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Iodotyrosine dehalogenase 1 (DEHAL1) is a transmembrane protein involved in the recycling of iodide close to the thyroglobulin iodination site

SÉDAMI GNIDEHOU^*,, BERNARD CAILLOU, MONIQUE TALBOT, RENÉE OHAYON^*, JACQUES KANIEWSKI^*, MARIE-SOPHIE NOËL-HUDSON^*, STANISLAS MORAND^*, DIANE AGNANGJI^*,, ALPHONSE SEZAN, FRANÇOISE COURTIN, ALAIN VIRION^* and CORINNE DUPUY^*,1

^* Unité 486 INSERM, Université Paris 11, Faculté de Pharmacie, Châtenay-Malabry, France;
UMR 8125 CNRS/CEA LRC 29V, Institut Gustave-Roussy, Villejuif, France;
Laboratoire de Biomembrane (FAST), Cotonou, Benin; and
Unité 488 INSERM, Gregory Pincus, Kremlin Bicêtre, France

¹Correspondence: Unite 486, INSERM, Universite Paris 11, Faculte de Pharmacie, 5 rue J. B. Clement, 92296 Chatenay-Malabry Cedex, France. E-mail: corinne.dupuy{at}cep.u-psud.fr

SPECIFIC AIMS

In the thyroid, iodotyrosine dehalogenase acts on the mono (L-MIT) and diiodotyrosines (L-DIT) released during thyroglobulin hydrolysis and liberates iodide, which can then reenter hormone-producing pathways. This deiodinating activity is so efficient that only negligible amounts of iodotyrosines are secreted by the thyroid. It has been reported that iodotyrosine deiodination occurs predominantly in the microsomes and is mediated by NADPH. Recently, two cDNAs, 7,401 and 7,513-base-pairs long, and encoding proteins with a conserved nitroreductase domain were published in GenBank as iodotyrosine dehalogenase 1 (DEHAL1) and iodotyrosine dehalogenase 1B (DEHAL1B). Because the DEHAL1 protein has a complete nitroreductase domain, we chose to study the localization and the activity of this isoform first.

PRINCIPAL FINDINGS

1. DEHAL1 belongs to the nitroreductase family
A 7401 base pair cDNA, isolated from human thyroid was published in GenBank as iodotyrosine dehalogenase protein mRNA (accession #AY259176). The cDNA sequence encoded a 289 amino acid protein with a theoretical molecular mass of 33 kDa. The SignalP prediction program indicated that the sequence contains a putative signal peptide. Sequence analysis showed that the protein is a member of the nitroreductase family, with a highly conserved domain comprised between Glu⁹² and Gly²⁴⁴. The hydropathy profile indicates a putative transmembrane segment between Asn²¹³ and Gln²²⁹. The presence of a peptide signal and of one transmembrane segment suggests that DEHAL1 has a large extracellular N-terminal region including most of the nitroreductase domain, plus a short C-terminal cytoplasmic tail.

2. DEHAL1 as a thyrocyte-differentiation marker
Using a probe recognizing the 3'-UTR to explore 23 different human tissues, an 7 kb mRNA was found to be present at a high level in the thyroid gland and at a lower level in the kidney and trachea. A search in the human expressed sequence tag (EST) database indicated that DEHAL1 mRNA is also found, although probably in smaller amounts, in liver and colon. Because a porcine counterpart was expected, we cloned porcine DEHAL1 cDNA encoding a protein 85% identical to the human DEHAL1 protein. A probe interacting with the 3'-UTR revealed two mRNA species, 1.35 and 2.4 kb long, respectively, present in high levels in the thyroid and enhanced by cyclic AMP, as are most of the genes involved in thyroid gland function.

