Identification of the Maturation Factor for Dual Oxidase

Abstract

Dual oxidase 2 (DUOX2), an NADPH:O 2 oxidoreductase flavoprotein, is a component of the thyroid H 2 O 2 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 coexpressed 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 H 2 O 2 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) 2 appear to constitute the catalytic core of the H 2 O 2 generator (1, 2). They are large homologs of the phagocyte gp91 phox /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 (3), 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) (4 -8), suggesting that an unidentified component, essential for DUOX maturation, may be specifically expressed in tissues containing the functional enzyme. EXPERIMENTAL PROCEDURESData Mining and Computational Analysis-Massively parallel signature sequencing (MPSS) data (9) 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. (9), was calculated for signatures with frequency Ͼ100 tags per million (ϳ30 mRNA copies/cell) in the thyroid/parathyroid library. Tags with scores ϾϪ1 were mapped to the human genome assembly using BLAST. DUOXA homologs were identified by tBLASTn searches against the NCBI nr data base and trace...

Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1

“…High level of p22 phox expression was detected in HaCaT cells, but A431 cells did not express any p22 phox mRNA. DuoxA1, which is the activator component of the Duox1 enzyme complex (13), was also present at high levels in both cells lines, while mRNAs encoding NoxO1 and NoxA1 were barely detected. These results pointed to Duox1 as the possible source of H 2 O 2 in these cell lines.…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…While mature N-glycosylated DUOX proteins are localized to the plasma membrane, substantial amounts of DUOX protein are also found intracellularly (27,32), presumably in association with the ER. Heterologous DUOX1/2 expression in non-epithelial cell types typically does not result in a functional H 2 O 2 -generating enzyme at the plasma membrane, presumably because these proteins are not fully glycosylated and are retained in the ER (10,93). This was recently resolved by the discovery of maturation factors of DUOX (DUOXA1/2), which are ER-resident transmembrane proteins that facilitate ER-to-Golgi transition and translocation of DUOX proteins to the plasma membrane (93).…”

“…Heterologous DUOX1/2 expression in non-epithelial cell types typically does not result in a functional H 2 O 2 -generating enzyme at the plasma membrane, presumably because these proteins are not fully glycosylated and are retained in the ER (10,93). This was recently resolved by the discovery of maturation factors of DUOX (DUOXA1/2), which are ER-resident transmembrane proteins that facilitate ER-to-Golgi transition and translocation of DUOX proteins to the plasma membrane (93). Analysis of particulate fractions of DUOX2-transfected HEK293 or Chinese hamster ovary cells, however, suggests that ERretained DUOX may not be without function, and is capable of producing H 2 O 2 /O 2 •− upon Ca 2+ stimulation in the absence of cytosolic activators or organizer subunits (10).…”

“…DUOX1 and its maturation factor DUOXA1 are both located on chromosome 15q15.3 in a complex locus in a head-to-head arrangement (92)(93)(94), and may share a bidirectional promoter. The same is true for their paralogs DUOX2/DUOXA2.…”

“…Bidirectionally promoted gene pairs represent ~10% of all transcriptional units and have a higher probability of coordinated expression than random pairs of genes (95). Accordingly, expression of DUOX1/DUOXA1 and DUOX2/DUOXA2 in airway epithelial cells or thyrocytes have been found to be closely coordinated (90,93). The promoter regions of human DUOX1 and DUOX2 are, however, markedly different.…”