Studies on the NADPH oxidase system responsible for anti-infectious activity of phagocytes

我们正在研究NADPH氧化酶系统负责吞噬细胞抗感染活性的机制。慢性肉芽肿病(CGD)是该系统中的一种遗传性疾病，患者不能产生超氧阴离子，并遭受反复感染。1.Gp91是NADPH氧化酶的氧化还原中心，被认为在其分子中同时含有血红素和FAD。然而，这种黄色素的概念只是基于gp91的氨基酸序列与其他黄素蛋白的相似性，没有任何直接的证据。其主要原因是gp91的大部分突变导致其蛋白质的完全缺失。我们发现一例罕见的CGD患者表达gp91载脂蛋白。CDNA分析显示His-338存在突变，位于FAD结合的预测区域。患者生化分析显示FAD完全消失，体外加入试剂FAD不能纠正其超氧化物歧化活性。这些结果表明，His-338对于FAD与NADPH氧化酶的结合非常关键。这是在CGD2中发现的第一个这样的突变。目前还没有研究探讨NADPH氧化酶与微生物来源的毒素的关系。在这里，我们研究了来自曲霉的胶质毒素对NADPH氧化酶的影响。这种毒素在结构上带有S-S键，能阻止中性粒细胞NADPH氧化酶对PMA的反应产生超氧化物。Gliooxin影响激活过程，但不影响激活酶的催化作用。推测其抑制方式为胶质毒素中的S-S键与NADPH氧化酶中邻近的SH基团的直接相互作用。

We are studying the mechanisms of the NADPH oxidase system responsible for anti-infectious activity of phagocytes. Patients with chronic granulomatous disease (CGD), a genetic disorder in this system, fail to generate superoxide anion and suffer from recurrent infections.1.Gp91, the redox center of the NADPH oxidase, had been considered to contain both heme and FAD in its molecule. However, this flavocytochrome concept was only based on the similarities between the amino acid sequences of gp91 and other flavoproteins, without any direct evidence. The main reason for this was that most of the mutations in gp91 lead complete absence of its protein. We discovered a rare CGD patient who expressed a gp91 apoprotein. Analysis of cDNA revealed the mutation of His-338 to Try, which resided in a predicted domain for FAD-binding. The biochemical analysis of the patient revealed that the lost FAD completely, and the addition of reagent FAD in vitro could not corrected his superoxide-generating activity. These resulted indicate that His-338 is a very critical for FAD incorporation into the NADPH oxidase. This was the first such mutation found in CGD.2. There have not been studies, which inquired the NADPH oxidase in relation to microorganism-derived toxins. Here, we investigated the effect of gliotoxin from Aspergillus on the NADPH oxidase. This toxin, bearing S-S bond in its structure, prevented the onset of superoxide generation by the neutrophil NADPH oxidase in response to PMA. Gliotoxin affected the activation process, but not the catalysis of the activated enzyme. The inhibitory mode was suggested to be direct interaction of S-S bond in gliotoxin with vicinal SH group in the NADPH oxidase.