Title: Purification of a Novel Flavoprotein Involved in the Thyroid NADPH Oxidase
Abstract: Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. 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 molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138Tox). Human and porcine p138Tox have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a CaenorhabditiscDNA and with rbohA, a protein involved in theArabidopsis NADPH oxidase. p138Tox shows also similarity to the p65Mox and to the gp91Phox in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91Phox, p138Tox shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91Phox with the p47Phox cytosolic factor are absent in p138Tox. The expression of porcine p138Tox mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138Tox is a differentiation marker of thyrocytes. The gene of human p138Tox has been localized on chromosome 15q15. Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. 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 molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138Tox). Human and porcine p138Tox have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a CaenorhabditiscDNA and with rbohA, a protein involved in theArabidopsis NADPH oxidase. p138Tox shows also similarity to the p65Mox and to the gp91Phox in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91Phox, p138Tox shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91Phox with the p47Phox cytosolic factor are absent in p138Tox. The expression of porcine p138Tox mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138Tox is a differentiation marker of thyrocytes. The gene of human p138Tox has been localized on chromosome 15q15. thyroperoxidase thyrotropin 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate polymerase chain reaction rapid amplification of cDNA ends fluorescence in situ hybridization polyacrylamide gel electrophoresis kilobase base pair phenylarsine oxide The synthesis of thyroid hormone is catalyzed by thyroperoxidase (TPO)1 in the presence of H2O2 (1Taurog A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid. 7th Ed. Lippincott-Raven Publishers, Philadelphia1996: 47-81Google Scholar) on the apical membrane of the thyroid follicular cells (2Ekholm R. Mol. Cell. Endocrinol. 1981; 24: 141-163Crossref PubMed Scopus (101) Google Scholar). The thyroid H2O2generator is found in the apical plasma membrane of rat and pig open follicles (3Björkman U. Ekholm R. Denef J.F. J. Ultrastruct. Res. 1981; 74: 105-115Crossref PubMed Scopus (59) Google Scholar, 4Björkman U. Ekholm R. Endocrinology. 1984; 115: 392-398Crossref PubMed Scopus (102) Google Scholar), and is an NAD(P)H oxidase in the pig (5Virion A. Michot J.L. Dème D. Kaniewski J. Pommier J. Mol. Cell. Endocrinol. 1984; 36: 95-105Crossref PubMed Scopus (55) Google Scholar, 6Dupuy C. Kaniewski J. Dème D. Pommier J. Virion A. Eur. J. Biochem. 1989; 185: 597-603Crossref PubMed Scopus (51) Google Scholar, 7Nakamura Y. Ogihara S. Ohtaki S. J. Biochem. 1987; 102: 1121-1132Crossref PubMed Scopus (47) Google Scholar) and human (8Leseney A.M. Dème D. Lègue O. Ohayon R. Chanson P. Sales J.P. Carvalho D.P. Dupuy C. Virion A. Biochimie ( Paris ). 1999; 81: 373-380Crossref PubMed Scopus (57) Google Scholar) thyroid, as proposed by Björkman et al. (3Björkman U. Ekholm R. Denef J.F. J. Ultrastruct. Res. 1981; 74: 105-115Crossref PubMed Scopus (59) Google Scholar). The thyroid NAD(P)H oxidase requires micromolar concentrations of calcium to acquire a functional conformation and to generate H2O2 (7Nakamura Y. Ogihara S. Ohtaki S. J. Biochem. 1987; 102: 1121-1132Crossref PubMed Scopus (47) Google Scholar, 9Dème D. Virion A. Aı̈t-Hammou N. Pommier J. FEBS Lett. 1985; 186: 107-110Crossref PubMed Scopus (60) Google Scholar). Thyrotropin (TSH) induces the expression of thyroid NAD(P)H oxidase through a cAMP-dependent pathway in the dog (10Raspé E. Dumont J.E. Endocrinology. 1995; 136: 965-973Crossref PubMed Scopus (56) Google Scholar) and pig (11Carvalho D.P. Dupuy C. Gorin Y. Lègue O. Pommier J. Haye B. Virion A. Endocrinology. 