Tetraspanin Is Required for Generation of Reactive Oxygen Species by the Dual Oxidase System in Caenorhabditis elegans

Abstract

Reactive oxygen species (ROS) are toxic but essential molecules responsible for host defense and cellular signaling. Conserved NADPH oxidase (NOX) family enzymes direct the regulated production of ROS. Hydrogen peroxide (H ₂O ₂) generated by dual oxidases (DUOXs), a member of the NOX family, is crucial for innate mucosal immunity. In addition, H ₂O ₂ is required for cellular signaling mediated by protein modifications, such as the thyroid hormone biosynthetic pathway in mammals. In contrast to other NOX isozymes, the regulatory mechanisms of DUOX activity are less understood. Using Caenorhabditis elegans as a model, we demonstrate that the tetraspanin protein is required for induction of the DUOX signaling pathway in conjunction with the dual oxidase maturation factor (DUOXA). In the current study, we show that genetic mutation of DUOX (bli-3), DUOXA (doxa-1), and peroxidase ( mlt-7) in C. elegans causes the same defects as a tetraspanin tsp-15 mutant, represented by exoskeletal deficiencies due to the failure of tyrosine cross-linking of collagen. The deficiency in the tsp-15 mutant was restored by co-expression of bli-3 and doxa-1, indicating the involvement of tsp-15 in the generation of ROS. H ₂O ₂ generation by BLI-3 was completely dependent on TSP-15 when reconstituted in mammalian cells. We also demonstrated that TSP-15, BLI-3, and DOXA-1 form complexes in vitro and in vivo. Cell-fusion-based analysis suggested that association with TSP-15 at the cell surface is crucial for BLI-3 activation to release H ₂O ₂. This study provides the first evidence for an essential role of tetraspanin in ROS generation.

Author Summary

ROS are highly reactive molecules, which can be inappropriately produced during aerobic metabolism or by exogenous stresses such as exposure to UV light and radiation. ROS interact with cellular components including nucleic acids, lipids, and proteins and irreversibly inhibit their functions. However, ROS are essential for innate host defense and multiple physiological processes and are generated by conserved NADPH oxidase (NOX) family enzymes. The release of ROS by ROS generator enzymes must be properly controlled, as chronic oxidative stress can cause an imbalance of the redox state and is often associated with disease and aging. Using C. elegans as a model, we identified a tetraspanin (TSP-15) protein as a new key component of the ROS generation system controlled by dual oxidase (BLI-3), a unique NOX isozyme in C. elegans. Mutants of both bli-3 and tsp-15 developed the same defects in extracellular matrix cross-linking. Using a combination of genetics and reconstitution experiments in mammalian cells, we have demonstrated a novel requirement of tetraspanin for dual oxidase-dependent ROS generation via complex formation at the cell surface.