Beaumel Sylvain, Grunwald Didier, Fieschi Franck, Stasia Marie José
*Chronic Granulomatous Disease Diagnosis and Research Centre, Therex-TIMC/Imag, UMR CNRS 5525, Université Grenoble Alpes, Grenoble, F-38041, France.
‡iRTS/CEA, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
Biochem J. 2014 Dec 15;464(3):425-37. doi: 10.1042/BJ20140555.
Cytochrome b558, the redox core of the NADPH oxidase (NOX) complex in phagocytes, is composed of NOX2 and p22phox, the synthesis of which is intimately connected but not fully understood. We reproduced 10 rare X-minus chronic granulomatous disease (CGD) mutations of highly conserved residues in NOX1-NOX4, in X0-CGD PLB-985 cells in order to analyse their impact on the synthesis of cytochrome b558. According to the impact of these mutations on the level of expression of NADPH oxidase 2 (NOX2) and its activity, mutants were categorized into group A (W18C, E309K, K315del and I325F), characterized by a linear relationship between NOX2 expression and NOX activity, and group B (H338Y, P339H, G389A and F656-F570del), showing an absence of NOX activity associated with variable levels of NOX2 expression. These last residues belong to the FAD-binding pocket of NOX2, suggesting that this functional domain also plays a role in the structural integrity of NOX2. Finally, we observed an abnormal accumulation of p65 (65-kDa monomer), the NOX2 precursor and p65-p22phox dissociation in the W18C, E309K, I325F and G389A mutants, pointing out a possible role of the first transmembrane domain (Trp18), and the region between the membrane and the dehydrogenase domain of NOX2 (Glu309, Ile325 and Gly389), in the binding with p22phox.
细胞色素b558是吞噬细胞中NADPH氧化酶（NOX）复合物的氧化还原核心，由NOX2和p22phox组成，它们的合成密切相关，但尚未完全了解。我们在X0-CGD PLB-985细胞中重现了NOX1-NOX4中10个高度保守残基的罕见X连锁慢性肉芽肿病（CGD）突变，以分析它们对细胞色素b558合成的影响。根据这些突变对NADPH氧化酶2（NOX2）表达水平及其活性的影响，突变体被分为A组（W18C、E309K、K315del和I325F），其特征是NOX2表达与NOX活性之间呈线性关系；以及B组（H338Y、P339H、G389A和F656-F570del），显示出与不同水平的NOX2表达相关的NOX活性缺失。这些最后的残基属于NOX2的FAD结合口袋，表明该功能域在NOX2的结构完整性中也起作用。最后，我们观察到W18C、E309K、I325F和G389A突变体中p65（65 kDa单体）、NOX2前体异常积累以及p65-p22phox解离，指出了第一个跨膜结构域（Trp18）以及NOX2的膜与脱氢酶结构域之间的区域（Glu309、Ile325和Gly389）在与p22phox结合中的可能作用。
