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Adenosine, a potent anticonvulsant, can be produced in the body by the hydrolysis of adenine nucleotides through the action of ecto- or soluble nucleotidases. Changes in nucleotide hydrolysis occur after pentylenetetrazol-induced epileptic events. We evaluated serum ATP, ADP and AMP hydrolysis rates and soluble nucleotide phosphodiesterase (PDEase) activity at 5, 10, 15, 30 and 60 min, and 12h following an epileptic event. Fifteen patients (seven female, eight male; mean age 15.5 years) were included in the study. The type of seizure was generalized in four patients and was localization related in the remaining 11. There were no differences in adenine nucleotide hydrolysis rates between patients and healthy subjects in the interictal stage. In comparison with controls, ATP, ADP and AMP hydrolysis rates were significantly increased at 5 min (53+/-1.4%, 79.2+/-2.8% and 37.0+/-2.6%, respectively) and up to 30 min following the epileptic event. In contrast to ADP and AMP, ATP hydrolysis remained significantly increased at 60 min (71.4+/-1.6%), returning to the basal level after 12h. Serum PDEase activity was also significantly higher in the patients than in healthy subjects, peaking at 15 min (61+/-2.9%) and remaining significantly increased up to 60 min (4.6+/-1.2%) following the epileptic episode. Globally, the variations in the postictal serum ADP hydrolysis rate almost overlapped those of AMP hydrolysis, whereas changes in the ATP hydrolysis rate overlapped those of PDEase activity. The clinical significance of this elevation in postictal soluble serum nucleotidase activity remains to be clarified. However, it is possible to hypothesize that the higher nucleotidase activity might play a role in the modulation of epileptic events.
Ecto-nucleotidase activity is involved in the infection process of Leishmania and various other parasites that enables modulation of host immune responses to promote disease progression. One of the enzymes responsible for this activity is the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase). The enzyme hydrolyzes nucleotides tri- and/or di-phosphate into monophosphate products, which are subsequently hydrolyzed into adenosine. These nucleotides can serve as purinergic signaling molecules involved in diverse cellular processes that govern immune responses. Given the importance of the extracellular metabolism of these nucleotides during intracellular pathogen infections, this study evaluates the role of ecto-nucleotidase activity during Leishmania infantum (L. infantum) infection in human macrophages. E-NTPDase protein expression and activity was evaluated in L. infantum during purine starvation, adenosine-enriched medium, or in the presence of an inhibitor of ecto-nucleotidases. Results show that E-NTPDase is expressed in L. infantum parasites, including on the cell membrane. Furthermore, functional activity of the enzyme was modulated according to the availability of adenosine in the medium. Purine starvation increased the hydrolytic capacity of nucleotides leading to higher infectivity, while growth in adenosine-enriched medium led to lower infectivity. Moreover, inhibiting E-NTPDase function decreased L. infantum infection in macrophages, suggesting the enzyme may serve as a ligand. Taken together, the ability of L. infantum to hydrolyze nucleotides is directly associated with increased infectivity in macrophages.
Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4⁺ T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24 hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5'-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5' nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4⁺ T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4⁺ T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.
One of the strategies used by tumors to evade immunosurveillance is the accumulation of extracellular adenosine, which has immunosupressive and tumor promoting effects. The study of the mechanisms leading to adenosine formation at the tumor interstitium are therefore of great interest in oncology. The dominant pathway generating extracellular adenosine in tumors is the dephosphorylation of ATP by ecto-nucleotidases. Two of these enzymes acting sequentially, CD39 and CD73, efficiently hydrolyze extracellular ATP to adenosine. They have been found to play a crucial role in a variety of tumors, but there were no data concerning endometrial cancer, the most frequent of the invasive tumors of the female genital tract. The aim of the present work is to study the expression of CD39 and CD73 in human endometrial cancer. We have analyzed protein and gene expression, as well as enzyme activity, in type I endometrioid adenocarcinomas and type II serous adenocarcinomas and their nonpathological endometrial counterparts. High levels of both enzymes were found in tumor samples, with significantly increased expression of CD39 in type II serous tumors, which also coincided with the higher tumor grade. Our results reinforce the involvement of the adenosinergic system in cancer, emphasizing the relevance of ecto-nucleotidases as emerging therapeutic targets in oncology.