3. DEHAL1 is a transmembrane protein localized in intracellular vesicles and the plasma membrane at the apical pole of thyrocytes
Antibodies were raised against the N-terminal domain to allow immunohistochemical and Western blot studies of DEHAL1 proteins. The antibody recognized a protein with an apparent molecular mass of 33 kDa in the membrane fraction of HEK293 cells stably transfected with DEHAL1 and in the human thyroid. Solubilization studies showed that DEHAL1 was solubilized with the nonionic detergent Triton X 100. Treatment of the membranes by salt or alkaline solution (pH 11) did not efficiently solubilize the protein, indicating that it is an integral membrane protein. Immunohistochemistry showed that the DEHAL proteins were diffusely localized in the cytoplasm of most of the tall, active columnar cells, as well as in the flattened, inactive thyrocytes (Fig. 1 A). In many cells, the labeling was much more marked on the apical membrane (Fig. 1B ). Cell-surface biotinylation experiments performed on the HEK293 cell line stably expressing human DEHAL1 showed that a fraction of the DEHAL1 protein was present at the cell surface.

View larger version (58K): [in this window] [in a new window]   Figure 1. Immunodetection of human DEHAL1. A) Immunostaining for DEHAL1 in normal human thyroid tissue. Most of the cells display diffuse intracytoplasmic staining. An apical increase in staining (arrows) can be seen in many cells (original magnification, x200). B) Immunostaining for DEHAL1 in normal human thyroid tissue. The protein is diffusely localized in the cytoplasm. Intracytoplasmic positively stained granules are sometimes visible (narrow arrows). There is obviously increased staining (thick arrows) at the apical pole of the thyrocytes (original magnification, x400). FL, follicular lumen; cp, capillary. C) Immunostaining with preimmune rabbit serum (original magnification, x200).

4. DEHAL1 is an NADPH-dependent iodotyrosine deiodinase
Iodotyrosine deiodinase was active if DEHAL1 cDNA was transfected into HEK293 cells, but not if it was transfected into CHO cells. The activity was completely dependent on NADPH. DEHAL1 catalyzed the deiodination of L-MIT more efficiently than that of L-DIT. The K_m value for L-DIT, determined by Lineweaver-Burk plots, was 2.67 x 10^–6 M) and that for L-MIT was half as great (1.35x10^–6 M). Moreover, the V_m value found with L-MIT (3.53 nmolx100 µg^–1x10 min^–1) was seven times that found with L-DIT (0.53 nmolx100 µg^–1x10 min^–1). The nitro analog 3,5-dinitro-L-tyrosine was a potent inhibitor of the NADPH dependent deiodinase activity. NADH was not a substrate of the enzyme.

