Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes.
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ABSTRACT:
SUBMITTER: Wiginton DA
PROVIDER: S-EPMC1162902 | biostudies-other | 1981 May
REPOSITORIES: biostudies-other
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Project description:The glutamate receptor subunit B (GluR-B) pre-mRNA is edited at two adenosine residues, resulting in amino acid changes that alter the electrophysiologic properties of the glutamate receptor. Previous studies showed that these amino acid changes are due to adenosine to inosine conversions in two codons resulting from adenosine deamination. Here, we describe the purification and characterization of an activity from human HeLa cells that efficiently and accurately edits GluR-B pre-mRNA at both of these sites. The purified activity contains a human homolog of the recently reported rat RED1 (rRED1) protein, a member of the family of double-stranded RNA-dependent deaminase proteins. Recombinant human RED1 (hRED1), but not recombinant dsRAD, another member of the family, efficiently edits both the Q/R and R/G sites of GluR-B RNA. We conclude that the GluR-B editing activity present in HeLa cell extracts and the recombinant hRED1 protein are indistinguishable.
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Project description:The aim of the present study was to investigate adenosine deaminase (ADA) and adenosine kinase (ADK) expression in human glioma and to explore its correlation with glioma?associated epilepsy. Tumor tissues (n=45) and peritumoral tissues (n=14) were obtained from glioma patients undergoing surgery. Normal control tissues (n=8) were obtained from brain trauma patients. The disease grade was determined by histological evaluation and the degree of tumor invasion was evaluated using immunofluorescence analyses. mRNA and protein expression of ADA and ADK were evaluated using reverse transcription quantitative polymerase chain reaction or western blot analysis, respectively. Based on histological evaluations, four cases were classified as Grade I gliomas, 18 cases as Grade II, 17 cases as Grade III and six cases were considered Grade IV. Increased ADA and ADK expression was observed in tumor tissues. ADA was predominantly distributed in the cytoplasm of tumor cells, whereas ADK was detected in the cytoplasm as well as in the nuclei. ADA and ADK levels were upregulated in patients with Grade II and Grade III gliomas compared to those in control subjects (p<0.05). In addition, tumor invasion was detected in peritumoral tissues. The number of ADA?positive or ADK?positive cells in tumor tissues was similar between glioma patients with and without epilepsy (p>0.05). However, ADA and ADK expression was upregulated in peritumoral tissues derived from patients with epilepsy compared to that in glioma patients without epilepsy. The results of the present study suggested that ADA and ADK are involved in glioma progression, and that increased ADA and ADK levels in peritumoral tissues may be associated with epilepsy in glioma patients.
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Project description:1. The partial purification of adenosine deaminase, types 1, 2 and 2-1, from human erythrocytes is described. 2. The isoenzyme components characteristic of the three forms of the enzyme were partially resolved by chromatography on DEAE-Sephadex. 3. Gel chromatography of the various forms of the enzyme gave estimates of the molecular weights in the range 30000-35000. 4. Electrophoresis in starch gels containing increasing percentages of starch did not reveal any differences in molecular weight between the genetic variants or their isoenzyme components. 5. Analytical isoelectric-focusing experiments in polyacrylamide gels gave the following pI values for the four isoenzyme components present in type 2-1 erythrocytes: 4.70, 4.83, 4.94 and 5.06. 6. All forms of the enzyme gave K(m) values for adenosine of about 30mum and K(i) values of about 8mum for the competitive inhibitor purine riboside. 7. Reaction rates of the type 1 and 2 enzymes were measured at different temperatures. Both enzymes gave values for the energy of activation for hydrolysis of adenosine of about 33.4kJ/mol (8kcal/mol). 8. Heat inactivation of all forms of the enzyme was markedly dependent on ionic strength, the rate of inactivation increasing with increasing ionic strength. The type 1 and type 2 forms of the enzyme differed significantly in their susceptibility to heat inactivation. From the variation of rates of inactivation with temperature, values were obtained for the energies of activation for the heat inactivation of both enzymes as follows: type 1 enzyme 275.5kJ/mol (65.9kcal/mol) and type 2 enzyme 241.6kJ/mol (57.8kcal/mol.).