Endometriosis, defined as the growth of endometrial tissue outside the uterus, is a common gynecologic condition affecting millions of women worldwide. It is an inflammatory, estrogen-dependent complex disorder, with broad symptomatic variability, pelvic pain, and infertility being the main characteristics. Ovarian endometriomas are frequently developed in women with endometriosis. Late diagnosis is one of the main problems of endometriosis; thus, it is important to identify biomarkers for early diagnosis. The aim of the present work is to evaluate the ecto-nucleotidases activities in the contents of endometriomas. These enzymes, through the regulation of extracellular ATP and adenosine levels, are key enzymes in inflammatory processes, and their expression has been previously characterized in human endometrium. To achieve our objective, the echo-guided aspirated fluids of endometriomas were analyzed by evaluating the ecto-nucleotidases activities and compared with simple cysts. Our results show that enzyme activities are quantifiable in the ovarian cysts aspirates and that endometriomas show significantly higher ecto-nucleotidases activities than simple cysts (5.5-fold increase for ATPase and 20-fold for ADPase), thus being possible candidates for new endometriosis biomarkers. Moreover, we demonstrate the presence of ecto-nucleotidases bearing exosomes in these fluids. These results add up to the knowledge of the physiopathologic mechanisms underlying endometriosis and, open up a promising new field of study.
Nucleotidases contribute to the regulation of inflammation, coagulation, and cardiovascular activity. Exercise promotes biological adaptations, but its effects on nucleotidase activities and expression are unclear. The objective of this study was to review systematically the effects of exercise on nucleotidase functionality in healthy and unhealthy subjects. The MEDLINE, EMBASE, Cochrane Library, and Web of Science databases were searched to identify, randomized clinical trials, non-randomized clinical trials, uncontrolled clinical trials, quasi-experimental, pre-, and post-interventional studies that evaluated the effects of exercise on nucleotidases in humans, and was not limited by language and date. Two independent reviewers performed the study selection, data extraction, and assessment of risk of bias. Of the 203 articles identified, 12 were included in this review. Eight studies reported that acute exercise, in healthy and unhealthy subjects, elevated the activities or expression of nucleotidases. Four studies evaluated the effects of chronic training on nucleotidase activities in the platelets and lymphocytes of patients with metabolic syndrome, chronic kidney disease, and hypertension and found a decrease in nucleotidase activities in these conditions. Acute and chronic exercise was able to modify the blood plasma and serum levels of nucleotides and nucleosides. Our results suggest that short- and long-term exercise modulate nucleotidase functionality. As such, purinergic signaling may represent a novel molecular adaptation in inflammatory, thrombotic, and vascular responses to exercise.
The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5'-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5'-Nucleotidase biological assay for nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT.
This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.
The concentrations of extracellularly released nucleotides are controlled by metabolism via ecto-nucleotidases, but the precise physiological roles of the ecto-nucleoside triphosphate diphosphohydrolases in the modulation of purinergic receptor signalling are still unclear. Bacterial endotoxin lipopolysaccharide (LPS) treatment (administered intraperitoneally, 2 mg/kg body weight) of rats resulted in no significant changes in the overall ecto-nucleotidase activities of the hippocampus, however, LPS treatment did cause transient changes in the morphology of endothelial cells and pericytes and in the localization pattern of ecto-ATPase activity in rat hippocampus. The transient decrease in NTPDase1 (ecto-nucleoside triphosphate diphosphohydrolase1) activity, located on the luminal side of the endothelial cells, was balanced by increases in ecto-nucleotidase activities in pericytes and at other sites, consistent with an unchanged overall ecto-ATPase activity of the hippocampus. Since the transient loss of NTPDase1 activity was not accompanied by a loss of NTPDase1 protein, we hypothesize that LPS caused transient alterations in the lipid membranes, since NTPDase1 activity is known to be sensitive to changes in membrane structure via its transmembrane domains. After 2-3 days, the LPS-induced changes in cell morphology and ecto-nucleotidase localization disappeared. We conclude that a low dose of LPS causes transient changes in the localization pattern of ecto-nucleotidases in endothelial cells and pericytes, which, coupled with the observed cellular morphological changes, may indicate modified cellular signalling in the hippocampus.
