Changes of frequency and expression level of CD161 in CD8+ T cells and natural killer T cells in peripheral blood of patients with systemic lupus erythematosus

Immunologic abnormalities of natural killer (NK) cells and T cells play a role in the pathogenesis of systemic lupus erythematosus (SLE). CD161 is expressed on most of the NK cells and on some T cells. The quantities of CD161-expressing cells and expression levels of CD161 were analyzed in T cells and NK cells from patients with SLE compared with normal controls. The expression of CD161 on NK cells, NKT cells, CD4⁺ T cells, and CD8⁺ T cells in peripheral blood from patients with inactive SLE and active SLE, and from the normal controls group were determined using flow cytometry. The frequency and expression level of CD161 in the lymphocyte subsets and its relationship with the quantity of regulatory T cells, anti-double stranded DNA antibody, and the titer of antinuclear antibody were evaluated. Both the percentages of the CD161⁺ subpopulation and the mean fluorescence intensities (MFIs) of CD161 in CD8⁺ T cells and NKT cells decreased significantly in SLE patients compared with normal controls (P < .001). The CD161 expression in CD8⁺ T cells and NKT cells also decreased in the anti-dsDNA (+) group (P < .05). The counts of Treg cells were lower in SLE patients and were weakly correlated with the percentage of the CD161 subpopulation (r = 0.229, P = .016) and the MFIs of CD161 expression in CD8⁺ T cells (r = .232, P = .014). The frequencies and levels of CD161 expression on CD8⁺ T cells and NKT cells were reduced in SLE patients, suggesting that an abnormality of these cells was related to the pathogenesis of SLE.     

CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases.     

Use of Tregs as a cell‐based therapy via CD39 for benign prostate hyperplasia with inflammation

Benign prostatic hyperplasia (BPH) occurs most commonly among older men, often accompanied by chronic tissue inflammation. Although its aetiology remains unclear, autoimmune dysregulation may contribute to BPH. Regulatory T cells (Tregs) prevent autoimmune responses and maintain immune homeostasis. In this study, we aimed to investigate Tregs frequency, phenotype, and function in BPH patients and to evaluate adoptive transfer Tregs for immunotherapy in mice with BPH via CD39. Prostate specimens and peripheral blood from BPH patients were used to investigate Treg subsets, phenotype and Treg‐associated cytokine production. Sorted CD39^+/? Tregs from healthy mice were adoptively transferred into mice before or after testosterone propionate administration. The Tregs percentage in peripheral blood from BPH patients was attenuated, exhibiting low Foxp3 and CD39 expression with low levels of serum IL‐10, IL‐35 and TGF‐β. Immunohistochemistry revealed Foxp3+ cells were significantly diminished in BPH prostate with severe inflammatory. Although the Tregs subset was comprised of more effector/memory Tregs, CD39 was still down‐regulated on effector/memory Tregs in BPH patients. Before or after testosterone propionate administration, no alterations of BPH symptoms were observed due to CD39‐ Tregs in mice, however, CD39⁺Tregs existed more potency than Tregs to regulate prostatic hyperplasia and inhibit inflammation by decreasing IL‐1β and PSA secretion, and increasing IL‐10 and TGF‐β secretion. Furthermore, adoptive transfer with functional Tregs not only improved prostate hyperplasia but also regulated muscle cell proliferation in bladder. Adoptive transfer with Tregs may provide a novel method for the prevention and treatment of BPH clinically.     

CD137 signaling interferes with activation and function of CD4+CD25+ regulatory T cells in induced tolerance to experimental autoimmune thyroiditis

Experimental autoimmune thyroiditis (EAT), a model for Hashimoto's thyroiditis, is a T cell-mediated disease inducible with mouse thyroglobulin (mTg). Pretreatment with mTg, however, can induce CD4+ T cell-mediated tolerance to EAT. We demonstrate that CD4+CD25+ regulatory cells are critical for the tolerance induction, as in vivo depletion of CD25+ cells abrogated established tolerance, and CD4+CD25+ cells from tolerized mice suppressed mTg-responsive cells in vitro. Importantly, administration of an agonistic CD137 monoclonal antibody (mAb) inhibited tolerance development, and the mediation of established tolerance. CD137 mAb also inhibited the suppression of mTg-responsive cells by CD4+CD25+ cells in vitro. Signaling through CD137 likely resulted in enhancement of the responding inflammatory T cells, as anti-CD137 did not enable CD4+CD25+ T cells to proliferate in response to mTg in vitro.     

