Binding and Internalization of Thyroglobulin: Selectivity, pH Dependence, and Lack of Tissue Specificity

International audienceThyroglobulin (Tg), the precursor of thyroid hormones, follows a unique secretion, storage, and recapture pathway. The first steps of recapture were studied by investigating the binding of 125I-labeled Tg on the apical surface of inside-out follicles and its internalization. The selectivity of the process was assessed by using molecules other than Tg and/or nonthyroid cells. Tg binding to the apical surface of thyroid inside-out follicles is selective relative to the binding of other molecules. It increases sharply over pH 8.0 and occurs through specific sites of moderately high affinity (Kd = approximately 200 nM; 2 x 10(4) sites/cells). At pH < 8.0 it occurs through numerous sites of very low affinity considered nonspecific. Endocytosis, although weak under our conditions, increases at pH 8.0 concomitantly with binding. Over pH 8.2, however, some inhibition occurs. Surprisingly, Tg binding and endocytosis are not tissue specific, as they showed the same properties on thyroid inside-out follicles and Madin-Darby canine kidney or Chinese hamster ovary cells. Thus, a selective uptake of Tg mediates its recapture by thyroid cells. This selectivity is an intrinsic Tg property, not dependent on the thyrocyte apical surface, as it was observed with Madin-Darby canine kidney and Chinese hamster ovary cells. Given the pH effect observed, we suggest that Tg binding is a regulated phenomenon and that, through luminal pH control, it can vary from a basal level at neutral pH to a stimulated level over pH 8.0

Calnexin and Calreticulin Binding to Human Thyroperoxidase Is Required for Its First Folding Step(s) But Is Not Sufficient to Promote Efficient Cell Surface Expression*

International audienceHuman thyroperoxidase (hTPO) is a type I transmembrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. In a previous study we stably expressed hTPO in Chinese hamster ovary cells and observed that after the synthesis, only 20% of the hTPO molecules were recognized by a monoclonal antibody (mAb 15) directed against a conformational structure, and that only 2% were able to reach the cell surface. In the present study it was proposed to determine how calnexin (CNX) and calreticulin (CRT) contribute to the folding of hTPO. Sequential immunoprecipitation was performed using anti-CNX or anti-CRT followed by anti-hTPO antibodies, and the results showed that CNX and CRT were associated with hTPO. Inhibiting the interactions between CNX or CRT and hTPO using castanospermine greatly reduced the first step(s) in the hTPO folding process. Under these conditions, the half-life of this enzyme was greatly reduced (2.5 vs. 17 h in the control experiments), and hTPO was degraded via the proteasome pathway. This reduced the rate of hTPO transport to the cell surface. Overexpression of CNX or CRT into the hTPO-CHO cells was found to enhance the first hTPO folding step(s) by 20-60%, but did not increase the level of hTPO present at the cell surface. All in all, these findings provide evidence that CNX and CRT are crucial to the first step(s) in hTPO folding, but that interactions with other molecular chaperones are required for the last folding steps to take place

Association of the thyrotropin receptor with calnexin, calreticulin and BiP. Efects on the maturation of the receptor.

International audienceThe thyrotropin receptor (TSHR) is a member of the G protein-coupled receptor superfamily. It has by now been clearly established that the maturation of the glycoproteins synthesized in the endoplasmic reticulum involves interactions with molecular chaperones, which promote the folding and assembly of the glycoproteins. In this study, we investigated whether calnexin (CNX), calreticulin (CRT) and BiP, three of the main molecular chaperones present in the endoplasmic reticulum, interact with the TSHR and what effects these interactions might have on the folding of the receptor. In the first set of experiments, we observed that in a K562 cell line expressing TSHR, about 50% of the receptor synthesized was degraded by the proteasome after ubiquitination. In order to determine whether TSHR interact with CNX, CRT and BiP, coimmunoprecipitation experiments were performed. TSHR was found to be associated with all three molecular chaperones. To study the role of the interactions between CNX and CRT and the TSHR, we used castanospermine, a glucosidase I and II inhibitor that blocks the interactions between these chaperones and glycoproteins. In K562 cells expressing the TSHR, these drugs led to a faster degradation of the receptor, which indicates that these interactions contribute to stabilizing the receptor after its synthesis. The overexpression of calnexin and calreticulin in these cells stabilizes the receptor during the first hour after its synthesis, whereas the degradation of TSHR increased in a cell line overexpressing BiP and the quantity of TSHR able to acquire complex type oligosaccharides decreased. These results show that calnexin, calreticulin and BiP all interact with TSHR and that the choice made between these two chaperone systems is crucial because each of them has distinct effects on the folding and stability of this receptor at the endoplasmic reticulum level