1996; 137: 1007-1012Crossref PubMed Scopus (40) Google Scholar) thyrocytes, and transforming growth factor β counteracts the effect of TSH on pig cells (8Leseney A.M. Dème D. Lègue O. Ohayon R. Chanson P. Sales J.P. Carvalho D.P. Dupuy C. Virion A. Biochimie ( Paris ). 1999; 81: 373-380Crossref PubMed Scopus (57) Google Scholar). Electron transfer from NAD(P)H to molecular oxygen involves a membrane-bound flavoprotein (12Dème D. Doussière J. De Sandro V. Dupuy C. Pommier J. Virion A. Biochem. J. 1994; 301: 75-81Crossref PubMed Scopus (39) Google Scholar) of unknown structure. Two flavoproteins involved in a mammalian NAD(P)H oxidase system have been cloned to date. One is the glycosylated flavoprotein gp91Phox involved in the respiratory burst oxidase, also called “phagocyte oxidase” (for a review, see Ref. 13Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar). The second is the p65Mox, which participates in the activity of the “mitogenic oxidase” found in vascular smooth muscle cells (14Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1299) Google Scholar). Both require the small membrane subunit p22Phox for their activity (13Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 15De Keulenaer G.W. Alexander R.W. Ushio-Fukai M. Ishizaka N. Griendling K.K. Biochem. J. 1998; 329: 653-657Crossref PubMed Scopus (278) Google Scholar). A functional NAD(P)H oxidase, generating H2O2 in a Ca2+-dependent manner, has been solubilized by treating pig thyroid plasma membranes with detergents at a high salt concentration (16Gorin Y. Ohayon R. Carvalho D.P. Dème D. Leseney A.M. Haye B. Kaniewski J. Pommier J. Virion A. Dupuy C. Eur. J. Biochem. 1996; 240: 807-814Crossref PubMed Scopus (28) Google Scholar). The flavoprotein became unable to reduce molecular oxygen after the first step of its purification by octyl-Sepharose chromatography, suggesting that it requires one or more additional component(s) to generate H2O2 (16Gorin Y. Ohayon R. Carvalho D.P. Dème D. Leseney A.M. Haye B. Kaniewski J. Pommier J. Virion A. Dupuy C. Eur. J. Biochem. 1996; 240: 807-814Crossref PubMed Scopus (28) Google Scholar). However, the partially purified flavoprotein is still able to catalyze the NADPH-dependent reduction of the electron scavengers nitroblue tetrazolium and potassium ferricyanide (K3Fe(CN)6). We used this property to detect and quantify the flavoprotein activity during its purification (16Gorin Y. Ohayon R. Carvalho D.P. Dème D. Leseney A.M. Haye B. Kaniewski J. Pommier J. Virion A. Dupuy C. Eur. J. Biochem. 1996; 240: 807-814Crossref PubMed Scopus (28) Google Scholar). We have now purified the thyroid oxidase flavoprotein as a first step in defining the molecular structure of the enzyme. Peptide microsequences were used to design a gene-specific primer for rapid amplification of the 3′ cDNA ends (3′-RACE). RACE by PCR provided full-length porcine and human cDNAs encoding a protein called p138Tox, which comprises 1207 (porcine) and 1210 (human) amino acids. p138Tox is similar to a predicted 1506-amino acids protein encoded by a Caenorhabditis elegans gene and with other flavoproteins involved in plant and mammalian NADPH oxidases. The p138Tox contains two Ca2+-binding motifs (EF-hand) which could be involved in the control of the thyroid NADPH oxidase by Ca2+. We also find that p138Tox is a new specific marker of thyrocyte differentiation. Finally, the gene of human p138Tox was located by FISH on chromosome 15q15. Forskolin and bovine serum albumin were from Calbiochem (La Jolla, CA); Triton X-100 was purchased from Serva (Heidelberg, Germany); FAD-agarose and CHAPS were from Sigma; trypsin and newborn calf serum were purchased from Life Technologies, Inc. (Gaithersburg, MD); [32P]dCTP (3000 Ci/mmol) was from DuPont NEN (Les Ulis, France); oligonucleotides were synthesized by Eurogentec Bel SA. Human thyroid Marathon-Ready cDNAs and Advantage 2 Taq Polymerase Mix were from CLONTECH(Palo Alto, CA). Restriction enzymes and T4 DNA ligase were from Ozyme (Montigny-le-Bretonneux, France). Endoproteinase Lys-C was from Roche Molecular Biochemicals (Meylan, France). NADPH oxidase was solubilized from pig thyroid plasma membrane, and the flavoprotein partially purified by chromatography on octyl-Sepharose CL 4B (16Gorin Y. Ohayon R. Carvalho D.P. Dème D. Leseney A.M. Haye B. Kaniewski J. Pommier J. Virion A. Dupuy C. Eur. J. Biochem. 