CONCLUSIONS AND SIGNIFICANCE

Five decades ago, an enzymatic activity that catalyzes the removal of iodine from the 3 and 5 positions of L-tyrosine was detected in various mammalian tissues, including thyroid, liver, and kidney. It was found that the iodotyrosine deiodination activity was localized in the microsomes and required NADPH as cofactor, in the thyroid, liver, and kidney. In 1979, a flavoprotein from the thyroid microsomal particulate fraction displaying iodotyrosine deiodinase activity was purified and characterized, but the corresponding gene was not identified. Recently, a novel thyroidal gene located at 6q24-25 and encoding proteins with a conserved nitroreductase domain that could catalyze iodotyrosine deiodination was identified. Two variants of the gene product were published in GenBank as DEHAL1 and DEHAL1B, for iodotyrosine dehalogenase 1 and iodotyrosine dehalogenase 1 isoform B, respectively. As DEHAL1 protein has a complete nitroreductase domain, we therefore chose to study this isoform first, and found that the expression of its activity was dependent of the cell type used: the activity was only obtained in HEK293 cells and not in CHO cells. Consequently, DEHAL1 was stably expressed in HEK293 cells and its biochemical characterization was undertaken. Until now, no enzymatic properties of the human protein have been published. For the first time, we show that human DEHAL1 efficiently deiodinates free L-MIT and L-DIT in the presence of NADPH and that it shows greater affinity for L-MIT. The analysis of the primary amino acid sequence of DEHAL1 protein predicted the presence of a signal peptide and of a transmembrane segment in the nitroreductase domain. Results reported here confirm that DEHAL1 is a transmembrane enzyme. It had been thought that the deiodination of iodotyrosines in the thyroid occurred predominantly after their release following the lysosomal degradation of Tg. According to this concept, one might expect lysosomal targeting of DEHAL1 to occur. However, by using antibodies directed against the N-terminal domain of DEHAL1, we found that it was essentially located at the apical pole of the thyrocytes. Although some of the DEHAL1 protein appeared to be localized on intracellular membranes, a pronounced labeling was observed in the sub-apical region and on the apical membrane. The targeting of the protein to the plasma membrane was demonstrated by cell surface biotinylation experiments and confocal microscopy, and is supported by the detection of the immunoreactive and functional enzyme in a preparation of porcine thyroid plasma membrane. These findings indicate that deiodination reaction occurs at the apical pole of the thyrocyte and is involved in a rapid recycling of iodide at and or close to the organification site. This implies that DIT and MIT would also be released from thyroglobulin (Tg) in the same cell region. In vitro, proteolysis of Tg is achieved by incubating it with cysteine-proteinase such as cathepsins B and L. Recently, however, it has been shown that thyroid epithelial cells also express and secrete cathepsin K which liberates T4 from Tg by limited extracellular proteolysis. The liberation of T4 is mediated by the combined action of cathepsins B, K, and L, that could also release L-MIT and L-DIT. The deiodination reaction could therefore occur during the proteolysis of Tg before and during its endocytosis (Fig. 2 ). In such a model, the catalytic domain of DEHAL1 would be expected to face the internal surface of the exocytotic vesicles membranes and, after fusion with the apical plasma membrane, to face toward the extracellular medium in contact with the colloid. After endocytosis, it would lie on the inside surface of the endosome and endolysosome membranes (Fig. 2) . This hypothesis is compatible with the predicted topology of DEHAL1, and implies that the NADPH binding site is located on the cytosolic region of the enzyme. As with bacterial nitroreductases, no canonical sequence for the binding of NADPH could be identified in the DEHAL1 sequence, thus raising the possibility that it may be located on a distinct component. It had been observed that treating bovine thyroid microsomes with steapsin yields a soluble enzyme preparation that catalyzes the deiodination of L-DIT in the presence of dithionite, but is unresponsive to NADPH. It was then suggested that NADPH-mediated deiodination might involve an electron carrier with a low oxidation-reduction potential, which is reduced by NADPH via a specific reductase. We observed that the iodotyrosine deiodination activity could be reconstituted by transfecting DEHAL1 cDNA into HEK293 cells, but not if it was transfected into CHO cells. Since DEHAL1 is expressed in the kidney, one cannot exclude the possibility that the human kidney cell line (HEK293), although devoid of DEHAL1 and iodotyrosine dehalogenase activity, could express the electron carrier intermediate and the specific NADPH-dependent reductase necessary for the functional reconstitution of iodotyrosine dehalogenase activity by DEHAL1. Further studies are needed to evaluate this hypothesis.

View larger version (34K): [in this window] [in a new window]   Figure 2. Enzymatic properties. A) Double reciprocal plot of deiodinase activity vs. the concentration of L-DIT and L-MIT. Labeled L-DIT and L-MIT were prepared by an exchange labeling method. Assays were performed with 100 µg and 20 µg of protein for the L-DIT and L-MIT experiments respectively. The iodide liberated was separated from [125I]DIT and [125MIT] by descending paper chromatography using ethanol/0.2 M ammonium carbonate (2:1) as the solvent. Each point represents the average of triplicate determinations using the same enzyme preparation, and the linear regression curve was fitted to the data by method of least squares. B) Iodotyrosine activity of DEHAL1-HEK293 cell membrane in the presence of 3,5 dinitro-L-tyrosine (DNT). Assays were performed with 100 µg of protein. Measurements were done after 10 min. The values represent mean ± SE of 3 estimations. C) NADPH and NADH-dependent iodotyrosine deiodinase activity catalyzed by DEHAL1-HEK293 cell membranes. Assays were performed with 100 µg of protein in the presence of 100 µM NADPH or NADH. Measurements were done after 10 min. The values represent mean ± SE of 3 estimations.

Congenital defects in the deiodination of mono- and diiodotyrosine, leading to excessive renal loss of iodine in the form of MIT and DIT, leading in turn to an iodine-deficient hypothyroid state and a goiter of variable size, were first reported more than four decades ago. Our demonstration that the DEHAL1 gene encodes an active iodotyrosine dehalogenase and its characterization open new prospects for elucidating the physiopathology and diagnosis of thyroid disease at the molecular level.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2023fje;

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