Role of Heme in Intracellular Trafficking of Thyroperoxidase and Involvement of H 2 O 2 Generated at the Apical Surface of Thyroid Cells in Autocatalytic Covalent Heme Binding

International audienceThyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule

Human Thyroperoxidase Is Largely Retained and Rapidly Degraded in the Endoplasmic Reticulum. Its N-Glycans Are Required for Folding and Intracellular Trafficking

International audienceHuman thyroperoxidase (hTPO), a type I transmembrane heme containing glycoprotein, catalyzes iodide organification and thyroid hormone synthesis and plays a major role in thyroid autoimmunity. Whereas hormonosynthesis occurs at the apical membrane of thyroid cells, TPO localizes mainly in the perinuclear membrane and the endoplasmic reticulum. To establish the intracellular trafficking and the structural characteristics of hTPO in the various cell compartments, hTPO was stably expressed in the Chinese hamster ovary cell line, and its folding was studied with two monoclonal antibodies (mAbs): mAb 47, recognizing a linear epitope; and mAb 15, recognizing a conformational epitope present in the mature protein. The results show that only 15-20% of hTPO molecules were able to acquire a conformation suitable for the recognition by mAb 15. On the other hand, only a part (approximately 15%) of the latter were able to reach the plasma membrane. The hTPO, unable to fold correctly, was more rapidly degraded than that recognized by mAb 15 (half-time, 2 h vs. 7 h). Study of the carbohydrate content of hTPO showed that N-glycans with complex-type structure were found only on hTPO at the cell surface, whereas intracellular hTPO bore high-mannose-type structures. Taken together, these data demonstrate that the intracellular pool of enzyme is formed of newly synthesized molecules and is not caused by recycling of mature hTPO from the cell surface. Complete inhibition of hTPO N-glycosylation with tunicamycin led to a 95% decrease in hTPO at the plasma membrane and, thus, to a decrease in enzymatic activity at the cell surface, emphasizing the role of N-glycans in the intracellular trafficking of hTPO. However, inhibition of formation of complex-type structures with deoxymannojirimycin and of O-glycans with phenyl-alpha-GalNAc did not influence the intracellular trafficking and enzymatic activity of hTPO

Human Thyroperoxidase in Its Alternatively Spliced Form (TPO2) Is Enzymatically Inactive and Exhibits Changes in Intracellular Processing and Trafficking

International audienceThyroid peroxidase (TPO1) is a membrane-bound heme-containing glycoprotein that catalyzes the synthesis of thyroid hormones. We generated stable cell lines expressing TPO1 and the alternatively spliced isoform TPO2. Pulse-chase studies showed that TPO2 half-life was dramatically decreased as compared with TPO1. The sensitivity of TPO2 to endo-beta-N-acetylglucosaminidase H indicated that the protein is processed through the endoplasmic reticulum and bears high mannose-type structures. Cell surface biotinylation experiments showed that the two isoforms also differ in their intracellular trafficking. TPO2 was totally retained in the cell, whereas 15% of TPO1 reached the cell surface. The inability of TPO2 to come out of the intracellular compartments was related to structural changes in the molecule. Evidence of these changes was obtained through the lack of recognition of TPO2 by half of the 13 TPO monoclonal antibodies tested in immunoprecipitation experiments. Our data suggest that because of an improper folding, TPO2 is trapped in the endoplasmic reticulum and rapidly degraded. The failure of incorporation of [14C]aminolevulinic acid in the cultured cells showed that TPO2 did not bind to heme, whereas TPO1 did. This result was confirmed through a guaiacol assay showing that TPO2 is enzymatically inactive