1996; 240: 807-814Crossref PubMed Scopus (28) Google Scholar) except that FAD was omitted from the washing buffer containing CHAPS and from the elution buffer containing 1% Triton X-100. Batches of eluted protein were stirred overnight at 4 °C with 350 μl of FAD-agarose equilibrated with 50 mm sodium phosphate, pH 7.2, containing 2 mm MgCl2, 0.25 msucrose, 1 mm NaN3, 0.5 mmCaCl2, 0.4 mm EGTA, 0.5 mmdithiothreitol, 0.1 m KCl, and 0.2% Triton X-100 (buffer A). 10 mm CHAPS was then added to the suspension, which was again gently stirred overnight at 4 °C. The mix was then centrifuged (2000 × g, 30 min at 4 °C). The FAD-agarose pellet was washed twice with 3 ml of buffer B (buffer A plus 10 mmCHAPS) and once with 1.5 ml of the same buffer and suspended in SDS-gel sample buffer (1.7% final) (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (218042) Google Scholar). The suspension was mixed gently overnight at room temperature and centrifuged. Proteins eluted by SDS-gel sample buffer were recovered in the supernatant. Analytical SDS-PAGE (8% acrylamide) was performed essentially as described by Laemmli (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (218042) Google Scholar). Gels were stained with 0.003% Amido Black in 45% methanol, 10% acetic acid and then extensively washed in distilled water. The band of interest (3.5 μg of protein) was removed from the gel, cut into small pieces, dried under vacuum, and incubated in 50 mm Tris (pH 8.6), 0.03% SDS, 0.2 μg/ml endoproteinase Lys-C (overnight at 35 °C) for amino acid sequencing. The supernatant volume was reduced to 200 μl under vacuum and injected onto an AX300 (2.1 × 30 mm) precolumn (Perkin-Elmer) followed by a 218TP52 Vydac reversed-phase high performance liquid chromatography column (2.1 × 250 mm). Peptides were eluted with a 2–35% (v/v) linear gradient of 0.1% trifluoroacetic acid in acetonitrile over 90 min at a flow rate of 200 μl/min. The major peaks were sequenced. RACE was performed using the SMART RACE cDNA amplification kit (CLONTECH, Palo Alto, CA) according to the protocol of the supplier with 1 μg total mRNA from porcine thyrocytes that had been cultivated for 4 days with 10 μm forskolin or with human thyroid Marathon-Ready cDNAs. PCR products were cloned in pCR-XL-TOPO (Invitrogen, Inc.) for sequencing. Sequencing was performed three times in the 5′ to 3′ direction and three times in the reverse direction. The porcine thyrocytes (2 × 106 cells/ml) were isolated by discontinuous trypsinization (18Haye B. Aublin J.L. Champion S. Lambert B. Jacquemin C. Mol. Cell. Endocrinol. 1985; 43: 41-50Crossref PubMed Scopus (31) Google Scholar) and cultivated at 37 °C in a 5% CO2, 95% air, water-saturated atmosphere as reconstituted follicles in suspension in 10-cm diameter untreated plastic Petri dishes (Falcon, Oxnard, CA). The cells were grown for 4 days in Eagle's minimal essential medium (pH 7.4) containing 10% (v/v) calf serum, penicillin (200 units/ml), and streptomycin sulfate (50 μg/ml), with or without 10 μm forskolin. The method of Chomczynski and Sacchi (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (66730) Google Scholar) was used to extract RNA from all cells and porcine tissues. For Northern blot analysis, total RNA (20 μg) was denaturated and electrophoresed in a 1% agarose gel containing formaldehyde. Denaturated RNAs were transferred by diffusion blotting onto a nylon membrane (Stratagene, La Jolla, CA) using 20 × SSC (1 × SSC = 0.15 m NaCl, 15 mm sodium citrate). The membrane was first incubated in 0.25 m sodium phosphate buffer (pH 6.8) containing 1 mm EDTA, 7% SDS, 10 mg/ml bovine serum albumin for 4 h at 65 °C and then was hybridized overnight at 65 °C with the heat-denatured probe cDNA. The cDNA probe was α-32P-labeled by random priming extension. The membrane was washed three times for 20 min in 2 × SSC, 0.1% SDS at 65 °C and then was autoradiographed at −80 °C using Hyperfilm MP (Amersham Pharmacia Biotech). The cDNA probe used was the porcine 5′-RACE cDNA EcoR1 insert (4 kb) of pCR-XL-TOPO. The sense and antisense 5′-RACE PCR primers (Eurogentec Bel SA) were 5′-CCCACACACACTTCATCTGGCACAGCCTGC-3′ and 5′-TGGCACAGCCTCTGAGCAGTTCTTCTC-3′. The probe used to locate the p138Tox gene was the pCR-XL-TOPO vector containing the human 4-kb product of the 5′-RACE. The probe preparation and FISH conditions were essentially as described previously (20Valent A. Meddeb M. Danglot G. Duverger A. Nguyen V.C. Bernheim A. Hum. Genet. 