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.     

Expression vectors for human adenosine deaminase gene therapy

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one fo these vectors (p delta NN2ADA), human ADA was detected in 60-85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.     

Expression of a functional human adenosine deaminase in transgenic tobacco plants

An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5′ UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves.     

Function of murine adenosine deaminase in the gastrointestinal tract

Adenosine deaminase (ADA) deficiency in humans leads to a combined immunodeficiency characterized by severe T and B cell lymphopenia. ADA-deficient humans also display defective development of gut-associated lymphoid tissues (GALT). They lack lymphoid cells, and the Peyer's patches are without germinal centers. In mice, ADA-deficient fetuses die perinatally due to liver damage, but they also exhibit pathology in the thymus, spleen, and the small intestine. The GI phenotype associated with ADA-deficient humans prompted us to examine the effect of ADA-deficiency on mouse small intestine tissue. The work presented here focuses on understanding the physiological role of ADA in the GI tract, using ADA-deficient mice rescued from perinatal lethality by restoring Ada expression to trophoblast cells. Histologically and immunologically, the GALT was compromised at all sites in ADA-/- mice, with the most dramatic changes seen in the Peyer's patches. Profound disturbances in purine metabolism were detected in all the gastrointestinal tissues. In particular, adenosine and deoxyadenosine, the ADA substrates, increased markedly while the product inosine decreased. The activity of S-adenosylhomocysteine hydrolase decreased throughout the GI tract, indicating a possible disruption of cellular transmethylation and activation of apoptotic pathways. There were also disturbances in the purine metabolic pathway with a decrease in the production of downstream nucleosides hypoxanthine and xanthine.     

腺苷酸脱氨酶在乳酸克鲁维酵母中构建表达

【目的】实现鼠灰链霉菌来源经密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母(Kluyveromyces lactis GG799)中组成型表达。【方法】以鼠灰链霉菌(Streptomyces murinus)来源的腺苷酸脱氨酶(AMP)基因经密码子优化后作为模板,设计特异性引物,PCR扩增AMP脱氨酶基因opt-AMPD,以p KLAC1为载体构建重组表达质粒p KLAC1-opt-AMPD,经Sac II线性化后电转化法转入K.lactis GG799,筛选得到重组菌株,测定酶活,经His TrapTM HP纯化后得到AMP脱氨酶,并优化重组菌的发酵培养基。【结果】对AMP脱氨酶基因进行了密码子优化后,构建了重组K.lactis GG799/p KLAC1-opt-AMPD,实现组成型表达,密码子优化后AMP脱氨酶酶活提高到586±50 U/m L。SDS-PAGE结果显示,纯化后的AMP脱氨酶为单一条带,蛋白大小约为60 k D。优化的发酵培养基为(g/L):葡萄糖40、蛋白胨20、酵母粉15、Na Cl 8、KCl 10、Mg SO4 2,30°C、200 r/min发酵120 h,酶活达到2 100±60 U/m L。【结论】实现了密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母GG799内的组成型表达,为实现腺苷酸脱氨酶的重组高效表达和发酵生产进行了有益探索。     

Preliminary X-ray analysis of crystals of murine adenosine deaminase

We have obtained single crystals of a cloned mammalian adenosine deaminase (Mr = 41,000), a key enzyme in purine degradation and in normal development of the immune system, that are suitable for high-resolution structural analysis. The crystals belong to the space group C2 with unit cell parameters a = 101.68 A (1 A = 0.1 nm), b = 94.38 A, c = 85.51 A, and beta = 96.54 degrees. The asymmetric unit contains two enzyme molecules.     

Refined 2.5 A structure of murine adenosine deaminase at pH 6.0.

The X-ray structure of murine adenosine deaminase complexed with the transition-state analogue 6-hydroxyl-1,6-dihydropurine ribonucleoside has been determined from a single crystal grown at pH 4.2 and transferred to mother liquor of increasing pH up to a final pH of 6.0 prior to data collection. The structure has been refined to 2.5 A to a final crystallographic R-factor of 20% using phases from the previously refined 2.4 A structure at pH 4.2. Kinetic measurements show that the enzyme is only 20% active at pH 4.2 whereas it is fully active between pH 6.0 and pH 8.5. The refined structures at either pH are essentially the same. Consideration of the pKa values of the key catalytic residues and the mechanism proposed on the basis of the structure suggests that the ionization state of these residues is largely responsible for the pH dependence on activity.     

