor progression
11.  Ectonucleotidases in the kidney 
Purinergic Signalling  2009;5(4):501-511.
Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-5′-nucleotidase and alkaline phosphatases, have been identified in the renal vasculature and/or tubular structures. In rats and mice, NTPDase1, which hydrolyses ATP through to AMP, is prominent throughout most of the renal vasculature and is also present in the thin ascending limb of Henle and medullary collecting duct. NTPDase2 and NTPDase3, which both prefer ATP over ADP as a substrate, are found in most nephron segments beyond the proximal tubule. NPPs catalyse not only the hydrolysis of ATP and ADP, but also of diadenosine polyphosphates. NPP1 has been identified in proximal and distal tubules of the mouse, while NPP3 is expressed in the rat glomerulus and pars recta, but not in more distal segments. Ecto-5′-nucleotidase, which catalyses the conversion of AMP to adenosine, is found in apical membranes of rat proximal convoluted tubule and intercalated cells of the distal nephron, as well as in the peritubular space. Finally, an alkaline phosphatase, which can theoretically catalyse the entire hydrolysis chain from nucleoside triphosphate to nucleoside, has been identified in apical membranes of rat proximal tubules; however, this enzyme exhibits relatively high Km values for adenine nucleotides. Although information on renal ectonucleotidases is still incomplete, the enzymes’ varied distribution in the vasculature and along the nephron suggests that they can profoundly influence purinoceptor activity through the hydrolysis, and generation, of agonists of the various purinoceptor subtypes. This review provides an update on renal ectonucleotidases and speculates on the functional significance of these enzymes in terms of glomerular and tubular physiology and pathophysiology.
PMCID: PMC2776140  PMID: 19333785
Renal tubule; Ectonucleotidases; Ectonucleoside triphosphate diphosphohydrolases; Ectonucleotide pyrophosphatase/phosphodiesterases; Ecto-5′-nucleotidase; Alkaline phosphatases
12.  Modulation of purinergic signaling by NPP-type ectophosphodiesterases 
Purinergic Signalling  2006;2(2):361-370.
Extracellular nucleotides can elicit a wide array of cellular responses by binding to specific purinergic receptors. The level of ectonucleotides is dynamically controlled by their release from cells, synthesis by ectonucleoside diphosphokinases and ectoadenylate kinases, and hydrolysis by ectonucleotidases. One of the four structurally unrelated families of ectonucleotidases is represented by the NPP-type ectophosphodiesterases. Three of the seven members of the NPP family, namely NPP1–3, are known to hydrolyze nucleotides. The enzymatic action of NPP1–3 (in)directly results in the termination of nucleotide signaling, the salvage of nucleotides and/or the generation of new messengers like ADP, adenosine or pyrophosphate. NPP2 is unique in that it hydrolyzes both nucleotides and lysophospholipids and, thereby, generates products that could synergistically promote cell motility. We review here the enzymatic properties of NPPs and analyze current evidence that links their nucleotide-hydrolyzing capability to epithelial and neural functions, the immune response and cell motility.
PMCID: PMC2254485  PMID: 18404476
alkaline sphingomyelinase; ectonucleotide pyrophosphatase; ectophosphodiesterase; ENPP; lysophospholipase D; NPP; NPP-type ectophosphodiesterase
13.  Positive feedback between vascular endothelial growth factor-A and autotaxin in ovarian cancer cells 
Molecular cancer research : MCR  2008;6(3):352-363.
Tumor cell migration, invasion, and angiogenesis are important determinants of tumor aggressiveness and these traits have been associated with the motility stimulating protein autotaxin (ATX). This protein is a member of the ecto-nucleotide pyrophosphatase and phosphodiesterase family of enzymes but unlike other members of this group, ATX possesses lysophospholipase D activity. This enzymatic activity hydrolyzes lysophosphatidylcholine (LPC) to generate the potent tumor growth factor and motogen, lysophosphatidic acid (LPA). In the current study, we demonstrate a link between ATX expression, LPA, and vascular endothelial growth factor (VEGF) signaling in ovarian cancer cell lines. Exogenous addition of VEGF-A to cultured cells induces ATX expression and secretion, resulting in increased extracellular LPA production. This elevated LPA, acting through LPA4, modulates VEGF responsiveness by inducing VEGFR2 expression. Down-regulation of ATX secretion in SKOV3 cells using antisense morpholino oligomers significantly attenuates cell motility responses to VEGF, ATX, LPA, and LPC. These effects are accompanied by decreased LPA4 and VEGFR2 expression as well as by increased release of soluble VEGFR1. Since LPA was previously shown to increase VEGF expression in ovarian cancer, our data suggest a positive feedback loop involving VEGF, ATX, and its product LPA that could affect tumor progression in ovarian cancer cells.