1996; 98: 12-15Crossref PubMed Scopus (7) Google Scholar). Labeled probe (40 ng) with competitor (120 ng of human placental DNA) were denatured and placed directly on the slides without incubation at 37 °C. The signal was detected as described by Valent et al. (21Valent A. Danglot G. Bernheim A. Eur. J. Hum. Genet. 1997; 5: 102-104Crossref PubMed Scopus (39) Google Scholar). Porcine flavoproteins bound to the FAD-agarose and eluted with SDS migrated in SDS-PAGE as a single major band of apparent molecular mass of 180 kDa, and two diffuse bands with apparent molecular masses of 150 and 130 kDa (Fig. 1). Digestion of the major 180-kDa protein (see “Experimental Procedures”) with endoproteinase Lys-C generated two peptides, peptide A: AVVPPPRLYTEALQEK, and peptide B: X(V)QLIN(R/P)QDQ(T)HFVHHYEN(P), whose sequences were not found in protein data bases. Peptide B was used to design a gene-specific primer for the 3′-RACE. We carried out 5′-RACE and then amplified porcine and human full-length cDNAs by reverse transcriptase-PCR. These were cloned and sequenced. The porcine cDNA (GenBankTMaccession number AF181973) is 4996-bp long, and the human cDNA (GenBankTM accession number AF181972) has 5160 bp; they are 78.1% identical (93.5% in coding region). The open reading frame (porcine: 3621 bp; human: 3630 bp) encodes a predicted protein with a theoretical molecular mass of 138 kDa. Kosak sequences (A/G)CCATGG were present at the translation start codon (porcine: CCACCATGG; human: CTACCATGG). The predicted protein, named p138Tox for “p138 thyroid-oxidase”, contains peptides A and B, confirming the cloning of the purified 180-kDa flavoprotein. Human and porcine p138Tox are 86.7% identical and contain 1210 and 1207 amino acids, respectively. p138Tox contained three main domains (Fig. 2 A). The N-terminal region had similarities to peroxidases. Residues 49–206 of the human p138Tox are 27% identical to a region of pig TPO that excludes the heme-linked proximal and distal histidines. There are four putative sites of N-glycosylation on the human p138Tox and three on the pig protein (Figs. 2 Aand 3 A), all in a predicted extracellular N-terminal domain. The difference between the 138-kDa theoretical molecular mass and the 180-kDa apparent mass of the flavoprotein could reflect the presence of complex sugars at these N-glycosylation sites. A median domain is similar to the Ca2+-binding proteins containing Ca2+ EF-hand motifs. The C-terminal domain is similar to gp91Phox and p65Mox, the large subunits of phagocyte (13Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar) and vascular smooth muscle cell NADPH oxidases (14Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1299) Google Scholar).FIG. 3Predicted amino acid sequences of human and porcine p138Tox and similarity to human gp91Phox. A, amino acid sequence alignments of porcine and human p138Tox N-terminal regions.Open boxes denote conserved amino acid residues. EF-hand motifs are underlined with continuous lines. Microsequenced peptide A is underlined with a dotted line. Asterisks denote potentialN-glycosylation sites. Adjacent cysteinyl residues are in bold. B, amino acid sequence alignments of porcine and human p138Tox C-terminal region and the whole human gp91Phox. Microsequenced peptides A and B areunderlined with dotted lines. Boxesdenote conserved amino acid residues. Dashed boxes indicate conserved histidine residues (in bold) potentially involved in heme binding and in the FAD- and NADPH-binding sites. Motifs involved in p47Phox binding are underlined withcontinuous lines. Adjacent cysteinyl residues in gp91Phox