Thyroperoxidase (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 ϳ30 and ϳ80%, 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 H 2 O 2 -dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H 2 O 2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 M H 2 O 2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H 2 O 2 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) H 2 O 2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule. Thyroperoxidase (TPO)1 is a membrane-bound, glycosylated hemoprotein that plays a key role in the biosynthesis of thyroid hormones. It catalyzes the iodination of thyroglobulin and the coupling of some of the iodotyrosyl residues to produce thyroxine and 3,3Ј,5-triiodothyronine (1-3 (2) showed significant differences between the pyridine hemochromogene of TPO and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. TPO, lactoperoxidase (LPO), myeloperoxidase (MPO), saliva peroxidase, eosinoperoxidase, and intestinal peroxidase belong to the mammalian peroxidase family. These proteins share related protein primary structure (10, 11) and are alike in spectral properties. Furthermore, their prosthetic heme moieties are not readily extracted by conventional approaches. Anderson et al. (12) considered that spectral similarities between LPO and other mammalian peroxidases indicated similar prosthetic heme moieties and a covalent attachment of the heme group to the protein. They suggested that the heme moiety of TPO is probably a type l heme, like LPO. This heme is covalently linked to the protein through ester bonds that link aspartate and glutamate residues (conserved in MPO, ...

Human thyroperoxidase (hTPO), a type I transmembrane glycoprotein, plays a key role in thyroid hormone synthesis. In a previous paper (Fayadat, L., Niccoli, P., Lanet, J., and Franc, J. L. (1998) Endocrinology 139, 4277-4285) we established that after the synthesis, only 15-20% of the hTPO molecules were recognized by a monoclonal antibody (mAb15) directed against a conformational structure and that only 2% were able to reach the cell surface. In the present study using pulse-chase experiments in the presence or absence of protease inhibitors followed by immunoprecipitation procedures with monoclonal antibodies recognizing unfolded or partially folded hTPO forms we show that: (i) unfolded hTPO forms are degraded by the proteasome and (ii) partially folded hTPO forms are degraded by other proteases. It was also established upon incubating endoplasmic reticulum (ER) membranes in vitro that the degradation of the partially folded hTPO was carried out by serine and cysteine integral ER membrane proteases. These data provide valuable insights into the quality control mechanisms whereby the cells get rid of misfolded or unfolded proteins. Moreover, this is the first study describing a protein degradation process involving two distinct degradation pathways (proteasome and ER cysteine/serine proteases) at the ER level, depending on the folding state of the protein.Thyroperoxidase (TPO) 1 is a type I transmembrane heme containing glycoprotein that plays a key role in thyroid hormone synthesis. Although TPO catalyzes both thyroglobulin iodination and hormone synthesis at the apical cell surface of thyroid cells, this enzyme is located mainly in the endoplasmic reticulum (ER) and the perinuclear membrane, and only a small proportion is present at the apical surface (reviewed in Ref. 1). Kuliawat et al. (2) and Penel et al. (3) report that no more than 30% of the immunoprecipitated TPO was detected at the cell surface of porcine thyrocytes cultured on porous filters. In a previous study (4), we proposed a model for the folding, degradation, and intracellular trafficking of human TPO (hTPO) expressed in CHO cells. hTPO was stably expressed in the CHO cell line, and its folding was studied with two monoclonal antibodies: mAb47, which recognizes a linear epitope, and mAb15, which recognizes a conformational epitope present in the mature protein. The results showed that only 15-20% of the hTPO molecules were able to acquire a suitable conformation to be recognized by mAb15 (4). On the other hand, only a fairly small proportion (ϳ15%) of the latter were able to reach the plasma membrane. These data indicated that after being synthesized, only 2% of the hTPO molecules were able to exit from the ER and reach the cell surface. In the steady state, the hTPO recovery rate at the cell surface ranged between 10 and 15%. This firmly suggests that the process involved in the folding and intracellular trafficking of this glycoprotein is the same in both thyroid and CHO cells and that the inefficient maturation of the wild type hTPO is a...

To understand the relationship between conformational maturation and quality control-mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the ␣-subunit of the T-cell antigen receptor (TCR␣) into the ␣-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ϳ50% of nascent mutant HA chains (HA ϩϩ ) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ϳ50% of nascent HA ϩϩ chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA ϩϩ molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HA wt . These apparently native HA ϩϩ molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane.

Thyroid 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-␤-N-acetylglucosaminidase H indicated that the protein is processed through the endoplasmic reticulum and bears high mannosetype 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 [ 14 C]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.

Human 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.