Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity
Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity
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DOI：
10.1074/jbc.m500516200
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发表时间：
2005-08-26
影响因子：
4.8
通讯作者：
Dupuy, C
中科院分区：
文献类型：
--
作者：
Ameziane-El-Hassani, R;Morand, S;Dupuy, C
DUOX2(也可能是DUX1)是一种糖黄素蛋白，参与甲状腺激素的生物合成，是甲状腺过氧化氢生成器，功能上与TPO(甲状腺过氧化物酶)相关。到目前为止，由于成熟和靶向过程的障碍，将DUOX导入非甲状腺细胞系并没有导致质膜上有功能的过氧化氢产生系统的表达。我们首次研究了转DUOX2和DUOX1基因的HEK293和中国仓鼠卵巢细胞颗粒组分中过氧化氢的产生活性。稳定或瞬时转染人或猪DUOX基因的这些细胞的颗粒部分显示出功能性的NADPH/钙依赖的H_2O_2产生活性。未成熟的DUOX蛋白的活性低于猪甲状腺细胞颗粒组分，其活性依赖于其一级结构。人DUOX2似乎比人DUOX1活性更高，但只有猪DUOX2活性的一半。TPO共转染组的酶活性略有升高，而白细胞NADPH氧化酶的22 kDa亚基p22(Phox)则没有影响。以往对H_2O_2形成机制的研究表明，成熟的甲状腺NADPH氧化酶不释放O-2。但是过氧化氢。使用自旋捕获技术结合电子顺磁共振光谱，我们证实了这一结果，但也证明了位于内质网的部分糖基化形式的DUOX2以钙依赖的方式产生超氧化物。这些结果表明，DUOX2成熟过程中的翻译后修饰可能通过促进分子内超氧化物歧化参与了过氧化氢的形成机制。
Duox2 ( and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O-2. but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.