Functional analysis of two-amino acid substitutions in gp91phox in a patient with X-linked flavocytochrome b558-positive chronic granulomatous disease by means of transgenic PLB-985 cells
Functional analysis of two-amino acid substitutions in gp91phox in a patient with X-linked flavocytochrome b558-positive chronic granulomatous disease by means of transgenic PLB-985 cells
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DOI：
10.1007/s00439-004-1173-z
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发表时间：
2004-10-01
期刊：
影响因子：
5.3
通讯作者：
Stasia, MJ
中科院分区：
文献类型：
--
作者：
Bionda, C;Li, XJ;Stasia, MJ
慢性肉芽肿病 (CGD) 是一种罕见的遗传性疾病，其中吞噬细胞缺乏 NADPH 氧化酶活性。最常见的形式是由编码 gp91phox 蛋白的 CYBB 基因突变引起，该蛋白是细胞色素 b(558) 的重链，是 NADPH 氧化酶的氧化还原元件。在一些罕见的情况下，突变的 gp91phox 正常表达，但检测不到 NADPH 氧化酶。这种类型的 CGD 称为 X91(+) CGD。我们之前报道过一例 X+ CGD 病例，其 gp91phox 存在双错义突变。现在已经使用转基因 PLB-985 细胞来研究每个单一突变对氧化酶活性和组装的影响，以排除 CYBB 基因中可能存在的新多态性。 His303Asn/Pro304Arg gp91phox 转基因 PLB-985 细胞完全模仿 X+ CGD 患者的中性粒细胞表型。 His303Asn 突变足以抑制完整细胞和破损细胞系统中的氧化酶活性，而在 Pro304Arg 突变体中，残余活性表明 Pro304Arg 取代对氧化酶活性的破坏性小于 His303Asn 突变。对细胞质因子 p47phox 和 p67phox 的体外和体内易位后的 NADPH 氧化酶组装的研究表明，在双突变体和 His303Asn 突变体中，NADPH 氧化酶组装被废除，尽管易位仅在 Pro304Arg 突变细胞中减弱。因此，尽管His303Asn突变比Pro304Arg突变对NADPH氧化酶活性和组装具有更严重的抑制作用，但这两种突变都不能被视为多态性。
Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. The most common form is caused by mutations in the CYBB gene encoding gp91phox protein, the heavy chain of cytochrome b(558), which is the redox element of NADPH oxidase. In some rare cases, the mutated gp91phox is normally expressed but no NADPH oxidase can be detected. This type of CGD is called X91(+) CGD. We have previously reported an X+ CGD case with a double-missense mutation in gp91phox. Transgenic PLB-985 cells have now been made to study the impact of each single mutation on oxidase activity and assembly to rule out a possible new polymorphism in the CYBB gene. The His303Asn/Pro304Arg gp91phox transgenic PLB-985 cells exactly mimic the phenotype of the neutrophils of the X+ CGD patient. The His303Asn mutation is sufficient to inhibit oxidase activity in intact cells and in a broken cell system, whereas in the Pro304Arg mutant, residual activity suggests that the Pro304Arg substitution is less devastating to oxidase activity than the His303Asn mutation. The study of NADPH oxidase assembly following the in vitro and in vivo translocation of cytosolic factors p47phox and p67phox has demonstrated that, in the double mutant and in the His303Asn mutant, NADPH oxidase assembly is abolished, although the translocation is only attenuated in Pro304Arg mutant cells. Thus, even though the His303Asn mutation has a more severe inhibitory effect on NADPH oxidase activity and assembly than the Pro304Arg mutation, neither mutation can be considered as a polymorphism.