are in bold. Asterisksdenote potential N-glycosylation sites of gp91Phox.View Large Image Figure ViewerDownload (PPT) The overall sequence of p138Tox is very similar to that of a predicted protein fromC. elegans (GenBankTM accession numberAF043697). In addition to its glycosylated N terminus being similar to peroxidases, the Caenorhabditis protein has two EF-hand motifs in the middle of the primary structure (Fig. 2 A) which are conserved in p138Tox(494DKDGNGYLSFREF506 and530DLDENGFLSKDEF542 in human sequence). These Ca2+-binding sites could be involved in the direct activation of the thyroid H2O2 generator by Ca2+. The flavoprotein p138Tox is also similar to rbohA, a homolog of gp91Phox involved in the respiratory burst oxidase of Arabidopsis thaliana, which also contains two Ca2+-binding EF-hand motifs in its hydrophilic N-terminal domain (22Keller T. Damude H.G. Werner D. Doerner P. Dixon R.A. Lamb C. Plant Cell. 1998; 10: 255-266PubMed Google Scholar). Hydrophilicity plots obtained by the method of Kyte and Doolittle (23Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (18532) Google Scholar) showed similar distributions of seven hydrophobic stretches inCaenorhabditis protein and in p138Tox, indicating that the proteins share a well conserved tertiary structure (Fig. 2 B). The first five hydrophobic stretches are probably membrane-spanning domains, according to the TMpred program (24Hofmann K. Stoffel W. Biol. Chem. Hoppe-Seyler. 1993; 374: 166Google Scholar), with the glycosylated N-terminal outside the cell and the EF-hand motifs logically located inside. However, p138Tox is also a truncated mammalian version of the Caenorhabditis protein, which has an additional 331 amino acids at its N terminus.Arabidopsis rbohA has a similar, but even shorter, N terminus (Fig. 2 A), so that the first membrane-spanning domain is absent as well as the peroxidase-like glycosylated domain. The C-terminal regions of p138Tox, rbohA, and the Caenorhabditis protein are very similar to the gp91Phox and p65Mox, with a similar distribution of six hydrophobic stretches. An isoalloxazine FAD-binding motif of gp91Phox(338HPFTLTS344) is conserved in human (978–984) and porcine (975–981) p138Tox as shown (Fig. 3 B) by the multiple alignment of human and porcine p138Tox sequences with human gp91Phox sequence made with the CLUSTALW program (25Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56741) Google Scholar). The ribose (410GIGVTPF416) and adenine (534VFXCGP539) NADPH-binding motifs of gp91Phox are also found in the C-terminal region of human (1048–1054) and porcine (1045–1051) p138Tox and should lie in a cytosolic domain. The four histidine residues, which are probably involved in heme binding in gp91Phox are conserved in p138Tox. Whereas the FAD and NADPH binding sites of gp91Phox are clearly present in p138Tox, alignment of the two sequences indicates some differences. For example, the N-glycosylation sites of p65Mox and gp91Phox are absent from the p138Tox, rbohA, and Caenorhabditis protein C-terminal regions. p138Tox also differs from gp91Phox in some other sequence features, which could be relevant to the different specific mechanisms involved in their activation. Previous biochemical studies (7Nakamura Y. Ogihara S. Ohtaki S. J. Biochem. 1987; 102: 1121-1132Crossref PubMed Scopus (47) Google Scholar, 9Dème D. Virion A. Aı̈t-Hammou N. Pommier J. FEBS Lett. 1985; 186: 107-110Crossref PubMed Scopus (60) Google Scholar) showed that micromolar concentrations of Ca2+ were necessary and sufficient to cause complete, reversible activation of thyroid oxidase, suggesting that interaction of the flavoprotein with cytosolic factors such as p47Phoxand p67Phox is not required, unlike the respiratory burst oxidase. Indeed, three of the four motifs involved in p47Phox binding to gp91Phox subunit are not present in p138Tox (Fig. 3 B). Furthermore, the tervalent arsenoxide phenylarsine oxide (PAO), a hydrophobic molecule that reacts with two vicinal thiol groups in proteins, alters the structure of the porcine thyroid oxidase so that electron transfer occurs more slowly but without Ca2+ (26Gorin