Identification of the Maturation Factor for Dual Oxidase
Dual oxidase 2 (DUOX2), an NADPH:O2 oxidoreductase flavoprotein, is a component of the thyroid H2O2 generator crucial for hormone synthesis at the apical membrane. Mutations in DUOX2 produce congenital hypothyroidism in humans. However, no functional DUOX-based NADPH oxidase has ever been reconstituted at the plasma membrane of transfected cells. It has been proposed that DUOX retention in the endoplasmatic reticulum (ER) of heterologous systems is due to the lack of an unidentified component required for functional maturation of the enzyme. By data mining of a massively parallel signature sequencing tissue expression data base, we identified an uncharacterized gene named DUOX maturation factor (DUOXA2) arranged head-to-head to and co-expressed with DUOX2. A paralog (DUOXA1) was similarly linked to DUOX1. The genomic rearrangement leading to linkage of ancient DUOX and DUOXA genes could be traced back before the divergence of echinoderms. We demonstrate that co-expression of DUOXA2, an ER-resident transmembrane protein, allows ER-to-Golgi transition, maturation, and translocation to the plasma membrane of functional DUOX2 in a heterologous system. The identification of DUOXA genes has important implications for studies of the molecular mechanisms controlling DUOX expression and the molecular genetics of congenital hypothyroidism. Dual oxidase 2 (DUOX2), an NADPH:O2 oxidoreductase flavoprotein, is a component of the thyroid H2O2 generator crucial for hormone synthesis at the apical membrane. Mutations in DUOX2 produce congenital hypothyroidism in humans. However, no functional DUOX-based NADPH oxidase has ever been reconstituted at the plasma membrane of transfected cells. It has been proposed that DUOX retention in the endoplasmatic reticulum (ER) of heterologous systems is due to the lack of an unidentified component required for functional maturation of the enzyme. By data mining of a massively parallel signature sequencing tissue expression data base, we identified an uncharacterized gene named DUOX maturation factor (DUOXA2) arranged head-to-head to and co-expressed with DUOX2. A paralog (DUOXA1) was similarly linked to DUOX1. The genomic rearrangement leading to linkage of ancient DUOX and DUOXA genes could be traced back before the divergence of echinoderms. We demonstrate that co-expression of DUOXA2, an ER-resident transmembrane protein, allows ER-to-Golgi transition, maturation, and translocation to the plasma membrane of functional DUOX2 in a heterologous system. The identification of DUOXA genes has important implications for studies of the molecular mechanisms controlling DUOX expression and the molecular genetics of congenital hypothyroidism. Generation of H2O2 at the apical membrane of thyroid follicular cells is essential for iodination of thyroglobulin by thyroid peroxidase and constitutes the rate-limiting step of thyroid hormone synthesis. Dual oxidases (DUOX1 and DUOX2) 2The abbreviations used are: DUOX, dual (domain) oxidase; BLAST, basic local alignment search tool; DPI, diphenyleneiodonium; EGFP, enhanced green fluorescent protein; ER, endoplasmatic reticulum; EST, expressed sequence tag; HA, hemagglutinin; MPSS, massively parallel signature sequencing; ORF, open reading frame; PNGase F, N-glycosidase F; Endo H, endoglycosidase H; contig, group of overlapping clones. appear to constitute the catalytic core of the H2O2 generator (1Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 2De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). They are large homologs of the phagocyte gp91phox/Nox2 NADPH-dependent oxidase with an N-terminal extension comprising a peroxidase-like domain. Although the crucial role of DUOX2 in thyroid hormonogenesis has been substantiated by reports of severe congenital hypothyroidism in patients with biallelic nonsense mutations (3Moreno J.C. Bikker H. Kempers M.J. van Trotsenburg A.S. Baas F. de Vijlder J.J. Vulsma T. Ris-Stalpers C. N. Engl. J. Med. 2002; 347: 95-102Crossref PubMed Scopus (398) Google Scholar), the understanding of structure, function, and regulation of DUOX has remained limited. The major obstacle for molecular studies of DUOX is the lack of a suitable heterologous cell system for DUOX-based functional NADPH oxidase expression. Transfected cells completely retain DUOX in the endoplasmatic reticulum (ER) (4De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar, 5Morand S. Chaaraoui M. Kaniewski J. Deme D. Ohayon R. Noel-Hudson M.S. Virion A. Dupuy C. Endocrinology. 