Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

Kaufman, Keith D.; Rapoport, Basil; Seto, Pui; Chazenbalk, Gregorio D.; Magnusson, Ronald P.

doi:10.1172/jci114179

Cited by 75 publications

(42 citation statements)

References 41 publications

(38 reference statements)

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“…However, these findings seem at odds both with the original studies of recombinant hTPO expression in other stably transfected CHO cells (31,33), as well as a recent suggestion of hTPO 933 surface expression upon transient transfection of CHO cells (42). We therefore decided to reinvestigate expression of hTPO in transiently transfected CHO cells.…”

Section: Stable Expression Of Htpo In Clonal Mdck Cells-thementioning

confidence: 91%

Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression

Zhang¹,

Arvan²

2000

Journal of Biological Chemistry

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Recently, it has been suggested that only ϳ2% of human thyroid peroxidase (hTPO 933 ) reaches the surface of stably transfected (Chinese hamster ovary) cells, most being degraded intracellularly, and this might be representative of thyroid peroxidase (TPO) behavior in thyrocytes (Fayadat, L., Siffroi-Fernandez, S., Lanet, J., and Franc, J.-L. (2000) In the past decade, cell type-dependent differences in membrane and secretory protein trafficking have been increasingly recognized. Certainly, the rate and efficiency of protein folding and export from the endoplasmic reticulum (ER) 1 varies between cell types (1). Further, in the distal secretory pathway, the expression of cDNAs encoding regulated secretory proteins has been found to result in storage in secretory granules in some regulated secretory cell types but not others (2-5). Additionally, cell surface polarity signals can be differentially interpreted by different polarized cell types (6 -10). The identification of cell type-dependent differences in protein sorting and trafficking represents a crucial first step in elucidating the underlying molecular mechanisms that account for such differences (11).Thyroid peroxidase (TPO) is a key enzyme responsible for iodination of thyroglobulin during the synthesis of thyroid hormones. Electron microscope autoradiography studies have established that iodination activity is localized primarily at the (extracellular aspect of the) apical surface of thyrocytes delimiting the thyroid follicle lumen; suggesting that under physiological circumstances, TPO is primarily an apical plasma membrane enzyme (12). TPO is neither the sole gene product responsible for the diaminobenzidine oxidation reaction (a technique that has been used to detect peroxidase activity cytochemically) nor the sole protein immunoreacting with thyroid autoimmune antisera (used to immunolocalize the "antimicrosomal antigen" (Ref. 13)). Nevertheless, TPO cytochemical activity in thyrocytes is associated with apical membrane vesicles and the outer surfaces of apical microvilli (14), while additional reaction product can be detected in the thyrocyte ER and Golgi complex. Moreover, using patients' antisera, the thyroid microsomal antigen is immunolocalized to the apical surface of thyrocytes (15), while additional immunoreaction is found in the cytoplasm (16,17). Acute thyroid-stimulating hormone stimulation of the thyroid gland causes additional TPO enzymatic activity and immunoreactivity at the apical cell surface (18 -23). These results all indicate that TPO travels via the secretory pathway to the plasma membrane.Despite this, lingering questions persist about the trafficking of TPO to the cell surface in the thyroid and in various cell culture systems (24). In primary porcine thyrocytes after 7 days in culture, only ϳ30% of endogenously expressed TPO could be chemically modified at the cell surface with a non-permeant biotinylation reagent (25). Further, it has recently been reported that only ϳ2% of recombinant wild-type hTPO (fulllength hTPO 933 ) re...

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Section: Stable Expression Of Htpo In Clonal Mdck Cells-thementioning

confidence: 91%

Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression

Zhang¹,

Arvan²

2000

Journal of Biological Chemistry

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“…Generation of recombinant human TPO displaying immunological and enzymatic activity has been reported (3)(4)(5). Also, a secreted form of TPO lacking the transmembrane domain was stably expressed in Chinese hamster ovary (CHO) cells (6).…”

Section: T Hyroid Peroxidase (Tpo) Is a Membrane-linkedmentioning

confidence: 99%

Molecular Analysis of Mutated Thyroid Peroxidase Detected in Patients with Total Iodide Organification Defects