It has long been known that the salivary glands of hematophagous (blood-feeding) arthropods secrete soluble apyrases, which are potent nucleotide hydrolyzing enzymes capable of hydrolyzing extracellular ATP and ADP, the latter being a major agonist contributing to platelet aggregation. Only recently, however, has the identification of proteins homologous to these apyrases been reported in non-blood-feeding organisms such as rodents and humans. In this review, we present an overview of the diverse family of apyrases first described in the blood-feeding arthropods, including the identification and characterization of the soluble and membrane-bound vertebrate enzymes homologous to these arthropod apyrases. We also describe the enzymatic properties and nucleotide specificities of the expressed enzymes, and insights gained into the structure and function of this calcium activated nucleotidase (CAN) family from biophysical, mutagenesis and crystallography studies. The potential therapeutic value of these proteins is also discussed.
Adenosine 5′-triphosphate (ATP) is released in the bladder lumen during filling. Urothelial ATP is presumed to regulate bladder excitability. Urinary ATP is suggested as a urinary biomarker of bladder dysfunctions since ATP is increased in the urine of patients with overactive bladder, interstitial cystitis or bladder pain syndrome. Altered urinary ATP might also be associated with voiding dysfunctions linked to disease states associated with metabolic syndrome. Extracellular ATP levels are determined by ATP release and ATP hydrolysis by membrane-bound and soluble nucleotidases (s-NTDs). It is currently unknown whether s-NTDs regulate urinary ATP. Using etheno-ATP substrate and HPLC-FLD detection techniques, we found that s-NTDs are released in the lumen of ex vivo mouse detrusor-free bladders. Capillary immunoelectrophoresis by ProteinSimple Wes determined that intraluminal solutions (ILS) collected at the end of filling contain ENTPD3 > ENPP1 > ENPP3 ≥ ENTPD2 = NT5E = ALPL/TNAP. Activation of adenylyl cyclase with forskolin increased luminal s-NTDs release whereas the AC inhibitor SQ22536 had no effect. In contrast, forskolin reduced and SQ22536 increased s-NTDs release in the lamina propria. Adenosine enhanced s-NTDs release and accelerated ATP hydrolysis in ILS and lamina propria. Therefore, there is a regulated release of s-NTDs in the bladder lumen during filling. Aberrant release or functions of urothelial s-NTDs might cause elevated urinary ATP in conditions with abnormal bladder excitability.
Prior studies suggest that urothelium-released adenosine 5'-triphosphate (ATP) has a prominent role in bladder mechanotransduction. Urothelial ATP regulates the micturition cycle through activation of purinergic receptors that are expressed in many cell types in the lamina propria (LP), including afferent neurons, and might also be important for direct mechanosensitive signaling between urothelium and detrusor. The excitatory action of ATP is terminated by enzymatic hydrolysis, which subsequently produces bioactive metabolites. We examined possible mechanosensitive mechanisms of ATP hydrolysis in the LP by determining the degradation of 1,N 6 -etheno-ATP (eATP) at the anti-luminal side of nondistended (empty) or distended (full) murine (C57BL/6J) detrusor-free bladder model, using HPLC. The hydrolysis of eATP and eADP was greater in contact with LP of distended than of nondistended bladders whereas the hydrolysis of eAMP remained unchanged during filling, suggesting that some steps of eATP hydrolysis in the LP are mechanosensitive. eATP and eADP were also catabolized in extraluminal solutions (ELS) that were in contact with the LP of detrusor-free bladders, but removed from the organ chambers prior to addition of substrate. The degradation of both purines was greater in ELS from distended than from nondistended preparations, suggesting the presence of mechanosensitive release of soluble nucleotidases in the LP. The released enzyme activities were affected differently by Ca2+ and Mg2+. The common nucleotidase inhibitors ARL67156, POM-1, PSB06126, and ENPP1 Inhibitor C, but not the alkaline phosphatase inhibitor (-)-p-bromotetramisole oxalate, inhibited the enzymes released during bladder distention. Membrane-bound nucleotidases were identified in tissue homogenates and in concentrated ELS from distended preparations by Wes immunodetection. The relative distribution of nucleotidases was ENTPD1 >> ENPP1 > ENTPD2 = ENTPD3 > ENPP3 = NT5E >> ENTPD8 = TNAP in urothelium and ENTPD1 >> ENTPD3 >> ENPP3 > ENPP1 = ENTPD2 = NT5E >> ENTPD8 = TNAP in concentrated ELS, suggesting that regulated ectodomain shedding of membrane-bound nucleotidases possibly occurs in the LP during bladder filling. Mechanosensitive degradation of ATP and ADP by membrane-bound and soluble nucleotidases in the LP diminishes the availability of excitatory purines in the LP at the end of bladder filling. This might be a safeguard mechanism to prevent over-excitability of the bladder. Proper proportions of excitatory and inhibitory purines in the bladder wall are determined by distention-associated purine release and purine metabolism.