2003; 144: 1241-1248Crossref PubMed Scopus (67) Google Scholar, 6Morand S. Agnandji D. Noel-Hudson M.S. Nicolas V. Buisson S. Macon-Lemaitre L. Gnidehou S. Kaniewski J. Ohayon R. Virion A. Dupuy C. J. Biol. Chem. 2004; 279: 30244-30251Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 7Wang D. De Deken X. Milenkovic M. Song Y. Pirson I. Dumont J.E. Miot F. J. Biol. Chem. 2005; 280: 3096-3103Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Ameziane-El-Hassani R. Morand S. Boucher J.L. Frapart Y.M. Apostolou D. Agnandji D. Gnidehou S. Ohayon R. Noel-Hudson M.S. Francon J. Lalaoui K. Virion A. Dupuy C. J. Biol. Chem. 2005; 280: 30046-30054Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), suggesting that an unidentified component, essential for DUOX maturation, may be specifically expressed in tissues containing the functional enzyme. Data Mining and Computational Analysis—Massively parallel signature sequencing (MPSS) data (9Jongeneel C.V. Delorenzi M. Iseli C. Zhou D. Haudenschild C.D. Khrebtukova I. Kuznetsov D. Stevenson B.J. Strausberg R.L. Simpson A.J. Vasicek T.J. Genome Res. 2005; 15: 1007-1014Crossref PubMed Scopus (133) Google Scholar) were obtained from the NCBI Gene Expression Omnibus repository (www.ncbi.nlm.nih.gov/geo/; records GSE1747 and GPL1443). A thyroid specificity score, as defined by Jongeneel et al. (9Jongeneel C.V. Delorenzi M. Iseli C. Zhou D. Haudenschild C.D. Khrebtukova I. Kuznetsov D. Stevenson B.J. Strausberg R.L. Simpson A.J. Vasicek T.J. Genome Res. 2005; 15: 1007-1014Crossref PubMed Scopus (133) Google Scholar), was calculated for signatures with frequency >100 tags per million (∼30 mRNA copies/cell) in the thyroid/parathyroid library. Tags with scores >–1 were mapped to the human genome assembly using BLAST. DUOXA homologs were identified by tBLASTn searches against the NCBI nr data base and trace archive and BLAT queries (at genome.ucsc.edu/) against assembled whole genome sequences. Orthologs were operationally defined as reciprocal best BLAST hits. Gene structures were deduced by spliced alignment maintaining maximum homolog similarity of the open reading frames (ORFs) and consensus splice junctions. Cladograms were constructed from ClustalX alignments (BLOSUM weight matrix, excluding gaps) using the Jones, Taylor, and Thornton (JTT) substitution model in PHYML 2.4.4 (10Guindon S. Gascuel O. Syst. Biol. 2003; 52: 696-704Crossref PubMed Scopus (14311) Google Scholar). SignalP 3.0 (11Bendtsen J.D. Nielsen H. von Heijne G. Brunak S. J. Mol. Biol. 2004; 340: 783-795Crossref PubMed Scopus (5660) Google Scholar) and Phobius (12Kall L. Krogh A. Sonnhammer E.L. J. Mol. Biol. 2004; 338: 1027-1036Crossref PubMed Scopus (1677) Google Scholar) were used to analyze signal peptides, transmembrane helices, and topology. Northern Blot Analysis—A human multiple tissue Northern blot (Origene) was hybridized with DUOXA2 (125–470 of DQ489734) and DUOXA1 (1244–1623 of BC020841) probes. Heterologous Expression of DUOX2 and DUOXA2 Constructs—cDNA was synthesized with Superscript reverse transcriptase (Invitrogen) by oligo(dT) priming of total RNA from a normal human thyroid gland. The DUOX2 and DUOXA2 ORFs were amplified using native Pfu polymerase (Stratagene) and cloned into pcDNA3.1 (Invitrogen). Epitope-tagged constructs and fusions with enhanced green fluorescent protein (EGFP) were prepared by replacement or splicing-by-overlap extension using specifically designed primers. All constructs were verified by sequencing. HeLa cells were cultured and transfected as described (13Grasberger H. Ringkananont U. Lefrancois P. Abramowicz M. Vassart G. Refetoff S. Mol. Endocrinol. 