Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family

Two cDNAs encoding NADPH oxidases and constituting the thyroid H2O2 generat ing system have been cloned. The strategy of cloning was based on the funct ional similarities between H2O2 generation in leukocytes and the thyroid, a ccording to the hypothesis that one of the components of the thyroid system would belong to the gp(91Phox)/Mox1 gene family and display sequence simil arities with gp91(Phox). Screening at low stringency with a gp91(Phox) prob e of cDNA libraries from thyroid cells in primary culture yielded two disti nct human cDNA clones harboring open reading frames of 1551 (ThOX1) and 154 8 amino acids (ThOX2), respectively. The encoded polypeptides display 83% s equence similarity and are clearly related to gp91(Phox) (53 and 47% simila rity). The theoretical molecular mass of 177 kDa is close to the apparent m olecular mass of 180 kDa of the native corresponding porcine flavoprotein a nd 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. The y show along their first 500 amino acids a similarity of 43% with thyropero xidase. The corresponding genes of ThOX1 and ThOX2 are closely linked on ch romosome 15q15.8, The dog mRNA expression is thyroid-specific and up-regula ted by agents activating the cAMP pathway as is the synthesis of the polype ptides they are coding for. In human thyroid the positive regulation by cAM P is less pronounced. The proteins ThOX1 and ThOX2 accumulate at the apical membrane of thyrocytes and are co-localized with thyroperoxidase.