A role for N-glycosylation in active adenosine deaminase 2 production

BackgroundAdenosine deaminase 2 (ADA2) regulates extracellular levels of adenosine and the optimal expression of ADA2 is essential for modulating the immune system. However, the mechanisms regulating the production of active ADA2 enzyme are not fully understood. In this study, we examined the role of N-glycosylation in the formation of functional structures and the secretory pathway of ADA2.MethodsWe investigated the roles of N-glycosylation in the activity, homodimerization, and secretion of ADA2 via site-directed mutagenesis and the application of N-glycosylation inhibitors. Subcellular localization of ADA2 along with the endoplasmic reticulum (ER) glucosidase inhibitor was observed under confocal fluorescence microscope.ResultsInhibiting the initial N-glycosylation of ADA2 in the ER via site-directed mutagenesis or treatment with N-glycosylation inhibitors reduced the intracellular ADA2 activity and secretion. At this time, decreases in the ADA2 homodimers and ADA2 aggregation were observed in the cells. Treating the cells with castanospermine, an inhibitor of N-glycan editing in the ER, resulted in a reduction of the localization rate to the Golgi and markedly suppressed the ADA2 secretion.ConclusionsThese data suggest that the initial N-glycosylation and N-glycan editing in the ER are essential for the production of an active ADA2 enzyme and proper trafficking to the extracellular space.General significanceWith sufficient N-glycosylation in the ER, ADA2 exerts its function and is secreted extracellularly.     

腺苷脱氨酶活性对自身免疫性肝病的临床意义研究

摘要 目的：探讨腺苷脱氨酶（ADA）在自身免疫性肝病患者血清中的变化及其临床意义。方法：利用酶法试剂盒检测自身免疫性肝病患者血清中的总ADA（tADA）及其同工酶ADA1和ADA2的活性变化,利用受试者工作曲线（ROC）分析总ADA、ADA1及ADA2活性的诊断价值。利用Spearman相关性分析自身免疫性肝病患者各指标之间的相关性。结果：与对照组相比,tADA和ADA2活性在自身免疫性肝病患者血清中均极显著升高（P<0.001）,ADA1活性有一定程度升高（P=0.035）。不同自身免疫性肝病亚型患者之间的血清tADA、ADA1及ADA2活性均无显著差异。ROC分析显示,血清tADA和ADA2活性具有诊断价值,ADA1活性无诊断价值。tADA活性截断值取13.5 U/L时,诊断特异性为93.3%,敏感性为81.2%。血清ADA2活性截断值取9.5 U/L时,诊断特异性为85.0%,敏感性为83.3%。而ADA1无显著的诊断价值,ADA活性与白蛋白球蛋白比值呈显著负相关（r=-0.41,P=0.004）,与球蛋白水平具有一定程度的正相关（r=0.34,P=0.018）,与ALT呈弱正相关（r=0.29,P=0.042）,与其他指标的相关性均无统计学显著性。结论：血清tADA及ADA2活性在自身免疫性肝病患者血清中显著升高,并且具有一定的诊断价值。     

结核感染T细胞斑点试验联合血清ADA、SAA、CA125对活动性肺结核诊断及治疗转归的评估价值

摘要 目的：探讨结核感染T细胞斑点试验（T-SPOT.TB）联合血清腺苷脱氨酶（ADA）、淀粉样蛋白A（SAA）、糖类抗原125（CA125）对活动性肺结核（APTB）诊断及治疗转归的评估价值。方法：选取2021年10月至2022年10月湖南省胸科医院收治的137例APTB患者（APTB组）和80例非APTB患者（对照组）,所有APTB患者接受常规抗结核治疗,根据治疗后转归情况分为转归组（92例）和未转归组（45例）。治疗前进行T-SPOT.TB,并检测血清ADA、SAA、CA125水平。受试者工作特征（ROC）曲线分析T-SPOT.TB联合血清ADA、SAA、CA125诊断APTB和预测治疗转归的效能。结果：APTB组T-SPOT.TB阳性率,血清ADA、SAA、CA125水平高于对照组（P＜0.05）。未转归组T-SPOT.TB阳性率,血清ADA、SAA、CA125水平高于转归组（P＜0.05）。T-SPOT.TB联合血清ADA、SAA、CA125诊断APTB以及预测其治疗转归的曲线下面积（AUC）分别为0.917、0.833,高于单一指标。结论：APTB患者T-SPOT.TB阳性率增加,血清ADA、SAA、CA125水平增高,与抗结核治疗后转归不良有关,T-SPOT.TB联合血清ADA、SAA、CA125在APTB诊断和治疗转归评估中具有较高价值。     