2005; 19: 1779-1791Crossref PubMed Scopus (72) Google Scholar). Confocal Laser Scanning Microscopy—Indirect immunofluorescence of permeabilized cells has been described previously (13Grasberger H. Ringkananont U. Lefrancois P. Abramowicz M. Vassart G. Refetoff S. Mol. Endocrinol. 2005; 19: 1779-1791Crossref PubMed Scopus (72) Google Scholar). For surface staining, cells were incubated with rat anti-HA clone 3F10 and/or mouse anti-c-myc clone 9E10 (both from Roche Applied Science) at 1 μg/ml in Hank's buffered saline solution/10 mm Hepes, pH 7.4, 1% bovine serum albumin at 4 °C. Rabbit anti-calnexin was obtained from StressGen. Images were captured on a Nikon Eclipse E800 equipped with PCM2000. Analysis of N-Glycosylation—Postnuclear supernatants (in 50 mm Tris/HCl, pH 8.0, 150 mm NaCl, and proteinase inhibitors) were adjusted to 0.5% SDS, 0.4 mm dithiotreitol and denatured, at room temperature, for 30 min. Samples were deglycosylated with N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) (both from New England Biolabs) according to manufacturer's recommendations, followed by SDS-PAGE under reducing conditions and Western blotting as described (13Grasberger H. Ringkananont U. Lefrancois P. Abramowicz M. Vassart G. Refetoff S. Mol. Endocrinol. 2005; 19: 1779-1791Crossref PubMed Scopus (72) Google Scholar). Measurement of H2O2 Generation—Release of H2O2 was determined by reaction with cell-impermeable 10-acetyl-3,7-dihydroxyphenoxazine (14Zhou M. Diwu Z. Panchuk-Voloshina N. Haugland R.P. Anal. Biochem. 1997; 253: 162-168Crossref PubMed Scopus (1126) Google Scholar) (Amplex Red reagent, Invitrogen) in the presence of excess peroxidase, producing fluorescent resorufin. Briefly, cell monolayers were incubated, with or without 10 μm diphenyleneiodonium (DPI), in Dulbecco's phosphate-buffered saline supplemented with 50 μm Amplex Red reagent and 0.1 unit/ml horseradish peroxidase for 1 h at 37°C. Relative fluorescence units (excitation/emission: 535/595) were corrected for Amplex Red oxidation in wells containing non-transfected cells and converted into H2O2 concentrations using a calibration curve. Renilla luciferase activity from co-transfected pRL-Tk plasmid (Promega) was used as internal control as described (13Grasberger H. Ringkananont U. Lefrancois P. Abramowicz M. Vassart G. Refetoff S. Mol. Endocrinol. 2005; 19: 1779-1791Crossref PubMed Scopus (72) Google Scholar). Identification of Novel Genes in the DUOX1/DUOX2 Intergenic Region—We used MPSS data for 32 normal human tissues (9Jongeneel C.V. Delorenzi M. Iseli C. Zhou D. Haudenschild C.D. Khrebtukova I. Kuznetsov D. Stevenson B.J. Strausberg R.L. Simpson A.J. Vasicek T.J. Genome Res. 2005; 15: 1007-1014Crossref PubMed Scopus (133) Google Scholar) to identify novel transcripts with predominant expression in thyroid gland. One of the extracted tags mapped to an uncharacterized locus (LOC405753) oriented head-to-head to DUOX2 in the ∼16-kbp DUOX1/DUOX2 intergenic region. For reasons outlined below, we called the corresponding gene DUOX maturation factor 2 (DUOXA2). 3The gene name and symbol have been approved by the HUGO Gene Nomenclature Committee. Based on human-mouse homology (Riken clone 9030623N16Rik), and supported by contig assembly of expressed sequence tags (ESTs), it comprises six exons, confirmed by reverse transcription-PCR amplification from human thyroid tissue (GenBank™ accession number DQ489734). The putative transcription start site defined by clone DKFZp686C04213 maps to a GpC rich region (Fig. 1A). This site is 135 bp from the 5′ terminus of a spliced DUOX2 EST (BI045475) on the opposite strand. A single polyadenylation signal (Fig. 1A) is supported by all mapped 3′ ESTs. We confirmed a specific transcript of the expected size (1.3 kbp) by Northern blot analysis (Fig. 1B), which also validated the MPSS-based expression profiling: DUOXA2 mRNA was by far most abundant in thyroid, with lower levels in salivary