SLE带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+FoxP3~+调节性T细胞的表达及相关性研究

目的:检测系统性红斑狼疮(systemic lupus erythematosus,SLE)合并带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+Fox P3~+调节性T细胞的表达及相关性,探讨其在SLE合并带状疱疹发病中的临床意义。方法:采用流式细胞术检测30例SLE患者、30例SLE合并带状疱疹患者及30例健康对照者外周血中CD4~+/CD8~+T淋巴细胞亚群表面CD28的表达及CD4~+CD25~+Fox P3~+Treg细胞的表达水平,并分析SLE合并带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+Fox P3~+调节性T细胞表达的相关性。结果:SLE合并带状疱疹组患者急性期外周血CD4~+T淋巴细胞比率、绝对计数显著降低,CD4~+、CD8~+T淋巴细胞表面的CD28表达下调,CD4~+CD25~+Fox P3~+Treg细胞水平显著高于SLE组及健康对照组,SLE合并带状疱疹组患者外周血CD4~+CD25~+Fox P3~+Treg水平与CD4~+CD28~+水平成负相关(P均0.05)。结论:SLE合并带状疱疹患者CD4~+、CD8~+T细胞活化异常,CD4~+CD25~+Fox P3~+Treg细胞可能参与抑制了T细胞的活化。     

狼疮肾炎患者外周血单个核细胞钙调神经磷酸酶活性的检测及CD40L表达变化

目的:检测活动性狼疮肾炎(LN)患者外周血单个核细胞(PBMC)钙调神经磷酸酶(calcineurin,CaN)活性及其与PBMC CD40L表达的关系.方法:体外培养活动性LN患者PBMC,应用发色底物法检测胞浆CaN活性,流式细胞仪检测细胞CD40L的表达.结果:①在单纯培养情况下,正常对照组和LN组PBMC均出现一定量CaN活化,活动性LN组显著高于正常对照组(46.08±5.58 nrmmol/mg pro vs 8.81±3.61nmol/mg pro,P＜0.01);在PMA Ionomycin刺激下,各组CaN活性均升高,活动性LN组CaN活性明显高于正常对照组(69.34±12.59 nmol/mg provs 37.12±11.57 hmol/mg pro,P＜0.01);②LN患者PBMC在单纯培养和PMA Ionomycin刺激时CD40L蛋白和mRNA表达均显著高于相应的对照组(P＜0.01);③在单纯培养和PMA Ionomycin刺激时,FK506对LNPBMC表达CD40L蛋白和mRNA均有显著抑制作用(P＜0.01).结论:LN患者PBMC存在CaN过度活化;LN患者PBMC高效表达CD40L与其CaN过度活化密切相关,通过阻断CaN活化可调控CD40-CD40L共刺激信号途径的活化.     

CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4 T cells

CD4⁺ T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4⁺ T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4⁺ T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4⁺CD25⁻ T cells into Rag2^−/− mice did not cause colitis accompanied by increasing Foxp3⁺ inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4⁺CD25⁻ T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4⁺ T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.     

Characterization of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) activity in mouse peritoneal cavity cells

This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E‐NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca²⁺. A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E‐NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.     

CD147在调节性T细胞中作用的研究进展

CD147是一种免疫球蛋白超家族,它与肿瘤侵袭和转染,类风湿性关节炎,病毒入侵,淋巴细胞的迁移和活化,老年痴呆症,疟原虫入侵等病理过程密切先关,已成为癌症等疾病的新型药物靶标分子。作为一个单次跨膜蛋白,CD147的多功能性依赖于它的胞外结构域以及细胞膜和细胞外多种分子的相互作用。有研究发现CD147可作为细胞膜受体并且在上皮细胞,肿瘤细胞和免疫T细胞中均有表达。其可作为T细胞亲环素受体并具有趋化T细胞的功能。近几年研究发现,CD147在调节性T细胞高表达并且具有标志性意义。该文就以作为活性调节性T细胞标志的CD147的潜在作用进展综述如下。     

Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer

Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.     

