Characterization of ThOX proteins as components of the thyroid H2O2-generating system
Characterization of ThOX proteins as components of the thyroid H2O2-generating system
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DOI：
10.1006/excr.2001.5444
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发表时间：
2002-02-15
影响因子：
3.7
通讯作者：
Miot, F
中科院分区：
文献类型：
--
作者：
De Deken, X;Wang, DT;Miot, F
我们最近克隆了两个甲状腺特异性cDNA编码NADPH氧化酶家族的新成员。ThOX 1和ThOX 2蛋白与甲状腺过氧化物酶共定位于人甲状腺细胞的顶膜。在本研究中，我们已经确定了它们的亚细胞定位和成熟的酶活性。大部分ThOX蛋白在细胞内积累，只有一小部分在表面表达。Western印迹证明ThOX是180，000和190，000的糖蛋白。当完全去糖基化时，ThOX 1和ThOX 2的分子量均下降至100，000。Ca ~（2+）刺激PC C13细胞基础H_2O_2生成的水平相当于PMA刺激白细胞H_2O_2生成的20%。用ThOX 1和ThOX 2转染的非甲状腺细胞系在Western印迹分析中仅显示单一免疫反应性条带，对应于180，000的蛋白。这种“未成熟”蛋白仅保留在细胞内，不存在任何酶活性。甲状腺过氧化物酶和p22（Phox）的共表达不会改变这一点。将ThOX cDNA转染入PLB-XCGD细胞不能重建其NADPH氧化酶活性。我们的结论是：（1）甲状腺含有白细胞H_2O_2生成系统的某些成分，但不是全部;（2）ThOX’s主要或专门位于甲状腺细胞或转染细胞的细胞内，因此它们是无活性的;（3）ThOX’s不能取代白细胞中的gp 91（Phox）;（4）甲状腺H_2O_2生成系统在ThOX’s和gp 91（Phox）方面与白细胞系统类似，但在其它方面非常不同。可能需要额外的甲状腺特异性成分来获得甲状腺中完整的蛋白质加工和完整的酶活性。(C)2002 Elsevier Science（美国）。
We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thyroperoxidase at the apical membrane of human thyroid cells. In the present study we have determined their subcellular localization and maturation in relation to their enzymatic activity. A majority of ThOX proteins accumulated inside the cell and only a small fraction was expressed at the surface. Western blots demonstrated that ThOX's are glycoproteins of 180,000 and 190,000. When totally deglycosylated the molecular weight of both ThOX1 and ThOX2 drops to 100,000. Ca2+ stimulates the basal H2O2 generation in PC C13 cells at a level corresponding to 20% of the leukocyte H2O2 production stimulated by PMA. Nonthyroid cell lines transfected with ThOX1 and ThOX2 show only a single immunoreactive band in Western blot analysis, corresponding to the protein of 180,000. This "immature" protein remains exclusively intracellular and does not present any enzymatic activity. This is not modified by coexpression of thyroperoxidase and p22(Phox). Transfection of ThOX cDNAs into PLB-XCGD cells does not reconstitute their NADPH oxidase activity. We conclude that (1) the thyroid contains some elements of the leukocyte H2O2-generating system but not all of them; (2) ThOX's are predominantly or exclusively located inside the cell in thyrocytes or in transfected cells, respectively, and as such they are inactive; (3) ThOX's cannot replace gp91(Phox) in the leukocyte; and (4) the thyroid H2O2-generating system is analogous to the leukocyte system with regard to ThOX's and gp91(Phox) but very different in other aspects. Additional thyroid-specific components are probably required to get complete protein processing and full enzymatic activity in the thyroid. (C) 2002 Elsevier Science (USA).