glands reflecting the known expression profile of DUOX2 (1Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 2De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 15Geiszt M. Witta J. Baffi J. Lekstrom K. Leto T.L. FASEB J. 2003; 17: 1502-1504Crossref PubMed Scopus (419) Google Scholar). The DUOXA2 ORF is initiated within a Kozak consensus (gcagcATGa) and spans all six exons. The encoded 320-amino acid protein was strongly predicted to comprise five membrane-integral regions, including a reverse signal anchor with external N terminus (type III) (Fig. 1C). The three NX(S/T) consensus sites for N-glycosylation are clustered within an extended external loop connecting the second and third transmembrane helices. We identified a single DUOXA2 paralog in the human genome. We will refer to this locus, annotated as “homolog of Drosophila Numb-interacting protein,” as DUOXA1. It is immediately adjacent, in tail-to-tail orientation to DUOXA2 and extends, via untranslated exons, into the DUOX1 promoter region. DUOXA1 mRNA was predominantly expressed in thyroid gland and, at lower level, in esophagus (Fig. 1B). Two transcripts of ∼2.9 and ∼3.5 kbp were detected, compatible with alternative splicing of 5′-untranslated exons and the use of alternative 3′-polyadenylation signals (data not shown). The DUOXA1 ORF was confirmed by sequencing from human thyroid cDNA (GenBank™ accession number DQ489735). By spliced alignment, we deduced the gene structures of all DUOXA homologs in 10 other vertebrate whole genome assemblies. The splicing sites of all structures were conserved at the single codon level (exon alignment shown in Fig. 2S in the on-line supplement). Remarkably, the bidirectional DUOX/DUOXA arrangement was conserved throughout the vertebrate lineage (Fig. 1E, accession numbers of genomic contigs available in Table 4S in the on-line supplement). Teleosts have a single DUOX/DUOXA arrangement, which has undergone tandem duplication to an inverted repeat (DUOX2/DUOXA2/DUOXA1/DUOX1) before the amphibian divergence. Analyzing unassembled genomic contigs, we mapped the evolutionary event leading to the bidirectional association of DUOX and DUOXA before the divergence of echinoderms, since linkage of the loci was present in Strongylocentrotus purpuratus. Thus, conserved microsynteny in deuterostomes was a strong predictor for cooperation between DUOX and DUOXA. The protostomes C. elegans and D. melanogaster lack a DUOXA homolog in the vicinity of their respective duox loci. They do, however, each harbor a single ancient DUOXA homolog. For instance, Drosophila moladietz (mol) encodes a 474-amino acid protein that exhibits 39% amino acid identity over 256 amino acids with human DUOXA1. Functional Rescue of DUOX2 by DUOXA2—To test whether DUOXA2 can reconstitute DUOX2 activity in a heterologous system, we expressed either DUOX2, DUOXA2, or both in HeLa cells and measured H2O2 released into the culture medium. Transfection of either DUOX2 or DUOXA2 alone did not result in increased H2O2 generation compared with nontransfected cells, confirming previous results for DUOX2 (2De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 4De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar). Remarkably, co-transfection of DUOX2 with DUOXA2 rescued DUOX2 activity as indicated by the significant amounts of H2O2 released from the cells (Fig. 2A). The H2O2 release triggered by DUOX2/DUOXA2 co-transfection was completely blocked by the flavoprotein inhibitor DPI (Fig. 2A). Co-expression of DUOXA2 Permits ER-Exit of DUOX2 and Plasma Membrane Targeting via the Secretory Pathway—Lack of DUOX2 activity in heterologous systems has been associated with absence of DUOX2 at the plasma membrane (4De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar). To directly test whether reconstitution of active DUOX2 by DUOXA2 is indeed due to translocation of DUOX2 to the plasma membrane, we HA-tagged DUOX2 at its extracellular domain (HA-DUOX2; tag inserted between Asp27 and Ala28). Non-permeabilized cells showed strong anti-HA