腺苷脱氨酶及同工酶对自身免疫病诊断和病情监测的作用

摘要 目的：检测腺苷脱氨酶(ADA)及其同工酶在自身免疫病患者血清中的变化,探讨其诊断和病情监测作用。方法：收集70例系统性红斑狼疮（SLE）、114例类风湿性关节炎(RA)、55例强直性脊柱炎(AS)、及其年龄性别对应的健康血清标本。测定血清总ADA（tADA）、ADA1及ADA2活性。ROC曲线分析其诊断价值。结果：与健康对照相比,ADA活性在SLE患者血清中显著升高[tADA：15（12,20）vs 8（7,10）U/L;ADA1：3.5（2,5）vs 3（2,3）U/L;ADA2：11（8,15）vs 6（5,7）U/L;P<0.01）]。与健康对照相比,RA患者血清中tADA和ADA1活性无显著变化(P>0.05),ADA2活性水平升高[8（5.25,10）vs 7（5,9）U/L,P<0.05）];与健康对照相比,AS患者血清中tADA和ADA1活性无显著变化(P>0.05),ADA2活性升高[7（5,9）vs 6（5,7）U/L,P<0.05）]。ROC分析显示tADA及ADA2对SLE具有较好诊断价值（tADA：88.6%特异性、77.1%敏感性;ADA2：92.9%特异性、68.6%敏感性）。ADA活性对RA及AS患者无诊断价值。spearman相关性分析显示,tADA活性与SLE患者疾病活动度有一定正相关性（r=0.303,P=0.011)。结论：血清tADA活性检测可作为SLE辅助诊断和病情监测指标。     

Biological Activity of 1-Deazapurine Nucleosides: Role of Deoxycytidine Kinase?

Abstract

Several 1-deazapurine nucleosides were tested for their biological activity; anti-HIV-1, cytotoxicity and inhibition of adenosine deaminase (ADA). A2780 human ovarian cancer cells and the deoxycytidine kinase (dCK) deficient variant AG6000, used to determine whether dCK plays a role in their activation, showed a similar sensitivity to the analogs. This is in line with substrate specificity tests, which revealed a very low affinity of dCK.     

Mercury Exposure: Protein Biomarkers of Mercury Exposure in Jaraqui Fish from the Amazon Region

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) is a water-soluble organogermanium compound that exerts various physiological effects, including anti-inflammatory activity and pain relief. In water, Ge-132 is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP), which in turn is capable of interacting with cis-diol compounds through its trihydroxy group, indicating that this compound could also interact with diol-containing nucleic acid constituents. In this study, we evaluated the ability of THGP to interact with nucleosides or nucleotides via nuclear magnetic resonance (NMR) analysis. In addition, we evaluated the effect of added THGP on the enzymatic activity of adenosine deaminase (ADA) when using adenosine or 2′-deoxyadenosine as a substrate. In solution, THGP indeed formed complexes with nucleotides or nucleosides through their cis-diol group. Moreover, the ability of THGP to form complexes with nucleotides was influenced by the number of phosphate groups present on the ribose moiety. Notably, THGP also inhibited the catalysis of adenosine by ADA in a concentration-dependent manner. Thus, interactions between THGP and important biological nucleic acid constituents might be implicated in the physiological effects of Ge-132.     

Synthesis of 1,5-Anhydro-2-(N 6-Cyclopentyladenin-9-Yl)-2-Deoxy-D-Altrohexitol

Abstract

The N ⁶-cyclopentyladenosine (CPA) analogue (4) was synthesized in 10 steps starting from glucose. The results of the radioligand binding assays are consistent with the thus far published findings that compounds containing a six-membered moiety at N ⁹ exhibit extremely weak affinity for adenosine receptors. Replacement of the ribofuranosyl moiety of CPA (2) by a 2-deoxy-D-altrohexitol moiety is sufficient to completely abolish its agonist activity.     

