Characterization of ThOX Proteins as Components of the Thyroid H2O2-Generating System

Abstract

We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thyroperoxidase at the apical membrane of human thyroid cells. In the present study we have determined their subcellular localization and maturation in relation to their enzymatic activity. A majority of ThOX proteins accumulated inside the cell and only a small fraction was expressed at the surface. Western blots demonstrated that ThOX's are glycoproteins of 180,000 and 190,000. When totally deglycosylated the molecular weight of both ThOX1 and ThOX2 drops to 160,000. Ca(2+) stimulates the basal H(2)O(2) generation in PC Cl3 cells at a level corresponding to 20% of the leukocyte H(2)O(2) production stimulated by PMA. Nonthyroid cell lines transfected with ThOX1 and ThOX2 show only a single immunoreactive band in Western blot analysis, corresponding to the protein of 180,000. This "immature" protein remains exclusively intracellular and does not present any enzymatic activity. This is not modified by coexpression of thyroperoxidase and p22(Phox). Transfection of ThOX cDNAs into PLB-XCGD cells does not reconstitute their NADPH oxidase activity. We conclude that (1) the thyroid contains some elements of the leukocyte H(2)O(2)-generating system but not all of them; (2) ThOX's are predominantly or exclusively located inside the cell in thyrocytes or in transfected cells, respectively, and as such they are inactive; (3) ThOX's cannot replace gp91(Phox) in the leukocyte; and (4) the thyroid H(2)O(2)-generating system is analogous to the leukocyte system with regard to ThOX's and gp91(Phox) but very different in other aspects. Additional thyroid-specific components are probably required to get complete protein processing and full enzymatic activity in the thyroid.

Nox enzymes, ROS, and chronic disease: An example of antagonistic pleiotropy

“…This reaction requires H 2 O 2 , which derives from a previously unidentified thyroid NADPH oxidase. Duox was first shown to be this longsought oxidase based on purification and partial cDNA cloning [187]; two forms of this oxidase were subsequently identified by molecular cloning [188][189][190], and both are expressed in thyroid [191]. The essential role for Duox2 in thyroid hormone biosynthesis is demonstrated conclusively by the occurrence of mutations in this gene in genetic variants of hypothyroidism [192,193].…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…The H 2 O 2 that is responsible for TPO activation originates from the NOX homologues DUOX1 and DUOX2 that are highly expressed within thyrocytes (27). The importance of DUOX2 in thyroid hormone synthesis is supported by clinical observations of hypothyroidism in association with missense mutations in the DUOX2 gene (28,29).…”

“…Both DUOX proteins are highly similar N-glycosylated proteins (with 83% homology) with a molecular weight of 180 kD. While mature N-glycosylated DUOX proteins are localized to the plasma membrane, substantial amounts of DUOX protein are also found intracellularly (27,32), presumably in association with the ER. Heterologous DUOX1/2 expression in non-epithelial cell types typically does not result in a functional H 2 O 2 -generating enzyme at the plasma membrane, presumably because these proteins are not fully glycosylated and are retained in the ER (10,93).…”

“…2+ binding domains within DUOX suggests that it can be directly activated by Ca 2+ -mobilizing stimuli. Indeed, Ca 2+ mobilization by various diverse stimuli were been found to promote apical H 2 O 2 production in airway epithelial cells (10,27,84,133). Moreover, in analogy to activation of NOX2 in phagocytes, H 2 O 2 production by airway epithelial cells or thyrocytes was also stimulated in response to activation of protein kinase C (PKC), and Ca 2+ and PKC stimulation appear to act synergistically in promoting H 2 O 2 production (82,105,134,135).…”

Targeting of the Dual Oxidase 2 N-terminal Region to the Plasma Membrane

“…The sequence of porcine Duox2 includes six putative N-glycosylated sites located in the N-terminal ectodomain, whereas only five such sites have been identified in the human protein. Western blots of membrane proteins from human and pig thyrocytes displayed Duox2 as two bands corresponding to two differently N-glycosylated states of Duox2 with oligosaccharide motifs accounting for 15 and 25 kDa of their molecular mass, respectively (13,14). Only the more highly glycosylated form was resistant to endoglycosidase H treatment, indicating that it passes through the GA and also suggesting that it constitutes the mature form of the NAD(P)H oxidase at the plasma membrane (14).…”