plasma membrane signals in cells co-transfected with HA-DUOX2 and DUOXA2 (Fig. 2B). Untransfected cells, or cells transfected with either DUOXA2 or HA-DUOX2 alone, were devoid of surface fluorescence (Fig. 2B and data not shown). To determine whether DUOXA2-induced surface expression of DUOX2 involved ER-to-Golgi transition of DUOX2, we analyzed the maturation of DUOX2 N-glycan moieties using specific glycosidases. Whereas all N-glycans are cleavable by PNGase F, the ER-derived N-glycans to Endo H have been by HA-DUOX2 expressed in HeLa cells as a single on SDS-PAGE and was to by Endo H, with data (4De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar). co-transfection with DUOXA2 in the of a second DUOX2 with and to by Endo H (Fig. previously obtained with DUOX2 protein (4De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar, 5Morand S. Chaaraoui M. Kaniewski J. Deme D. Ohayon R. Noel-Hudson M.S. Virion A. Dupuy C. Endocrinology. 2003; 144: 1241-1248Crossref PubMed Scopus (67) Google Scholar, N. M. J.C. V. Morand S. Gnidehou S. Agnandji D. Ohayon R. Kaniewski J. Noel-Hudson M.S. M. Virion A. Dupuy C. J. 2005; PubMed Scopus Google Scholar), that expression of DUOX2 in reconstituted system involved normal maturation of DUOX2 within the of DUOXA2 as ER-resident could be an of a DUOX2 a with the to the and the plasma membrane. determined whether DUOXA2 alone or in with DUOX2 be at the plasma membrane. constructs DUOXA2 with or tags were functional in DUOX2 activity as by H2O2 generation and HA-DUOX2 plasma membrane (data not shown). However, was at the plasma membrane (data not the expected size on Western blot analysis (Fig. and with HA-DUOX2 (Fig. To that this was due to a between the and DUOXA2 membrane or due to of the N-terminal we to the terminus of DUOXA2 N-terminal was not shown in Fig. did not with HA-DUOX2 at the plasma membrane, the by anti-HA surface The of of showed a as the (Fig. To that DUOXA2 is indeed an ER-resident protein, we analyzed the maturation of N-glycosylation in cells We that the N-glycans of were of the (Fig. of HA-DUOX2 in the that of DUOX2 protein been to of its (data not Fig. results that DUOXA2 is not an of a DUOX2 at the plasma membrane an ER-resident protein and maturation of DUOX2. It be that N-glycosylation of DUOXA2 model (Fig. since the molecular weight of the N-glycan moieties N-glycosylation of all three consensus on the of DUOX/DUOXA is an of bidirectional transcription of linked genes that are not are involved in the an arrangement to a A. J.E. H. Mol. Biol. PubMed Scopus Google Scholar). to the in the analyzed were not linked to in is also for a functional The Drosophila (mol) encodes a plasma membrane protein in the of a signal to the plasma membrane H. A. C. G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). may not with Drosophila is also by the by of as in J. A.S. L. J. S. J. P. F. R. U. C. T. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) or Drosophila duox 2005; PubMed Scopus Google Scholar). Remarkably, this functional divergence to a of the region crucial for functional of and DUOXA. the second and third transmembrane are by an extended loop with N-glycosylation of DUOXA2 and the region a crucial for of H. A. C. G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), have to be Analysis of the for a in the Phobius indeed a of membrane the profile of transmembrane in not vertebrate the for (Fig. which the in its as we DUOX expression in Drosophila not could have been the of a DUOX/DUOXA system that its in deuterostomes over million F. H. U. S. A. 2004; PubMed Scopus Google Scholar) of For in J.L. R. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and in follicular thyroid cells (4De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar), the of DUOX protein is not at the cell surface in which could a Thus, from an evolutionary the of DUOXA may have an level of DUOX the control of DUOX translocation to the plasma membrane. The to reconstitute active DUOX will the to the molecular mechanisms DUOX expression in model with