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Project description:BACKGROUND:Improved survival in ADA-SCID patients is revealing new aspects of the systemic disorder. Although increasing numbers of reports describe the systemic manifestations of adenosine deaminase deficiency, currently there are no studies in the literature evaluating genital development and pubertal progress in these patients. METHODS:We collected retrospective data on urogenital system and pubertal development of 86 ADA-SCID patients followed in the period 2000-2017 at the Great Ormond Street Hospital (UK) and 5 centers in Italy. In particular, we recorded clinical history and visits, and routine blood tests and ultrasound scans were performed as part of patients' follow-up. RESULTS AND DISCUSSION:We found a higher frequency of congenital and acquired undescended testes compared with healthy children (congenital, 22% in our sample, 0.5-4% described in healthy children; acquired, 16% in our sample, 1-3% in healthy children), mostly requiring orchidopexy. No urogenital abnormalities were noted in females. Spontaneous pubertal development occurred in the majority of female and male patients with a few cases of precocious or delayed puberty; no patient presented high FSH values. Neither ADA-SCID nor treatment performed (PEG-ADA, BMT, or GT) affected pubertal development or gonadic function. CONCLUSION:In summary, this report describes a high prevalence of cryptorchidism in a cohort of male ADA-SCID patients which could represent an additional systemic manifestation of ADA-SCID. Considering the impact urogenital and pubertal abnormalities can have on patients' quality of life, we feel it is essential to include urogenital evaluation in ADA-SCID patients to detect any abnormalities, initiate early treatment, and prevent long-term complications.
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Project description:Two distinct adenosine deaminases, ADA1 and ADA2, are found in humans. ADA1 has an important role in lymphocyte function and inherited mutations in ADA1 result in severe combined immunodeficiency. The recently isolated ADA2 belongs to the novel family of adenosine deaminase growth factors (ADGFs), which play an important role in tissue development. The crystal structures of ADA2 and ADA2 bound to a transition state analogue presented here reveal the structural basis of the catalytic/signaling activity of ADGF/ADA2 proteins. In addition to the catalytic domain, the structures discovered two ADGF/ADA2-specific domains of novel folds that mediate the protein dimerization and binding to the cell surface receptors. This complex architecture is in sharp contrast with that of monomeric single domain ADA1. An extensive glycosylation and the presence of a conserved disulfide bond and a signal peptide in ADA2 strongly suggest that ADA2, in contrast to ADA1, is specifically designed to act in the extracellular environment. The comparison of catalytic sites of ADA2 and ADA1 demonstrates large differences in the arrangement of the substrate-binding pockets. These structural differences explain the substrate and inhibitor specificity of adenosine deaminases and provide the basis for a rational design of ADA2-targeting drugs to modulate the immune system responses in pathophysiological conditions.
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Project description:Adenosine deaminase 2 (ADA2) belongs to the novel family of adenosine deaminase growth factors (ADGFs), which play an important role in tissue development. The deficiency of adenosine deaminase 2 (DADA2) is a recently recognized autosomal recessive autoinflammatory disease, characterized by various systemic vascular and inflammatory manifestations, which is associated with ADA2 mutations. Considering that a recent screening of an international registry of children with systemic primary vasculitis revealed novel and already known variants in ADA2, this study aimed to further investigate the functional significance of the rare variants detected, namely p.Gly47Arg, p.Gly47Ala, p.Arg8Trp, p.Leu351Gln and p.Ala357Thr, by using a structural biological approach. Three‑dimensional models of the mutants were developed and their three‑dimensional (3D) structures were subjected to detailed interaction and conformational analyses. This led to suggestions that the novel mutations found may affect the formation/stability of the homodimer or may influence the activity of the enzyme. It was thus concluded that the Arg8Trp and Gly47Arg mutations affect the position and interaction of the dimer‑associated HN1 helical structure and therefore, dimer formation and stabilization, while Leu351Gln and Ala357Thr influence the metal coordination in the active site. These findings shed further light onto the structural consequences of the mutations under investigation.
