TY - JOUR
T1 - Dual oxidase-2 has an intrinsic Ca2+-dependent H 2O2-generating activity
AU - Ameziane-El-Hassani, Rabii
AU - Morand, Stanislas
AU - Boucher, Jean Luc
AU - Frapart, Yves Michel
AU - Apostolou, Daphné
AU - Agnandji, Diane
AU - Gnidehou, Sédami
AU - Ohayon, Renée
AU - Noël-Hudson, Marie Sophie
AU - Francon, Jacques
AU - Lalaoui, Khalid
AU - Virion, Alain
AU - Dupuy, Corinne
PY - 2005/8/26
Y1 - 2005/8/26
N2 - 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 H 2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the participate 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 p22phox, the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H 2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2 .- but H2O 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 H2O 2 formation by favoring intramolecular superoxide dismutation.
AB - 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 H 2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the participate 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 p22phox, the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H 2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2 .- but H2O 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 H2O 2 formation by favoring intramolecular superoxide dismutation.
UR - http://www.scopus.com/inward/record.url?scp=24044546151&partnerID=8YFLogxK
U2 - 10.1074/jbc.M500516200
DO - 10.1074/jbc.M500516200
M3 - Article
C2 - 15972824
AN - SCOPUS:24044546151
SN - 0021-9258
VL - 280
SP - 30046
EP - 30054
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 34
ER -