Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family
Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family
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DOI：
10.1074/jbc.m000916200
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发表时间：
2000-07-28
影响因子：
4.8
通讯作者：
Miot, F
中科院分区：
文献类型：
--
作者：
De Deken, X;Wang, DT;Miot, F
已克隆了两个编码NADPH氧化酶并构成甲状腺H_2O_2产生系统的cDNAs。克隆策略是基于白细胞和甲状腺中H2 O2生成之间的功能相似性，根据甲状腺系统的一个组成部分属于gp（91 Phox）/Mox 1基因家族并显示与gp 91（Phox）的序列相似性的假设。用gp 91（Phox）探针对原代培养的甲状腺细胞的cDNA文库进行低严格性筛选，得到两个不同的人cDNA克隆，其分别具有1551（ThOX 1）和1548（ThOX 2）的开放阅读框。编码的多肽显示83%的序列相似性，并明确相关的gp 91（Phox）（53和47%的相似性）。177 kDa的理论分子量接近于天然相应猪黄素蛋白以及通过蛋白质印迹法在犬和人甲状腺中检测到的蛋白质的180 kDa的表观分子量。ThOX 1和ThOX 2显示的序列相似性分别为53%和61%，预测的蛋白质的秀丽隐杆线虫在其全长。它们显示沿着它们的前500个氨基酸与甲状腺过氧化物酶有43%的相似性。ThOX 1和ThOX 2的相应基因在染色体15q15.8上紧密连锁。犬mRNA表达具有甲状腺特异性，并通过激活cAMP途径的试剂上调，因为它们编码的多肽合成也是如此。在人甲状腺中，cAMP的正调节不太明显。蛋白质ThOX 1和ThOX 2积累在甲状腺细胞的顶膜，并与甲状腺过氧化物酶共定位。
Two cDNAs encoding NADPH oxidases and constituting the thyroid H2O2 generating system have been cloned. The strategy of cloning was based on the functional similarities between H2O2 generation in leukocytes and the thyroid, according to the hypothesis that one of the components of the thyroid system would belong to the gp(91Phox)/Mox1 gene family and display sequence similarities with gp91(Phox). Screening at low stringency with a gp91(Phox) probe of cDNA libraries from thyroid cells in primary culture yielded two distinct human cDNA clones harboring open reading frames of 1551 (ThOX1) and 1548 amino acids (ThOX2), respectively. The encoded polypeptides display 83% sequence similarity and are clearly related to gp91(Phox) (53 and 47% similarity). The theoretical molecular mass of 177 kDa is close to the apparent molecular mass of 180 kDa of the native corresponding porcine flavoprotein and the protein(s) detected by Western blot in dog and human thyroid. ThOX1 and ThOX2 display sequence similarities of 53% and 61%, respectively, with a predicted protein of Caenorhabditis elegans over their entire length. They show along their first 500 amino acids a similarity of 43% with thyroperoxidase. The corresponding genes of ThOX1 and ThOX2 are closely linked on chromosome 15q15.8, The dog mRNA expression is thyroid-specific and up-regulated by agents activating the cAMP pathway as is the synthesis of the polypeptides they are coding for. In human thyroid the positive regulation by cAMP is less pronounced. The proteins ThOX1 and ThOX2 accumulate at the apical membrane of thyrocytes and are co-localized with thyroperoxidase.