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Missense Mutations of Dual Oxidase 2 (DUOX2) Implicated in Congenital Hypothyroidism Have Impaired Trafficking in Cells Reconstituted with DUOX2 Maturation Factor

Departments of Medicine (H.G., S.R.) and Pediatrics (S.R.), and Committee on Genetics (S.R.), The University of Chicago, Chicago, Illinois 60637; Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (X.D.D., F.M.), Université Libre de Bruxelles, B-1070 Brussels, Belgium; and Children’s Hospital of the Johannes Gutenberg University (J.P.), D-55101 Mainz, Germany

Address all correspondence and requests for reprints to: Helmut Grasberger, The University of Chicago, MC3090, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: hgrasber{at}uchicago.edu.

Dual oxidase 2 (DUOX2), a reduced NAD phosphate:O₂ oxidoreductase flavoprotein, is a component of the thyrocyte H₂O₂ generator required for hormone synthesis at the apical plasma membrane. We recently identified a specific DUOX2 maturation factor (DUOXA2) that is necessary and sufficient for expression of functional DUOX2 in mammalian cell lines. We have now used a DUOXA2 reconstituted system to provide the first characterization of natural DUOX2 missense variants (Q36H, R376W, D506N) at the molecular level, analyzing their impact on H₂O₂ generation, trafficking, stability, folding, and DUOXA2 interaction. The Q36H and R376W mutations completely prevent routing of DUOX2 to the cell surface. The mutant proteins are predominantly present as core N-glycosylated, thiol-reduced folding intermediates, which are retained by the quality control system within the endoplasmic reticulum (ER) as indicated by increased complexation with the lectin calnexin. D506N displays a partial deficiency phenotype with reduced surface expression of a mutant protein with normal intrinsic activity in generating H₂O₂. D506N N-glycan moieties are not subject to normal modification in the Golgi apparatus, suggesting that nonnative protein can escape the quality control in the ER. Oxidative folding of DUOX2 in the ER appears to be the rate-limiting step in the maturation of DUOX2, but is not facilitated by DUOXA2. Rather, DUOXA2 allows rapid ER exit of folded DUOX2 or enhanced degradation of mutant DUOX2 proteins not competent for ER exit. DUOXA2 may thus be part of a secondary quality control system specific for DUOX2.

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