PMCID: PMC2442564  PMID: 18337445
14.  Npp1 promotes atherosclerosis in ApoE knockout mice 
Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PPi), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in humans. NPP1 and PPi have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification.
Methods and Results
Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high fat/high cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE−/− and ttw/+ ApoE−/− mice demonstrated decreased Opn expression. The 28 weeks old ttw/ttw ApoE−/− and ttw/+ ApoE−/− had significantly smaller atherosclerotic lesions compared with wild type congenic ApoE−/− mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE−/− mice without Npp1 deficiency.
We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.
PMCID: PMC3154990  PMID: 21477221
atherosclerosis; plaque calcification; osteopontin; inorganic pyrophosphate
15.  Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production 
The Journal of Cell Biology  2002;158(2):227-233.
Autotaxin (ATX) is a tumor cell motility–stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5′-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein–coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.
PMCID: PMC2173129  PMID: 12119361
lysoPLD; EDG receptor; lysophosphatidylcholine; chemotaxis; cell proliferation
16.  Carba analogs of cyclic phosphatidic acid are selective inhibitors of autotaxin and cancer cell invasion and metastasis 
The Journal of biological chemistry  2006;281(32):22786-22793.
Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2, NPP2) is an autocrine motility factor initially characterized from A2058 melanoma cell conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.
PMCID: PMC3505596  PMID: 16782709
17.  Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics* 
The Journal of Biological Chemistry  2010;285(10):7598-7609.
We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5′-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5′-monophosphate, and PPi by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5′-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PPi were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PPi by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.
PMCID: PMC2844207  PMID: 20048161
Calcium/ATPase; Cell/Compartmentation; Enzymes/ATPases; Membrane/Enzymes; Membrane/Reconstitution; Methods/Liposomes; Subcellular Organelles/Vesicles
18.  L-histidine inhibits production of lysophosphatidic acid by the tumor-associated cytokine, autotaxin 
Autotaxin (ATX, NPP-2), originally purified as a potent tumor cell motility factor, is now known to be the long-sought plasma lysophospholipase D (LPLD). The integrity of the enzymatic active site, including three crucial histidine moieties, is required for motility stimulation, as well as LPLD and 5'nucleotide phosphodiesterase (PDE) activities. Except for relatively non-specific chelation agents, there are no known inhibitors of the ATX LPLD activity.
We show that millimolar concentrations of L-histidine inhibit ATX-stimulated but not LPA-stimulated motility in two tumor cell lines, as well as inhibiting enzymatic activities. Inhibition is reversed by 20-fold lower concentrations of zinc salt. L-histidine has no significant effect on the Km of LPLD, but reduces the Vmax by greater than 50%, acting as a non-competitive inhibitor. Several histidine analogs also inhibit the LPLD activity of ATX; however, none has greater potency than L-histidine and all decrease cell viability or adhesion.
L-histidine inhibition of LPLD is not a simple stoichiometric chelation of metal ions but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. The inhibitory effect of L-histidine requires all three major functional groups of histidine: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Because of LPA's involvement in pathological processes, regulation of its formation by ATX may give insight into possible novel therapeutic approaches.
PMCID: PMC554093  PMID: 15737239
19.  High-resolution analysis of Zn2+ coordination in the alkaline phosphatase superfamily by EXAFS and x-ray crystallography 
Journal of molecular biology  2011;415(1):102-117.
Comparisons among evolutionarily related enzymes offer opportunities to reveal how structural differences produce different catalytic activities. Two structurally-related enzymes, E. coli alkaline phosphatase (AP) and X. axonopodis nucleotide pyrophosphatase/phosphodiesterase (NPP) have nearly identical binuclear Zn2+ catalytic centers, but show tremendous differential specificity for hydrolysis of phosphate monoesters or phosphate diesters. To determine if there are differences in Zn2+ coordination in the two enzymes that might contribute to catalytic specificity, we analyzed both x-ray absorption spectroscopic and x-ray crystallographic data. We report a 1.29 Å crystal structure of alkaline phosphatase with bound phosphate, allowing evaluation of interactions at the AP metal site with high resolution. To make systematic comparisons between AP and NPP, we measured zinc extended x-ray absorption fine structure (EXAFS) for AP and NPP in the free enzyme forms, with AMP and inorganic phosphate ground-state analogs, and with vanadate transition state analogs. These studies yielded average zinc-ligand distances in AP and NPP free-enzyme forms and ground-state analog forms that were identical within error, suggesting little difference in metal ion coordination among these forms. Upon binding of vanadate to both enzymes, small increases in average metal-ligand distances were observed, consistent with an increased coordination number. Slightly longer increases were observed in NPP relative to AP, which could arise from subtle rearrangements of the active site or differences in the geometry of the bound vanadyl species. Overall, the results suggest that the binuclear Zn2+ catalytic site remains very similar between AP and NPP during the course of a reaction cycle.
