Purification of a novel flavoprotein involved in the thyroid NADPH oxidase- Cloning of the porcine and human cDNAs

Hydrogen peroxide is the final electron acceptor for the biosynthesis of th yroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocy tes, Pig and human thyroid plasma membrane contain a Ca2+-dependent NAD(P)H oxidase that generates H2O2 by transferring electrons from NAD(P)H to mole cular oxygen. We purified from pig thyroid plasma membrane a flavoprotein w hich constitutes the main, if not the sole, component of the thyroid NAD(P) H oxidase, Microsequences permitted the cloning of porcine and human full-l ength cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138(Tox)). Human and porcine p138( Tox) have 86.7% identity. The strongest similarity was to a predicted polyp eptide encoded by a Caenorhabditis cDNA and with rbohA, a protein involved in the Arabidopsis NADPH oxidase. p138(Tox) shows also similarity to the p6 5(Mox) and to the gp91(Phox) in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91(Phox), p138( Tox) shows an extended N-terminal containing two EF-hand motifs that may ac count for its calcium-dependent activity, whereas three of four sequences i mplicated in the interaction of gp91(Phox) With the p47(Phox) cytosolic fac tor are absent in p138(Tox). The expression of porcine p138(Tox) mRNA analy zed by Northern blot is specific of thyroid tissue and induced by cyclic AM P showing that p138(Tox) is a differentiation marker of thyrocytes. The gen e of human p138(Tox) has been localized on chromosome 15q15.