Expression of different isoenzymes of adenylate deaminase in cultured human muscle cells. Relation to myoadenylate deaminase deficiency.

Adenylate deaminase activity was determined in cultured muscle cells of different maturation grades and muscle biopsies from normal subjects and four patients with a primary myoadenylate deaminase (MAD) deficiency. Adenylate deaminase activity was much lower in cultured human muscle cells than in normal muscle. The activity increased with maturation. The ratio of activities measured at 5 and 2 mM AMP decreased in the order: immature muscle cells greater than more mature muscle cells greater than muscle. Adenylate deaminase activity was detectable in muscle cell cultures of MAD-deficient patients. However, both at 2 and 5 mM AMP this activity was significantly lower than in cultured cells with the same high maturation grade obtained from control subjects, whereas the ratio between the activities at 5 and 2 mM AMP was higher. The observations indicate that transition from a fetal to an adult muscle isoenzyme of adenylate deaminase takes place in human cultured muscle cells during maturation. In cultures obtained from MAD-deficient patients this transition does not occur and only the fetal isoenzyme is present.     

The role of divalent cations in structure and function of murine adenosine deaminase.

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.     

Lack of adenosine deaminase activity in cultured murine cytotoxic T lymphocytes

The level of adenosine deaminase (ADA) activity was investigated in various populations of IL 2-dependent, cultured cytotoxic T lymphocytes (CTL), from bulk cultures as well as from CTL lines (CTL-A and CTL-B types). The study of C57BL/6 derived, cytotoxic bulk cultures yielded the following mean values of ADA activity: 12,500 U/mg in the cortical, immature region of the thymus, 1500 U/mg in the immunocompetent, cortisone-resistant medullary thymocytes, and 2000 U/mg in the T cell population from the spleen. These results are in agreement with previous studies on separated T lymphocyte populations of known origin and further indicate that a fall in ADA activity accompanies T cell maturation. ADA activity was measured in C57BL/6-derived CTL-A lines obtained from the thymic and splenic bulk cultures. All lines were characterized by a very low level of ADA activity, compared with the T cell bulk cultures freshly initiated from the thymic medulla or from the spleen, and to a variety of T tumor lines established in long term culture. Some showed undetectable ADA activity (less than or equal to 20 units/mg), whereas others maintained significant activity (50 to 500 U/mg). No correlation was found between the residual ADA activity level and the killing activity, at the time of the enzyme assay. Identical properties were observed for CTL-B cloned lines of various genetic backgrounds. These results suggest that the level of ADA activity of the CTL in the mouse is lower than the average value of mature T cells of the thymic medulla, and might constitute a differentiation marker specific to the CTL population. A possibility remains that low ADA activity levels in these CTL lines may be the consequence of an extinction of the ADA gene during in vitro growth, as it is observed for the cytotoxic activity itself. In either case, a low ADA activity level is a remarkable property of IL 2-dependent CTL clones, when compared to various established T tumor lines, which exhibit high and stable ADA levels during long term in vitro growth (5000 to 15,000 U/mg).     

Different functional subsets of cultured murine T cells express characteristic levels of adenosine deaminase activity

The level of adenosine deaminase (ADA) activity in mouse T-lymphocyte cultures was studied under different growth-supporting conditions and in mixed lymphocyte culture-derived long-term T-cell lines and clones. Early after the initiation of in vitro culture, the levels of ADA (2000 U/mg) were similar in bulk cultures either depleted or not depleted in Lyt-2+ T cells. Enrichment for cytolytic T lymphocytes (CTL) obtained by addition of exogenous interleukin 2 (IL-2), was accompanied by a net decrease of ADA activity (110 +/- 15 U/mg). All the tested CTL-A lines derived from such cultures were also characterized by a low or undetectable level of this enzyme (at best 160 +/- 70 U/mg) as previously observed. In contrast, "Lyt-2-" T-cell bulk cultures grown, without addition of exogenous IL-2, in the presence of gamma-irradiated H-2d stimulators maintained a constant level of ADA activity (1770 +/- 340 U/mg) for at least 3 months. Functionally distinct types of Lyt-2- T-cell lines were also analyzed: T-cell lines competent to activate B lymphocytes to growth and terminal maturation as well as others devoid of detectable functions showed a stable ADA level comparable to that expressed by the original bulk culture 1685 +/- 620 U/mg). The present results demonstrate that, like tumor cell lines, most normal T lymphocytes express a high level of ADA activity in culture, which strongly suggests that the low level of ADA activity exhibited by CTL is a characteristic of this functional subset.