PMCID: PMC3249517  PMID: 22056344
x-ray absorption spectroscopy; crystal structure; nucleotide pyrophosphatase/phosphodiesterase; catalytic promiscuity; phosphoryl transfer
20.  Altered Bone Development and an Increase in FGF-23 Expression in Enpp1−/− Mice 
PLoS ONE  2012;7(2):e32177.
Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PPi), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1−/−) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1−/− mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1−/− mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1−/− mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1−/− tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1−/− null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1−/− mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1−/− mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1−/− mice compared to controls. These results indicate that Enpp1−/− mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization.
PMCID: PMC3281127  PMID: 22359666
21.  Structural and Functional Characterization of the C-terminal Domain of the Ecdysteroid Phosphate Phosphatase from Bombyx mori reveals a new enzymatic activity 
Biochemistry  2008;47(46):12135-12145.
Here, we present the crystal structure of the ecdysone phosphate phosphatase (EPPase) phosphoglycerate mutase (PGM) homology domain, the first structure of a steroid phosphate phosphatase. The structure reveals a α/β-fold common to members of the two histidine (2H)-phosphatase superfamily with strong homology to the suppressor of T-cell receptor signaling-1 (Sts-1PGM) protein. The putative EPPasePGM active site contains signature residues shared by 2H-phosphatase enzymes, including a conserved histidine (His80) that acts as a nucleophile during catalysis. The physiological substrate ecdysone 22-phosphate was modeled in a hydrophobic cavity close to the phosphate-binding site. EPPasePGM shows limited substrate specificity with an ability to hydrolyze steroid phosphates, the phospho-tyrosine (pTyr) substrate analogue para-nitrophenylphosphate (pNPP) and pTyr-containing peptides and proteins. Altogether, our data demonstrate a new protein tyrosine phosphatase (PTP) activity for EPPase. They suggest that EPPase and its closest homologues can be grouped into a distinct subfamily in the large 2H-phosphatase superfamily of proteins.
PMCID: PMC2722925  PMID: 18937503
EPPase; ecdysone; E22P; ecdysteroid; phosphatase; phospho-histidine; PTP; Sts-1
22.  Purinergic system ecto-enzymes participate in the thromboregulation of patients with indeterminate form of Chagas disease 
Purinergic Signalling  2012;8(4):753-762.
Chagas disease (CD) is a chronic and endemic illness caused by the parasite Trypanosoma cruzi. Microvascular disturbances play an important role in the progress of the disease. The purinergic signaling system participates in regulatory functions, such as immunomodulation, neuroprotection, and thromboregulation. This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface and the platelet aggregation profile from patients with indeterminate form of Chagas disease (IFCD). Thirty patients diagnosed with IFCD and 30 healthy subjects were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), ecto-5′-nucleotidase (E-5′-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals as well as the platelet aggregation. Results demonstrated an increase of 21 % in the E-NPP activity and 30 % in the E-5′-NT activity in IFCD group (P < 0.05); however, a decrease of 34 % in the E-ADA activity was determined in the same group (P < 0.001). A significant decrease of 12.7 % and 12.8 % in the platelet aggregation of IFCD group in two different concentrations of ADP (5 and 10 μM) was observed, respectively (P < 0.05). Increased E-NPP and E-5-NT activities as well as decreased E-ADA activity in platelets of patients with IFCD contributed to decrease platelet aggregation, suggesting that the purinergic system is involved in the thromboregulation process in these patients, since adenosine (the final product of ATP hydrolysis) has cardioprotective and vasodilator effects that prevent the clinical progress of the disease.