Y. Leseney A.M. Ohayon R. Dupuy C. Pommier J. Virion A. Dème D. Biochem. J. 1997; 321: 383-388Crossref PubMed Scopus (24) Google Scholar). The porcine and human p138Tox contain only two adjacent cysteinyl residues (Fig. 3 A), which are located in the first predicted membrane-spanning domain (268C and 269C), just before the intracellular segment containing the two EF-hand motifs. They are probably the binding site of PAO, reinforcing the idea that this region, absent from gp91Phox, is crucial for the control of the thyroid NAD(P)H oxidase activity by Ca2+. PAO also affects the respiratory burst oxidase, but in a different manner, because it essentially prevents the human (27Le Cabec V. Maridonneau-Parini I. J. Biol. Chem. 1995; 270: 2067-2073Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and bovine (28Doussière J. Poinas A. Blais C. Vignais P.V. Eur. J. Biochem. 1998; 251: 649-658Crossref PubMed Scopus (42) Google Scholar) oxidase activation by cytosolic factors through its binding to gp91Phox. This particular effect could result from the binding of PAO to the only two adjacent cysteinyl residues of gp91Phox, located just before the first peptide involved in activation by p47Phox(85CCSTRVRRQL94), a sequence which is absent from p138Tox (Fig. 3 B). Fig. 4 A shows that the 5-kb p138Tox mRNA was detected by Northern blot only in thyroid tissue. This strongly supports the idea that the cloned flavoprotein actually participates in the specific function of thyrocytes, the synthesis of thyroid hormones, as an essential component the H2O2 generator associated with TPO. The thyroid-specific expression of the p138 mRNA also justifies the designation of the NADPH oxidase as “thyroid oxidase” or “tox.” Fig. 4 B shows that porcine thyrocytes cultured without forskolin do not contain the 5-kb p138ToxmRNA, whereas its concentration is maintained, or even enhanced, by cyclic AMP. These data are in good agreement with those of Raspéand Dumont (10Raspé E. Dumont J.E. Endocrinology. 1995; 136: 965-973Crossref PubMed Scopus (56) Google Scholar), who reported that TSH induces H2O2 generator activity in dog thyrocytes by activating the adenylate cyclase cascade. They also corroborate our results showing that the TSH and/or forskolin stimulate NADPH oxidase activity in pig thyrocytes (11Carvalho D.P. Dupuy C. Gorin Y. Lègue O. Pommier J. Haye B. Virion A. Endocrinology. 1996; 137: 1007-1012Crossref PubMed Scopus (40) Google Scholar), which seems to be correlated with a higher expression of the flavoprotein (16Gorin Y. Ohayon R. Carvalho D.P. Dème D. Leseney A.M. Haye B. Kaniewski J. Pommier J. Virion A. Dupuy C. Eur. J. Biochem. 1996; 240: 807-814Crossref PubMed Scopus (28) Google Scholar). FISH was used to locate the p138Tox gene in the human genome (Fig. 5). We analyzed 50 normal metaphases and detected no fluorescent signal in 10 metaphases because of FISH efficiency. But a fluorescent signal was observed at chromosomal band 15q15 in 40 of them. The fluorescent signal was present on only one of the two chromosomes 15 in 10% of positive metaphases, due again to the efficiency of FISH or to the detection procedure used. 15q15 is not presently considered to be a susceptibility locus for any thyroid disease. We have demonstrated that the thyroid NAD(P)H oxidase is different from other known mammalian NADPH oxidases. Its flavoprotein is not only a new homolog of gp91Phox and p65Mox, but it also has specific structural features that can account for its biochemical properties. Among them is the presence of two calcium binding EF-hand motifs, which have only been found in non-mammalian NADPH oxidases to date. Its tissue specificity and cAMP-regulated expression makes p138Tox a new marker of thyrocytes, which could be relevant in thyroid disorders. We thank Dr. J. P. Blondeau for critical reading of the manuscript and helpful discussions, Prof. B. Haye and O. Legué for kindly providing the dispersed porcine thyrocytes, and J. d'Alayer (Laboratoire de Microséquençage des protéines, Institut Pasteur) for microsequencing peptides of the porcine flavoprotein. The English text was edited by Dr. O. Parkes.