Schistosomiasis is a debilitating disease caused by flatworm parasites of the Schistosoma genus and remains a high public health impact disease around the world, although effective treatment with Praziquantel (PZQ) has been available since the 1970s. Control of this disease would be greatly improved by the development of a vaccine, which could be combined with chemotherapy. The sequencing of the Schistosoma mansoni transcriptome and genome identified a range of potential vaccine antigens. Among these, three nucleotidases from the tegument of the parasite, presumably involved in purinergic signaling and nucleotide metabolism, were proposed as promising vaccine candidates: an alkaline phosphatase (SmAP), a phosphodiesterase (SmNPP-5) and a diphosphohydrolase (SmNTPDase). Herein, we evaluate the potential of these enzymes as vaccine antigens, with or without subcurative PZQ treatment. Immunization of mice with the recombinant proteins alone or in combination demonstrated that SmAP is the most immunogenic of the three. It induced the highest antibody levels, particularly IgG1, associated with an inflammatory cellular immune response characterized by high TNF-α and a Th17 response, with high IL-17 expression levels. Despite the specific immune response induced, immunization with the isolated or combined proteins did not reduce the worm burden of challenged mice. Nonetheless, immunization with SmAP alone or with the three proteins combined, together with subcurative PZQ chemotherapy was able to reduce the worm burden by around 40%. The immunogenicity and relative exposure of SmAP to the host immune system are discussed, as key factors involved in the apparently synergistic effect of SmAP immunization and subcurative PZQ treatment.
Nucleotide-activated P2X channels and P2Y metabotropic receptors participate in nociceptive signaling. Agonist availability is regulated by nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3, and -8, a family of enzymes that hydrolyze extracellular ATP to generate ADP (a P2Y agonist) and AMP. They provide a major source of extracellular AMP, the substrate for adenosine production by ecto-5'-nucleotidase (NT5E), and thereby regulate adenosine (P1) receptor signaling. NTPDases vary in their efficiency of tri- and diphosphate hydrolysis; therefore, which family members are expressed impacts nucleotide availability and half-life. This study employed enzyme activity histochemistry to examine the distribution of ATPase activity and immunohistochemistry for NTPDase1, 2, 3, and 8 in dorsal root ganglion (DRG) and spinal cord. Nucleotidase activity was robust in spinal dorsal horn, confirming that nociceptive pathways are a major site of nucleotide transmission. In DRG, extensive staining revealed ATPase activity in a subset of neurons and in non-neuronal cells. mRNA for NTPDase1-3, but not NTPDase8, was detected in lumbar DRG and spinal cord. Immunoreactivity for NTPDase3 closely matched the distribution of ATPase activity, labeling DRG central projections in the dorsal root and superficial dorsal horn, as well as intrinsic spinal neurons concentrated in lamina II. In DRG, NTPDase3 co-localized with markers of nociceptors and with NT5E. In addition, labeling of a subset of larger-diameter neurons in DRG was consistent with intense staining of Meissner corpuscle afferents in glabrous skin. Merkel cells and terminal Schwann cells of hair follicle afferents were also labeled, but the axons themselves were negative. We propose that NTPDase3 is a key regulator of nociceptive signaling that also makes an unexpected contribution to innocuous tactile sensation.
1. ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio. 2. Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities. 3. ATP did not raise intracellular Ca2+ in B10 cells. Addition of apyrases III or VII (1 u ml(-1)) to ATP treated cells induced large intracellular Ca2+ transients. Apyrases had no action in the absence of ATP. 4. A 1 u ml(-1) apyrase III solution generated 20 microM ADP from 0.1 mM ATP within 15 s. This concentration of ADP was sufficient to produce maximal activation of P2Y1 receptors. 5. ATP was a full agonist of P2Y1 receptors in the presence of 1 u ml(-1) apyrase III. Dose response curves for the apparent actions of ATP were bell shaped in the presence of 0.1 u ml(-1) apyrase III. Apyrase III did not alter ADP dose response curves when coincubated with ADP for 15 s. 6. Apyrase VII (1 u ml(-1)) shifted dose response curves for the actions of ADP to larger concentrations. It induced a bell shaped ATP dose response curve. 7. Results suggest that ATPDases prevent P2Y1 receptor activation by degrading ADP but may contribute to P2Y1 receptor activation by generating ADP from ATP.