PMCID: PMC3486159  PMID: 22752606
Adenosine triphosphate; Chagas disease; Trypanosoma cruzi; Platelet aggregation; Adenosine
23.  Novel point mutations attenuate autotaxin activity 
The secreted enzyme autotaxin (ATX) stimulates tumor cell migration, tumorigenesis, angiogenesis, and metastasis. ATX hydrolyzes nucleotides, but its hydrolysis of lysophospholipids to produce lysophosphatidic acid (LPA) accounts for its biological activities. ATX has been identified only as a constitutively active enzyme, and regulation of its activity is largely unexplored. In spite of its presence in plasma along with abundant putative substrate LPC, the product LPA is found in plasma at unexpectedly low concentrations. It is plausible that the LPA-producing activity of ATX is regulated by its expression and by access to substrate(s). For this reason studying the interaction of enzyme with substrate is paramount to understanding the regulation of LPA production.
In this study we determine ATX hydrolytic activities toward several artificial and natural substrates. Two novel point mutations near the enzyme active site (H226Q and H434Q) confer attenuated activity toward all substrates tested. The Vmax for LPC compounds depends upon chain length and saturation; but this order does not differ among wild type and mutants. However the mutant forms show disproportionately low activity toward two artificial substrates, pNpTMP and FS-3. The mutant forms did not significantly stimulate migration responses at concentrations that produced a maximum response for WT-ATX, but this defect could be rescued by inclusion of exogenous LPC.
H226Q-ATX and H434Q-ATX are the first point mutations of ATX/NPP2 demonstrated to differentially impair substrate hydrolysis, with hydrolysis of artificial substrates being disproportionately lower than that of LPC. This implies that H226 and H434 are important for substrate interaction. Assays that rely on hydrolyses of artificial substrates (FS-3 and pNpTMP), or that rely on hydrolysis of cell-derived substrate, might fail to detect certain mutated forms of ATX that are nonetheless capable of producing LPA in the presence of sufficient exogenous substrate. H420Q-ATX could not be differentiated from WT-ATX, indicating that histidine at position 420 is not required for any of the activities of ATX tested in this study.
PMCID: PMC2649126  PMID: 19222837
24.  Molecular modeling of human alkaline sphingomyelinase 
Bioinformation  2011;6(2):78-82.
Alkaline sphingomyelinase, which is expressed in the human intestine and hydrolyses sphingomyelin, is a component of the plasma and the lysosomal membranes. Hydrolase of sphingomyelin generates ceramide, sphingosine, and sphingosine 1-phosphate that have regulatory effects on vital cellular functions such as proliferation, differentiation, and apoptosis. The enzyme belongs to the Nucleotide Pyrophosphatase/Phosphodiesterase family and it differs in structural similarity with acidic and neutral sphingomyelinase. In the present study we modeled alkaline sphingomyelinase using homology modeling based on the structure of Nucleotide Pyrophosphatase/Phosphodiesterase from Xanthomonas axonopodis with which it shares 34% identity. Homology modeling was performed using Modeller9v7. We found that Cys78 and Cys394 form a disulphide bond. Further analysis shows that Ser76 may be important for the function of this enzyme, which is supported by the findings of Wu et al. (2005), that S76F abolishes the activity completely. We found that the residues bound to Zn2+ are conserved and geometrically similar with the template. Molecular Dynamics simulations were carried out for the modeled protein to observe the effect of Zinc metal ions. It was observed that the metal ion has little effect with regard to the stability but induces increased fluctuations in the protein. These analyses showed that Zinc ions play an important role in stabilizing the secondary structure and in maintaining the compactness of the active site.
PMCID: PMC3082857  PMID: 21544170
alkaline sphingomyelinase; homology modeling; NPP; gromacs
25.  Structure-Based Design of Novel Boronic Acid-Based Inhibitors of Autotaxin 
Journal of Medicinal Chemistry  2011;54(13):4619-4626.
Autotaxin (ATX) is a secreted phosphodiesterase that hydrolyzes the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in inflammation, fibrosis, and tumor progression, rendering ATX an attractive drug target. We recently described a boronic acid-based inhibitor of ATX, named HA155 (1). Here, we report the design of new inhibitors based on the crystal structure of ATX in complex with inhibitor 1. Furthermore, we describe the syntheses and activities of these new inhibitors, whose potencies can be explained by structural data. To understand the difference in activity between two different isomers with nanomolar potencies, we performed molecular docking experiments. Intriguingly, molecular docking suggested a remarkable binding pose for one of the isomers, which differs from the original binding pose of inhibitor 1 for ATX, opening further options for inhibitor design.
PMCID: PMC3131786  PMID: 21615078

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