Dual Oxidase-2 Has an Intrinsic Ca super(2+)-dependent H sub(2)O sub(2)-generating Activity

• Published in: The Journal of biological chemistry Vol. 280; no. 34; pp. 30046 - 30054
• Main Authors: Ameziane-El-Hassani, Rabii, Morand, Stanislas, Boucher, Jean-Luc, Frapart, Yves-Michel, Apostolou, Daphne, Agnandji, Diane, Gnidehou, Sedami, Ohayon, Renee, Noel-Hudson, Marie-Sophie, Francon, Jacques, Lalaoui, Khalid, Virion, Alain, Dupuy, Corinne
• Format: Journal Article
• Language: English
• Published: 01-08-2005
• ISSN: 0021-9258; 1083-351X

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H sub(2)O sub(2) generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H sub(2)O sub(2)-generating system at the plasma membrane. For the first time, we investigated the H sub(2)O sub(2)-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca super(2+)-dependent H sub(2)O sub(2)-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22 super(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H sub(2)O sub(2) formation, it was shown that mature thyroid NADPH oxidase does not release [Formula: see text] but H sub(2)O sub(2). Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H sub(2)O sub(2) formation by favoring intramolecular superoxide dismutation.