Is the anomeric effect an important factor in the rate of adenosine deaminase catalyzed hydrolysis of purine nucleosides? A direct comparison of nucleoside analogues constructed on ribose and carbocyclic templates with equivalent heterocyclic bases selected to promote hydration

The aglycone of (North)-methanocarbadeoxyadenosine [(N)-MCdA, (5)], a relatively weak substrate for adenosine deaminase (ADA)-relative rate of deamination ca. 100 times lower than adenosine-was modified with substitutions at positions 6 (6-fluoro, compound 6) and 8 (8-aza, compound 7) with the intent to improve the level of hydration and hence hydrolysis by ADA. In these substrates the fused cyclopropane moiety constrains the cyclopentane ring to mimic the conformation of a furanose sugar in the North hemisphere of the pseudorotational cycle, which matches the conformation of the ribose ring of adenosine in complex with ADA. The order of susceptibility to ADA hydrolysis was adenosine>(N)-MCdA (5) approximately equal to(N)-6F-MCdP (6)>(N)-8-aza-MCdA (7). Despite the known fact that 8-azaadenosine is hydrolyzed twice as fast as adenosine, the corresponding carbocyclic analogue 7 was hydrolyzed at approximately half the rate of the parent 5. These results argue in favor of the critical role of the O(4') oxygen atom and its associated anomeric effect in assisting hydrolysis by ADA.     

Anti-Inflammatory Activity of Purine Nucleoside Analogs

Abstract

It is well known that adenosine plays an important role in inflammatory processes. A collection of adenosine analogs modified in the base and/or sugar functional moiety have been synthesized and submitted for biological testing. Each purine nucleoside analog was tested for inhibition of endothelial cell activation by various inflammatory stimuli. A number of analogs exhibited potent anti-inflammatory activity. Animal studies have been carried out on AMG002370 which was found to potently inhibit adjuvant induced arthritis in the Lewis rat.     

Interaction of Adenosine and Guanine Derivatives in the Rat Hippocampus: Effects on Cyclic AMP Levels and on the Binding of Adenosine Analogues and GMP

Guanine nucleotides (GN) have been implicated in many intracellular mechanisms. Extracellular actions, probably as glutamate receptor antagonists, have also been recently attributed to these compounds. GN may have a neuroprotective role by inhibiting excitotoxic events evoked by glutamate. Effects of extracellular GN on adenosine-evoked cellular responses have also been reported. However, the exact mechanism of such interaction is not known. In the present study, we showed that GN potentiated adenosine-induced cAMP accumulation in slices of hippocampus from young rats. However, neither GMP nor the metabotropic glutamate receptor agonist, 1S,3R-ACPD, inhibited the binding of the adenosine receptor agonist [³H]NECA (when binding to adenosine A2 receptors), or the binding of the adenosine A2a receptor agonist [³H]CGS 21680 in hippocampal membrane preparations. GppNHp, probably by interacting with G-proteins, decreased [³H]CGS 21680 binding. [³H]GMP binding was assayed in order to evaluate the GN sites which are not G-proteins. [³H]GMP binding was inhibited by GMP and GppNHp, but not by 1S,3R-ACPD. The interaction of endogenous adenosine with the GMP-binding sites was determined by incubating membranes in the presence or absence of adenosine deaminase (ADA). NECA, CADO, CGS 21680 and CPA (only at the highest concentration used) increased GMP binding in the presence of ADA. However, in the absence of ADA, the control levels of GMP binding were as high as in the presence of added ADA plus adenosine agonists, indicating that endogenous adenosine modulates the binding of GMP. If this site has a neuroprotective role, adenosine may be increasing its neuromodulator and proposed protective action.     

A Role for Adenosine Deaminase in Drosophila Larval Development

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals.     

Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: Implications for modulation of the membrane-associated protein kinase A

In this work, the metabolism of adenosine by isolated BLM associated-enzymes and the implications of this process for the cAMP-signaling pathway are investigated. Inosine was identified as the major metabolic product, suggesting the presence of adenosine deaminase (ADA) activity in the BLM. This was confirmed by immunoblotting and ADA-specific enzyme assay. Implications for the enzymatic deamination of adenosine on the receptor-modulated cAMP-signaling pathway were also investigated. We observed that inosine induced a 2-fold increase in [³⁵S] GTPγS binding to the BLM and it was inhibited by 10⁻⁶ M DPCPX, an A₁ receptor-selective antagonist. Inosine (10⁻⁷ M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and G_αi-3 protein-coupled A₁ receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A₁ receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A₁ receptor-mediated inhibition of adenylyl cyclase.     