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Project description:The mammalian adenosine deaminases acting on RNA (ADARs) constitute a family of sequence-related proteins involved in pre-mRNA editing of nuclear transcripts through site-specific adenosine modification. We report here the identification and characterization of a human ADAR protein, hADAT1, that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala). It represents the functional homologue of the recently identified yeast protein Tad1p [Gerber, A., Grosjean, H., Melcher, T. & Keller, W. (1998) EMBO J. 17, 4780-4789]. The hADAT1 cDNA predicts a protein of 502 aa whose sequence displays strongest overall homology to a Drosophila melanogaster ORF (50% similarity, 32% identity), and the catalytic domain is closely related to the other ADAR proteins. In vitro, the recombinantly expressed and purified hADAT1 protein efficiently and specifically deaminates A(37) in the anticodon loop of tRNA(Ala) from higher eukaryotes and with lower efficiency from lower eukaryotes. It does not modify adenosines residing in double-stranded RNA or in pre-mRNAs that serve as substrates for ADAR1 or ADAR2. The anticodon stem-loop of tRNA(Ala) alone is not a functional substrate for hADAT1. The enzyme is expressed ubiquitously in human tissues and is represented by a single gene. The identification and cloning of hADAT1 should help to elucidate the physiological significance of this unique modification in tRNA(Ala), which is conserved from yeast to man.
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Project description:A panel of lectins was used to analyse glycoproteins of normal granulocytes and leukaemic myeloid cells. The glycoproteins of detergent-solubilized whole cells were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their lectin-binding properties determined by incubation of the fixed gels with radioiodinated lectins. Normal granulocytes and leukaemic myeloid cells in different stages of maturation possess a cell-surface sialic acid-rich glycoprotein of apparent mol.wt. 115 000 (GP115), that can be labelled by both the lactoperoxidase and periodate/NaB3H4 cell-surface labelling techniques. The sialoglycoprotein of leukaemic myeloblasts has a slightly lower apparent mol.wt., 112000 (GP112). After neuraminidase treatment before cell solubilization, both GP115 and GP112 bind the lectins from Arachis hypogaea (peanut) and Helix pomatia (snail) and have an increased apparent molecular weight of 125000. Two concanavalin A-binding glycoproteins of apparent mol.wts. 98000 and 90000 are present in leukaemic myeloblasts. Concanavalin A binding to these glycoproteins is decreased in more mature leukaemic cells and absent in granulocytes. As concanavalin A binding decreases in the maturer forms, there is a concomitant increase in the binding of Ricinus communis (castor bean) and Maclura aurantiaca (osage orange) lectins to these glycoproteins. Whole granulocytes, but not leukaemic myeloblasts, contain a major cell-surface concanavalin A binding glycoprotein of apparent mol.wt. 130000, which is labelled by the periodate/NaB3H4 technique. Concanavalin A binding to this glycoprotein increases as the morphology of leukaemic cells approaches that of mature granulocytes.
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Project description:Deficiency of adenosine deaminase type 2 (DADA2) was first described in 2014 as a monogenic cause of polyartertitis nodosa (PAN), early onset lacunar stroke and livedo reticularis. The clinical phenotype of DADA2 is, however, very broad and may involve several organ systems. Apart from vasculitis, children may present with i) Hematological manifestations (ii) Lymphoproliferation and iii) Immunodeficiencies. Patients with DADA2 can have variable patterns of cytopenias and bone marrow failure syndromes. Patients with DADA2 who have predominant haematological manifestations are associated with ADA2 gene variants that result in minimal or no residual ADA2 activity. Lymphoproliferation in patients with DADA2 may range from benign lymphoid hyperplasia to lymphoreticular malignancies. Patients may present with generalized lymphadenopathy, splenomegaly, autoimmune lymphoproliferative syndrome (ALPS) like phenotype, Hodgkin lymphoma, T-cell large granular lymphocytic infiltration of bone marrow and multicentric Castleman disease. Immunodeficiencies associated with DADA are usually mild. Affected patients have variable hypogammaglobulinemia, decrease in B cells, low natural killer cells, common variable immunodeficiency and rarely T cell immunodeficiency. To conclude, DADA2 has an extremely variable phenotype and needs to be considered as a differential diagnosis in diverse clinical conditions. In this review, we describe the evolving clinical phenotypes of DADA2 with a special focus on haematological and immunological manifestations.
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Project description:Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.
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