Bikker¹

1997

Journal of Clinical Endocrinology & Metabolism

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Molecular analysis of mutated thyroid peroxidase detected in patients with total iodide organification defects Bikker, H.; Baas, F.; de Vijlder, J.J.M. General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. ABSTRACTWild-type and mutant thyroid peroxidase (TPO) was expressed in a Semliki Forest Virus (SFV)-based transient expression system in Chinese hamster ovary-K1 cells. Twenty four hours after transfection, proteins immunoreactive with TPO antibodies could be detected on a Western blot. Peroxidase activity was assayed using both the guaiacol and the I 3 Ϫ assay. Addition of hematin was necessary to obtain enzymatic active TPO. Thyroid peroxidase complementary DNA constructs containing mutations originally found in patients with hereditary congenital hypothyroidism caused by total iodide organification defects were analyzed using these techniques. In all cases TPO was expressed as shown by Western blotting and immunostaining. Enzymatic activity (measured by guaiacol and iodide oxidation assay) was below the detection level in four out of five mutants. The only mutant yielding TPO with enzymatic activity was G 1858 A (Gly 590 Ser). However, the mutation could affect splicing of TPO messenger RNA, leading to inactive TPO, because it is located at the exon 10/intron 10 border.

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“…For analysis of patients' sera, cells were processed as described previously for TPO expression on CHO cells using patients' sera and human monoclonal autoantibodies (21,22), with modifications. In brief, after detachment by mild trypsinization, cells were pelleted and rinsed (5 min at 100 ϫ g) in Ham's F-12 medium supplemented with 10% dialyzed FCS and antibiotics as described above.…”

Section: Flow Cytometric Analysis [Fluorescein-activated Cell Sortingmentioning

confidence: 99%

Thyrotropin Receptor Autoantibodies in Serum Are Present at Much Lower Levels Than Thyroid Peroxidase Autoantibodies: Analysis by Flow Cytometry*

Jaume

Kakinuma

Chazenbalk

et al. 1997

The Journal of Clinical Endocrinology &Amp; Metabolism

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Using Chinese hamster ovary (CHO) cells that express high numbers of TSH receptor (TSHR) on their surface, we studied the feasibility of detecting directly by flow cytometry the binding of autoantibodies in patients' sera to the native TSHR. After using a serum (BBl) with high potency in the TSH binding inhibition (TBI) assay to establish the protocol, we studied an additional 38 sera: 10 without TBI activity (1-4.2% inhibition), 10 with moderately high TBI values (17.3-39.4% inhibition), 10 with high TBI levels (52-95.1% inhibition), 4 from normal individuals without autoimmune thyroid disease, and 4 from patients with systemic lupus erythematosus. We observed that a number of sera, including some without thyroid autoantibodies, contain antibodies against unknown antigens on CHO cells. Preadsorption with untransfected CHO cells before addition to the TSHR-10,000 cells eliminated or greatly reduced this nonspecific background. None of the sera from normal individuals, subjects with negative TBI values, or patients with systemic autoimmunity generated a positive signal on flow cytometry with TSHR-10,000 cells relative to the signal on untransfected cells. Remarkably, only 4 of 21 TBI-positive sera (including BBl) unequivocally recognized the TSHR on flow cytometry. In contrast, when thyroid peroxidase (TPO) autoantibodies in the same sera were studied using CHO cells overexpressing TPO on their surface, all 20 sera with TPO autoantibodies clearly elicited positive net fluorescence relative to untransfected cells. Study of the potent serum, BBl, revealed similar fluorescence (ϳ250 U) for TPO autoantibodies and TSHR autoantibodies at dilutions of 1:1000 and 1:10, respectively. Thus, by flow cytometry, the titer of TPO autoantibodies in the BBl serum is about 100-fold higher than that for TSHR autoantibodies in the same serum.In conclusion, the present data provide the strongest support for the idea that TSHR autoantibodies in the sera of patients with autoimmune thyroid disease are present at much lower levels than are TPO autoantibodies. This finding has important implications for the diagnostic detection of TSHR autoantibodies and for understanding the pathogenesis of Graves' disease. (J Clin Endocrinol Metab 82: 500 -507, 1997)

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Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

Abstract: A full-length cDNA clone for human thyroid peroxidase (TPO) In conclusion, we expressed enzymaticaliy active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen.

Cited by 75 publications

References 41 publications

Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression

Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression

Molecular Analysis of Mutated Thyroid Peroxidase Detected in Patients with Total Iodide Organification Defects

Thyrotropin Receptor Autoantibodies in Serum Are Present at Much Lower Levels Than Thyroid Peroxidase Autoantibodies: Analysis by Flow Cytometry*

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