Probing the expression and function of the P2X7 purinoceptor with antibodies raised by genetic immunization
Probing the expression and function of the P2X7 purinoceptor with antibodies raised by genetic immunization
复制标题
DOI：
10.1016/j.cellimm.2005.08.011
复制
发表时间：
2005-07-01
影响因子：
4.3
通讯作者：
Koch-Nolte, F
中科院分区：
文献类型：
--
作者：
Adriouch, S;Dubberke, G;Koch-Nolte, F
细胞溶解性P2 X7嘌呤受体广泛表达于白细胞上，并且由于其在用外ATP处理细胞后诱导大的非选择性膜孔的独特能力而引起了人们的兴趣。针对合成P2 X7肽产生的抗体通常在蛋白质印迹分析中工作良好，但不能识别细胞表面上的天然蛋白质。基因免疫是一种有用的技术，用于产生针对天然构象蛋白质的抗体。使用这种技术，我们已经产生了高度特异性的多克隆（兔）和单克隆（大鼠）抗P2 X7抗体，很容易检测小鼠P2 X7的活细胞表面的免疫荧光分析和流式细胞术。这些抗体与P2 X7的结合在用ATP处理细胞后数秒内减少，表明配体结合诱导P2 X7的构象转变和/或脱落。通过定点突变，我们突变了P2 X7细胞外环第二跨膜区附近的三个保守精氨酸残基（R294 A，R307 A，R316 A）。这些突变中的每一种都会导致ATP反应的丧失。流式细胞仪和免疫印迹分析表明，R294 A突变体在转染细胞中的表达水平高于野生型P2 X7，而R307 A和R316 A突变体几乎检测不到，因为这些构建体没有或很少有蛋白质合成。与其对ATP诱导的活化的抗性一致，R294 A突变体不被ATP处理从细胞表面下调。(c)2005年爱思唯尔公司All rights reserved.
The cytolytic P2X7 purinoceptor is widely expressed on leucocytes and has sparked interest because of its peculiar ability to induce a large nonselective membrane pore following treatment of cells with ecto-ATP. Antibodies raised against synthetic P2X7 peptides generally work well in Western-Blot analyses but fail to recognize the native protein on the cell surface. Genetic immunization is a useful technique to raise antibodies directed against proteins in native conformation. Using this technique we have generated highly specific polyclonal (rabbit) and monoclonal (rat) anti-P2X7 antibodies that readily detect mouse P2X7 on the surface of living cells by immunofluorescence analyses and flow cytometry. Binding of these antibodies to P2X7 is reduced within seconds after treatment of cells with ATP, suggesting that ligand binding induces a conformational shift and/or the shedding of P2X7. By site directed mutagenesis we have mutated three conserved arginine residues (R294A, R307A, R316A) in the extracellular loop of P2X7 near the second transmembrane region. Each of these mutations results in loss of ATP response. FACS and immunoblot analyses reveal that the R294A mutant is expressed at higher levels than wild-type P2X7 in transfected cells, whereas the R307A and R316A mutants are barely detectable because there is no or very little protein synthesis of these constructs. In accord with its resistance to ATP-induced activation the R294A mutant is not down-modulated from the cell surface by ATP-treatment. (c) 2005 Elsevier Inc. All rights reserved.