Characterization of ThOX Proteins as Components of the Thyroid H 2 O 2 -Generating System Xavier De Deken, 1,2,3 Dantong Wang, 1,4 Jacques E. Dumont, and Franc ¸oise Miot Institut de Recherche Interdisciplinaire, Universite ´ Libre de Bruxelles, Campus Erasme, 808, Route de Lennik, 1070 Brussels, Belgium We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thy- roperoxidase at the apical membrane of human thy- roid cells. In the present study we have determined their subcellular localization and maturation in rela- tion 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 molecu- lar weight of both ThOX1 and ThOX2 drops to 160,000. Ca 21 stimulates the basal H 2 O 2 generation in PC Cl3 cells at a level corresponding to 20% of the leukocyte H 2 O 2 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 “imma- ture” 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 activ- ity. We conclude that (1) the thyroid contains some elements of the leukocyte H 2 O 2 -generating system but not all of them; (2) ThOX’s are predominantly or exclu- sively located inside the cell in thyrocytes or in trans- fected cells, respectively, and as such they are inac- tive; (3) ThOX’s cannot replace gp91 Phox in the leukocyte; and (4) the thyroid H 2 O 2 -generating system is analogous to the leukocyte system with regard to ThOX’s and gp91 Phox but very different in other as- pects. Additional thyroid-specific components are probably required to get complete protein processing and full enzymatic activity in the thyroid. © 2002 Elsevier Science (USA) Key Words: thyroid; H 2 O 2 -generating system; NADPH oxidase; NOX; ThOX; protein maturation. INTRODUCTION The iodination of thyroglobulin is catalyzed by thy- roperoxidase (TPO) in the presence of H 2 O 2 at the apical membrane of the thyroid follicular cells [1]. Io- dine enters the thyroid as iodide through an active process mediated by the Na 1 /I 2 symporter (NIS) lo- cated at the basal pole of the thyrocyte [2– 4]. Three crucial enzymes are colocalized within the apical plasma membrane and cooperate in the hormonogenic reaction: an iodide transporter corresponding to the product of the pendrin gene [5, 6], the TPO [1, 7], and the H 2 O 2 -generating system, which constitutes the limiting step of thyroid hormone synthesis [8]. The thyroid H 2 O 2 -generating system consists of Ca 21 -dependent flavoprotein presenting an NADPH oxidase activity. Its expression is stimulated by the cyclic-AMP pathway through the thyrotropin (TSH) 5 receptor and its enzymatic activity is essentially trig- gered by the Ca 21 -phosphatidylinositol cascade [9]. A flavoprotein has been partially purified from solubi- lized pig thyroid plasma membrane [10] and a partial cDNA coding for a 138-kDa truncated protein has been cloned [11]. Based on the functional homology with the leukocyte H 2 O 2 -generating system, we have cloned two new human thyroid-specific cDNAs. They code for 1551- and 1548-amino-acid proteins called ThOX1 and ThOX2, respectively [12] (also called DUOX1 and 2 [13] or LNOX1 and 2 [14]). ThOX1 and ThOX2 show 53 and 47% sequence similarity with the gp91 Phox protein, one of the catalytic subunits of the leukocyte NADPH oxidase complex. They also present 43% similarity along their first 500 amino acids with TPO, two poten- tial EF-hand motifs, five putative N-glycosylation sites, and high sequence similarities with gp91 Phox re- 1 These authors contributed equally to the work. 2 Fellow of the Fonds pour la Formation a ` la Recherche dans l’Industrie et l’Agriculture. 3 To whom correspondence and reprint requests should be ad- dressed at IRIBHN, Universite ´ Libre de Bruxelles, Campus Erasme, Bat. C, 808, Route de Lennik, B-1070 Brussels, Belgium. Fax: 32-2- 555 46 55. E-mail: xaddken@ulb.ac.be. 4 Fellow of the Bourse de la Fondation David et Alice Van Buuren. 5 Abbreviations used: TSH, thyrotropin hormone; PMA, phorbol 12-myristate 13-acetate; ThOX, thyroid oxidase; DPI, diphenylene- iodonium. 0014-4827/02 $35.00 187 © 2002 Elsevier Science (USA) All rights reserved. Experimental Cell Research 273, 187–196 (2002) doi:10.1006/excr.2001.5444, available online at http://www.idealibrary.com on