Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity.
Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 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 H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-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/Ca2+-dependent H2O2-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(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. 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 H2O2 formation by favoring intramolecular superoxide dismutation.
Unité 486 INSERM, Université Paris 11, Faculté de Pharmacie, 5, rue J. B. Clément, 92296 Châtenay-Malabry Cedex, France.
Rabii
R
Ameziane-El-Hassani
Stanislas
S
Morand
Jean-Luc
JL
Boucher
Yves-Michel
YM
Frapart
Daphné
D
Apostolou
Diane
D
Agnandji
Sédami
S
Gnidehou
Renée
R
Ohayon
Marie-Sophie
MS
Noël-Hudson
Jacques
J
Francon
Khalid
K
Lalaoui
Alain
A
Virion
Corinne
C
Dupuy
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D000074623:Dual Oxidases; D004578:Electron Spin Resonance Spectroscopy; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism / Q000502:physiology*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D007962:Leukocytes / Q000201:enzymology; D008280:Magnetics; D008954:Models, Biological; D009154:Mutation; D019255:NADPH Oxidases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D019097:Spin Trapping; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D013961:Thyroid Gland / Q000378:metabolism; D013963:Thyroid Hormones / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D005420:Flavoproteins; D013963:Thyroid Hormones; D013481:Superoxides; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431515:DUOX2 protein, human; D002118:Calcium