A role for adenosine deaminase in Drosophila larval development

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals.     

血清腺苷脱氨酶、糖类抗原199、红细胞沉降率与活动性肺结核患者病情严重程度及预后的关系研究

摘要 目的：探讨血清腺苷脱氨酶（ADA）、糖类抗原199（CA199）、红细胞沉降率（ESR）与活动性肺结核（ATB）患者病情严重程度及预后的关系。方法：选取2021年1月～2022年10月湖南省胸科医院收治的75例ATB患者作为观察组,另外选取60例同期在我院体检健康的体检者作为对照组。比较两组血清ADA、CA199及ESR水平。根据病情严重程度将ATB患者分为轻度组、中度组和重度组。比较不同病情严重程度ATB患者的血清ADA、CA199及ESR水平。观察组接受抗结核治疗,根据疗效分为预后良好组和预后不良组,比较不同预后ATB患者的血清ADA、CA199及ESR水平。收集ATB患者的临床资料,多因素Logistic回归分析ATB患者预后的影响因素。受试者工作特征（ROC）曲线分析血清ADA、CA199、ESR对ATB患者预后的预测价值。结果：观察组血清ADA、CA199、ESR均高于对照组（P＜0.05）。将观察组患者分为轻度组26例、中度组34例和重度组15例。中度组和重度组血清ADA、CA199、ESR高于轻度组（P＜0.05）,重度组血清ADA、CA199、ESR高于中度组（P＜0.05）。治疗后,根据疗效将观察组患者分为预后良好组50例和预后不良组25例,预后良好组和预后不良组在性别、年龄、BMI、吸烟史、饮酒史、糖尿病史、居住地、哮喘、支气管扩张、乙型肝炎、胸片改变等方面比较差异无统计学意义（P＞0.05）,预后不良组患者血清PCT、白细胞计数、ADA、CA199、ESR高于预后良好组（P＜0.05）。经多因素Logistic回归分析结果显示,血清ADA、CA199、ESR升高是ATB患者预后不良的危险因素（P＜0.05）。绘制ROC曲线结果显示,血清ADA、CA199、ESR单独预测ATB患者预后不良的AUC分别为0.655、0.675、0.699,血清ADA、CA199、ESR联合预测ATB患者预后不良的AUC为0.828,大于各指标单独检测。结论：血清ADA、CA199、ESR升高可提示ATB患者的病情严重程度,血清ADA、CA199、ESR联合检测对预测ATB患者预后不良具有较高的价值。     

Expression of murine adenosine deaminase (ADA) in transgenic maize

A murine adenosine deaminase (ADA) gene, driven by the maize ubi-1 promoter and intron region, was transformed into embryogenic maize callus, along with a bar and gusA gene-containing plasmid, using microparticle bombardment. Selection in the presence of either the herbicide Basta® or the adenosine analogue 2-deoxyadenosine resulted in transgenic cultures that expressed GUS and accumulated a 41kD protein that immunoprecipated with an ADA-specific polyclonal antibody. ADA enzyme activity was observed in extracts from transgenic callus as well as regenerated plants and progeny. Cultures expressing ADA grew in the presence of 200mg/l 2-deoxyadenosine, a concentration which completely inhibited the growth of non-transgenic cultures. ADA activity appeared to segregate in progeny of regenerated plants as a single, dominant Mendelian trait. These results suggest that ADA, in combination with adenosine analogue selection, represents a potentially viable selectable marker system for transgenic maize production.     

Erythrocytenenzyme der Primaten

Zusammenfassung Die Adenosindesaminasen der Primaten zeigen eine genetisch determinierte Variabilität. Bei der Untersuchung von 327 subhumanen Primaten (289 Simiae der Alten Welt, 38 Prosimiae) konnten wir neun Adenosindesaminase-Varianten nachweisen, die auf Grund ihrer unterschiedlichen elektrophoretischen Wandergeschwindigkeit als ADA 6, ADA 4, ADA 2, ADA 2, ADA 1, ADA 3, ADA 5, ADA 5 und ADA 7 bezeichnet werden. Die Verteilung der verschiedenen Phänotypen wurde ermittelt.