Ecto-5'-nucleotidase (CD73) is well known for its implication in cancer. Inhibition of ecto-5'-nucleotidases is thought to provide an attractive approach to cancer therapy. This study identifies sulfonic acid compounds as efficient inhibitors of ecto-5'-nucleotidases. The compounds were tested against recombinant human and rat ecto-5'-nucleotidases. The most potent new sulfonic acid inhibitor 6-amino-4-hydroxynaphthalene-2-sulfonic acid (1) of ecto-5'-nucleotidase had an IC₅₀ of 1.32 ± 0.09 μM for the human and 10.4 ± 3.3 μM for the rat enzyme. Generally, all compounds were more active against the human enzyme. Plausible binding mode models were developed for this new class of inhibitors. Furthermore, several sulfonic acid inhibitors were efficient cytotoxic agents when tested on H157 cancer cell lines. Hence, new ecto-5'-nucleotidases inhibitors displayed significant potential for further development as compounds for anti-cancer therapy.
Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3'-phosphoadenosine-5'-phosphate pAp, inhibits the activity of the Xrn1p 5'-3' ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.
Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi ) to pyrophosphate (PPi ) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi , a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto-nucleotidases. This study investigated the expression and activity of ecto-nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto-nucleotidases including NTPdase 1-6 (ecto-nucleoside triphosphate diphosphohydrolase) and NPP1-3 (ecto-nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto-5-nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8-fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto-nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5-fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions.
5'-nucleotidases are widespread among all domains of life. The enzymes hydrolyze phosphate residues from nucleotides and nucleotide derivatives. In some pathobiontic bacteria, 5'-nucleotidases contribute to immune evasion by dephosphorylating adenosine mono-, di-, or tri-phosphates, thereby either decreasing the concentration of pro-inflammatory ATP or increasing the concentration of anti-inflammatory adenosine, both acting on purinergic receptors of phagocytic cells. The strict human pathogen Streptococcus pyogenes expresses a surface-associated 5'-nucleotidase (S5nA) under infection conditions that has previously been discussed as a potential virulence factor. Here we show that deletion of the S5nA gene does not significantly affect growth in human blood, evasion of phagocytosis by neutrophils, formation of biofilms and virulence in an infection model with larvae of the greater wax moth Galleria mellonella in S. pyogenes serotypes M6, M18 and M49. Hence, the surface-associated 5'-nucleotidase S5nA seems dispensable for evasion of phagocytosis and biofilm formation in S. pyogenes.
5'-nucleotidases (EC 3.1.3.5) catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides as well as complex nucleotides, such as uridine 5'-diphosphoglucose (UDP-glucose), nicotinamide adenine dinucleotide and flavin adenine dinucleotide, to their corresponding nucleosides plus phosphate. These enzymes have been found in diverse species in intracellular and membrane-bound, surface-localized forms. Soluble forms of 5'-nucleotidases belong to the ubiquitous haloacid dehalogenase superfamily (HADSF) and have been shown to be involved in the regulation of nucleotide, nucleoside and nicotinamide adenine dinucleotide (NAD+) pools. Despite the important role of 5'-nucleotidases in cellular metabolism, only a few of these enzymes have been characterized in the Gram-positive bacterium Bacillus subtilis, the workhorse industrial microorganism included in the Food and Drug Administration's GRAS (generally regarded as safe) list. In the present study, we report the identification of a novel 5'-nucleotidase gene from B. subtilis, yutF, which comprises 771 bp encoding a 256-amino-acid protein belonging to the IIA subfamily of the HADSF. The gene product is responsible for the major p-nitrophenyl phosphatase activity in B. subtilis. The yutF gene was overexpressed in Escherichia coli, and its product fused to a polyhistidine tag was purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity. The recombinant YutF protein was found to hydrolyze various purine and pyrimidine 5'-nucleotides, showing preference for 5'-nucleoside monophosphates and, specifically, 5'-XMP. Recombinant YutF also exhibited phosphohydrolase activity toward nucleotide precursors, ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate. Determination of the kinetic parameters of the enzyme revealed a low substrate specificity (Km values in the mM concentration range) and modest catalytic efficiencies with respect to substrates. An initial study of the regulation of yutF expression showed that the yutF gene is a component of the yutDEF transcription unit and that YutF overproduction positively influences yutDEF expression.
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