Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity

Area of Science:

• Biochemistry
• Molecular Biology
• Endocrinology

Background:

• Thyroid hormone biosynthesis relies on Duox2 (and Duox1), a glycoflavoprotein functioning as the thyroid H2O2 generator, associated with thyroperoxidase (TPO).
• Previous attempts to express functional H2O2-generating systems by transfecting DUOX into nonthyroid cell lines were hindered by maturation and targeting impairments.

Purpose of the Study:

• To investigate and characterize the H2O2-generating activity in DUOX1- and DUOX2-transfected nonthyroid cell lines.
• To explore the role of post-translational modifications in the H2O2 formation mechanism.

Main Methods:

• Utilized HEK293 and Chinese hamster ovary cells stably or transiently transfected with human or porcine DUOX cDNA.
• Analyzed H2O2-generating activity in particulate fractions.
• Employed spin-trapping technique with electron paramagnetic resonance (EPR) spectroscopy.

Main Results:

• Demonstrated functional NADPH/Ca2+-dependent H2O2-generating activity in particulate fractions of transfected cells.
• Observed varying activity levels among immature Duox proteins, with human Duox2 being more active than human Duox1 but less than porcine Duox2.
• Confirmed H2O2 release by mature thyroid NADPH oxidase and identified superoxide generation by partially glycosylated Duox2 in the endoplasmic reticulum.

Conclusions:

• Established a functional H2O2-generating system in DUOX-transfected nonthyroid cells.
• Post-translational modifications during Duox2 maturation are suggested to be crucial for the H2O2 formation mechanism, potentially via intramolecular superoxide dismutation.