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Red cell enzymes of primatesAdenosine deamisnase (EC: 3.5.4.4.)

Summary The polymorphism of adenosine deaminase has been investigated in 327 subhuman Primates (289 Old World Monkeys and 38 Prosimians). Nine adenosine deaminase variants were found to be present, which on the basis of their different electrophoretic mobilities were designated ADA 6, ADA 4, ADA 2, ADA 2, ADA 1, ADA 3, ADA 5, ADA 5 and ADA 7. The distribution of these various phenotypes has been estimated.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Adenosine and 2-Chloroadenosine Deaza-Analogues as Adenosine Receptor Agonists1

Abstract

Several deaza-analogues of adenosine and 2-chloro-adenosine have been examined for their adenosine receptor affinity. It was found that the relative contribution of the nitrogen atoms of the purine moiety to binding at A₁ rat brain adenosine receptor, follows the order N⁷ > N³ > N¹. The affinity of the adenosine analogues for the adenosine rat brain receptor was besides compared with their activity as inhibitors of platelet aggregation. A synthesis of 2-chloro-1-deazaadenosine by two alternative routes starting from 7-nitroimidazo[4,5-b]pyridine-4-oxide is also reported.     

Erythrocyte acid phosphatase (ACP1) activity

Summary Erythrocyte acid phosphatase (ACP₁) activity was determined in the absence of modulators and in the presence of either adenosine or inosine as modulators in 154 samples of red blood cells collected from adult donors. Adenosine and inosine showed modulating effects (activation), that were genotype dependent in the allele order p^bac; the activation by inosine was much higher than by adenosine. The modulating effect was dependent on adenosine deaminase (ADA) genotype: In carriers of ADA² allele the activation with ACP₁ phenotype A was lower and that with phenotypes CA and CB was higher than in ADA¹/ADA¹ subjects. In addition, the basic ACP₁ activity (i.e., without modulators) also appeared to be dependent on ADA genotype: The lowest ACP₁ activity was observed in A and BA subjects carrying the ADA² allele. Since the deamination of adenosine to inosine associated with ADA2-1 phenotype is slower than that associated with ADA1, the interaction of ADA on ACP₁ activity may in fact be explained by a lower intracellular concentration of inosine in ADA² carriers and, therefore, by a lower modulating effect of this on acid phosphatase activity.     

Roles of nucleotide substructures in the regulation of cystathionine β-synthase domain-containing pyrophosphatase

BackgroundRegulatory cystathionine β-synthase (CBS) domains are ubiquitous in proteins, yet their mechanism of regulation remains largely obscure. Inorganic pyrophosphatase which contains regulatory CBS domains as internal inhibitors (CBS-PPase) is activated by ATP and inhibited by AMP and ADP; nucleotide binding to CBS domains and substrate binding to catalytic domains demonstrate positive co-operativity.Methods: Here, we explore the ability of an AMP analogue (cAMP) and four compounds that mimic the constituent parts of the AMP molecule (adenine, adenosine, phosphate, and fructose-1-phosphate) to bind and alter the activity of CBS-PPase from the bacterium Desulfitobacterium hafniense.ResultsAdenine, adenosine and cAMP activated CBS-PPase several-fold whereas fructose-1-phosphate inhibited it. Adenine and adenosine binding to dimeric CBS-PPase exhibited high positive co-operativity and markedly increased substrate binding co-operativity. Phosphate bound to CBS-PPase competitively with respect to a fluorescent AMP analogue.ConclusionsProtein interactions with the adenine moiety of AMP induce partial release of the internal inhibition and determine nucleotide-binding co-operativity, whereas interactions with the phosphate group potentiate the internal inhibition and decrease active-site co-operativity. The ribose moiety appears to enhance the activation effect of adenine and suppress its contribution to both types of co-operativity.General significanceOur findings demonstrate for the first time that regulation of a CBS-protein (inhibition or activation) is determined by a balance of its interactions with different chemical groups of the nucleotide and can be reversed by their modification. Differential regulation by nucleotides is not uncommon among CBS-proteins, and our findings may thus have a wider significance.     

Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.