Small numbers of residual tumor cells at the site of primary inoculation are critical for anti-tumor immunity following challenge at a secondary location

Luciferase-transduced B16 murine melanoma cells (luc-B16) inoculated in ear skin do not form tumors but prevent tumor formation by luc-B16 cells injected into the footpad. To determine the requirements for such immunity, we followed the fate of luc-B16 cells following ear injection. Surprisingly, small numbers of viable luc-B16 cells were detected in tumor-free mouse skin for up to 60 days post-inoculation. After 1 week, the number of Foxp3⁺CD4⁺CD25⁺ T cells (along with foxp3 mRNA expression) increased rapidly in the injected ear skin. Residual tumor cells in ears were reduced in mice treated with anti-CD25 mAb and in CD4-deficient mice, but increased in CD8-deficient mice. Strikingly, the loss of luc-B16 cells in the ear skin, either spontaneously or following amputation of the injected ear, resulted in significantly enhanced tumor formation by parental and luciferase-expressing B16 cells after footpad injection. These studies suggest that small numbers of tumor cells (possibly regulated by CD4⁺CD25⁺ regulatory T cells expressing Foxp3) are required for effective host anti-tumor responses at alternate inoculation sites.     

The role of CD4 T cell help for CD8 CTL activation

CD4 T cells play an important role in the initiation and persistence of CD8 T cells responses. In this review, we report on and evaluate the mechanisms by which CD4 T cells contribute to activation of CD8 T cells and the signal pathways of the down-streaming events after CD4 T cell help.     

Gamma irradiation alters the phenotype and function of CD4CD25 regulatory T cells

To examine the effects of gamma irradiation on Tregs, changes in phenotype and suppression function in Tregs treated with or without gamma ray were analyzed. Purified CD4⁺CD25⁺ regulatory T cells were irradiated at different dosages with a ¹³⁷Cs source gamma ray at 4.8 Gy/min. After culture, the phenotype and function changes were determined by flow cytometry and [³H]-thymidine incorporation, respectively. A dose-dependent reduction of Tregs proliferation in response to gamma irradiation was noted, which paralleled the apoptosis induction of Tregs. Gamma irradiation downregulated the Tregs expression of CD45RO, CD62L, FOXP3, membrane TGF-β, but upregulated Bax and GITR. High dose gamma irradiation (30 Gy) significantly abolished the suppression of Tregs on CD4⁺CD25⁻ T cells proliferation. Thus Tregs not only influences the phenotype but also alters their suppressive capacities. Our findings suggest that radiotherapy may be an important strategy to alter the immunologic balance of Tregs and effector cells in cancer therapy.     

Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

Cell membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are homooligomeric, with native quaternary structure required for maximal enzyme activity. In this study, we mutated lysine 79 in human ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3). The residue corresponding to lysine 79 in NTPDase3 is conserved in all known cell surface membrane NTPDases (NTPDase1, 2, 3, and 8), but not in the soluble, monomeric NTPDases (NTPDase5 and 6), or in the intracellular, two transmembrane NTPDases (NTPDase4 and 7). This conserved lysine is located between apyrase conserved region 1 (ACR1) and an invariant glycosylation site (N81), in a region previously hypothesized to be important for NTPDase3 oligomeric structure. This lysine residue was mutated to several different amino acids, and all mutants displayed substantially decreased nucleotidase activities. A basic amino acid at this position was found to be important for the increase of nucleotidase activity observed after treatment with the lectin, concanavalin A. After solubilization with Triton X-100, mutants showed little or no decrease in activity, unlike the wild-type enzyme, suggesting that the lysine at this position may be important for maintaining proper folding and for stabilizing the quaternary structure. However, mutation at this site did not result in global changes in tertiary or quaternary structure as measured by Cibacron blue binding, chemical cross linking, and native gel electrophoretic analysis, leaving open the possibility of other mechanisms by which mutation of this conserved lysine residue might decrease enzyme activity.     

