New insights into the membrane topology of the phagocyte NADPH oxidase: Characterization of an anti-gp91-phox conformational monoclonal antibody
New insights into the membrane topology of the phagocyte NADPH oxidase: Characterization of an anti-gp91-phox conformational monoclonal antibody
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DOI：
10.1016/j.biochi.2007.01.010
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发表时间：
2007-09-01
期刊：
影响因子：
3.9
通讯作者：
Morel, Francoise
中科院分区：
文献类型：
--
作者：
Campion, Yannick;Paclet, Marie-Helene;Morel, Francoise
细胞色素B（558）是吞噬细胞NADPH氧化酶的催化核心，介导杀菌活性氧的产生。细胞色素B（558）由两个非共价结合的亚基gp 91-phox和p22-phox（1/1）形成。其活化依赖于与胞质调节蛋白（p67-phox、p47-phox、p40-phox和Rac）的相互作用，导致电子从NADPH转移到分子氧并释放超氧阴离子。一些研究表明，激活过程与细胞色素b558构象的变化有关。最近，我们通过分离组成型活性形式的细胞色素B（558）证实了这一假设。为了表征活性和非活性细胞色素b558构象，我们制备了四种新的单克隆抗体（7A 2，13 B6，15 B12和8 G11），这些抗体针对从静息和刺激的中性粒细胞中纯化的细胞色素B（558）的混合物。四种抗体标记gp 91-phox，并结合天然和变性的细胞色素b558。有趣的是，它们对蛋白质的胞外结构域具有特异性。噬菌体展示作图结合重组gp 91-phox截短形式的研究允许识别表位区域。然后使用这些抗体研究NADPH氧化酶活化过程。特别是，他们被证明几乎完全抑制NADPH氧化酶活性重建在体外与膜和胞质溶胶。此外，对刺激的中性粒细胞进行的流式细胞术分析和共聚焦显微镜检查指出，单克隆抗体13136能够优先结合细胞色素B的活性形式（558）。所有这些数据表明，这四种新型抗体是检测完整细胞中细胞色素B（558）表达和分析其膜拓扑结构的潜在有力工具。此外，抗体13 B6可以是构象敏感的，并且可以用作用于在体内鉴定活性NADPH氧化酶复合物的探针。(c)2007年，Elsevier Masson SAS。All rights reserved.
Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b558 conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b558 conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated nentrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b558. Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13136 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformation ally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo. (c) 2007 Elsevier Masson SAS. All rights reserved.