Tolerance and immunity in a mathematical model of T-cell mediated suppression

Regulatory CD4+CD25+ T cells play a major role in natural tolerance to body components and therefore are relevant to understand the self-non-self discrimination by the immune system. The most pressing theoretical question, regarding the fact that these regulatory cells perform their function through linked recognition of the APCs, is how this "non-specific" mechanism permits a proper balance between tolerance and immunity that is compatible with an effective self-non-self discrimination. To tackle this issue, we develop a numerical simulation, which extends a previous mathematical model of T-cell-mediated suppression to include the thymic generation and the peripheral dynamics of many T cell clones. This simulation can mimic the capacity of the immune system to establish natural tolerance to self-antigens and reliably mount immune responses to foreign antigens. Natural tolerance is based on ubiquitous and constitutive self-antigens, which select and sustain clones of specific regulatory cells. Immune responses to foreign antigens are only achieved if they displace the self-antigens from the APCs, leading to a loss of the regulatory cells, and/or if the foreign antigen introduction entails a sharp increase in the total number of APCs. Meaningful behavior is obtained even if differentiation of regulatory cells in the thymus is antigen non-specific, but requires that a minimum number of new T cells enter the periphery per unit of time, and that the repertoire is selected so that anti-self-affinities are within a proper interval. We conclude that positive selection is required to generate a sufficiently high frequency of self-antigen specific regulatory cells that reliably mediate natural tolerance. Negative selection is required to avoid the emergence at the periphery of very high affinity anti-self-regulatory cells that will make the tolerant state so robust that it could no be broken by the introduction of a foreign antigen. This result highlights the importance of repertoire selection in dominant tolerance proposing a novel role for the processes of positive and negative selection within this framework.     

Heightened NTPDase-1/CD39 expression and angiogenesis in radiation proctitis

Radiation proctitis is an inflammatory process associated with persistent and refractory lower gastrointestinal bleeding. Purinergic signaling regulates hemostasis, inflammation, and angiogenesis. For example, CD39, the vascular ectonucleotidase, blocks platelet activation and is required for angiogenesis. Whether CD39 expression is affected by radiation injury is unknown. The aim of this work was to study CD39 expression patterns after clinical radiation injury to the rectum. We prospectively enrolled eight patients with radiation proctitis and five gender-matched controls. Biopsies were taken from normal-appearing rectal mucosa of controls and from the normal sigmoid and abnormal rectum of patients. Expression patterns of CD39, P2Y2 receptor, CD31, CD61 integrin, and vascular endothelial growth factor receptor 2 were examined by immunostaining; levels of CD39 were further evaluated by Western blots. Chronic inflammatory lesions of radiation proctitis were associated with heightened levels of angiogenesis. Immunohistochemical stains showed increased vascular expression of CD39, as confirmed by Western blots. CD39 was co-localized with vascular endothelial markers CD31 and CD61 integrin, as well as expressed by stromal tissues. Development of neovasculature and associated CD39 expression in radiation proctitis may be associated with the chronic, refractory bleeding observed in this condition.     

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Vα14Jα18 T cell receptor α-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver (∼0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-γ (IFN-γ) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-γ in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.     

CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers

Purpose: Regulatory T cells (T regs) can inhibit immune responses mediated by T cells. It has been shown that there is an increased proportion of T regs in several different human malignancies, although the actual mechanism remains unclear. In the present study, we evaluated the prevalence of CD4(+)CD25^high T regs in PBMCs from patients with gastric and esophageal cancers in relation to the clinical outcome. Methods: PBMCs in 72 patients with gastric cancer and 42 patients with esophageal cancer were evaluated for the proportion of CD4(+)CD25^high T cells, as a percentage of the total CD4(+) cells, by flow cytometric analysis with triple-color staining. Actuarial overall survival rates of the patients were analyzed by the Kaplan–Meier method. Results: The percentages of CD4(+)CD25^high T cells for cases of gastric cancer (4.9±1.2%) and esophageal cancer (5.2±2.1%) were significantly higher than those for healthy donors (1.9±1.1%, P<0.01). There were significant differences in the prevalence of CD4(+)CD25^high T cells between the early and advanced disease stages, both in gastric cancer (stage I vs. III, P<0.05; stage I vs. IV, P<0.05) and esophageal cancer (stage I vs. IV, P<0.05). The patients with a high proportion of CD4(+)CD25^high T cells showed poorer survival rates in comparison to those with a low proportion, in both gastric and esophageal cancers. After patients received curative resections of gastric cancers (n=57), the increased proportions of CD4(+)CD25^high T cells were significantly reduced, and the levels were almost equal to those in normal healthy donors. In addition, studies of gastric cancer patients with postoperative recurrent tumors (n=6) revealed that the prevalence of CD4(+)CD25^high T cells individually increased compared to 2 months after the operations. CD4(+)CD25^high T cells expressed FOXP3 mRNA and had abundant CD45RO and intracellular CTLA-4 molecules. Conclusions: These results strongly suggest that tumor-related factors induce and expand CD4(+)CD25^high T regs.     

Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants

Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.