Since 1985 we have known that human thyroid peroxidase (TPO), previously known as the thyroid microsomal antigen, is a target for the autoimmune response in autoimmune thyroid diseases (1). The majority of patients’ autoantibodies to thyroid peroxidase are directed to two discontinuous determinants on the protein surface. Several approaches have been taken by our and other laboratories to localize the autoantibody binding regions on TPO. These studies provided a greater insight into the nature of the autoimmune response to the thyroid. In a few recent reviews the current knowledge on TPO as an autoantigen was summarized (2-5). Here, we focus on the biochemical aspects of thyroid peroxidase, especially on its maturation process, trafficking and participation in thyroid hormone biosynthesis.

The human TPO gene, that was cloned in 1987 (6-8), is located on chromosome 2 (region pter-p12) and it is composed of 17 exons and 16 introns (9). The TPO expression is controlled by thyroid-specific factors, namely TTF-1, TTF-2, and Pax-8 (reviewed in Ref. 10-12). Till now several isoforms of TPO cDNA were identified, cloned and characterized (reviewed in Ref. 2, 10).

TPO belongs to the animal peroxidase protein family together with myeloperoxidase (MPO), lactoperoxidase (LPO), eosinophil peroxidase (EPO) (reviewed in Ref. 13, 14). TPO, a 933 amino-acid residues polypeptide, contains ectodomain, single membrane-spanning region and short intracellular tail (15). The dominant part of the protein, called ectodomain, consists of three distinct domains, namely MPO-like domain with high homology to myeloperoxidase (residues 142-738), CCP-like domain similar to complement control protein (residues 739-795), and EGF-like domain that is homologous to the epidermal growth factor (residues 796-841) (15). A three-dimensional computer model of TPO ectodomain was constructed in our laboratory, however, the localization of all TPO domains relatively to each other is still unknown (15). Till now the attempts to solve the TPO structure by X-ray crystallography ended in failure (16, 17). On the contrary, the crystal structure of other two members of the animal peroxidase family, namely MPO (18, 19) and LPO (20), has been already determined.

The TPO molecule undergoes a few modifications while biosynthesis, that are glycosylation, heme incorporation, propeptide removal, and dimer formation (fig. 1). TPO ectodomain contains five predicted N-glycosylation sites in the amino-acid sequence (Asn129, Asn307, Asn342, Asn478, and Asn569) (6-8). However, glycosylation at position 478 probably does not occur because the X residue of the Asn-X-Thr consensus sequence is proline (21). It is well documented that N-linked oligosaccharides are added to the nascent protein in the endoplasmic reticulum (ER) (reviewed in Ref. 22). At this stage, the glycoprotein contains high mannose oligosaccharides that are sensitive to endoglycosidase H (Endo H) digestion. During the passage through the Golgi apparatus, glycoprotein undergoes a final process of N-glycosylation resulting in the transformation of Endo H-sensitive high mannose oligosaccharides to Endo H–resistant complex oligosaccharides. Thus, susceptibility to Endo H digestion is used to distinguish between high-mannose and complex oligosaccharides and help in differentiation whether the protein is before or after transit through the Golgi apparatus (reviewed in Ref. 22).

N-glycans content in human TPO was debated. The hypothesis, that TPO acquires complex oligosaccharides during maturation, was confirmed in research on TPO purified from human thyroid (23, 24), full-length TPO produced in insect (25) and CHO cells (26, 27), TPO ectodomain expressed in insect (24, 28) and CHO cells (28). Deglycosylation analysis of intracellular and cell surface fractions of TPO expressed in CHO cells showed that, inside the cell, this protein contains only high mannose-type structures, whereas TPO at plasma membrane bears complex-type structures (26). However, there are some reports suggesting that TPO contains only high mannose structures regardless of whether the protein is expressed in CHO cells (29) or is purified from thyroid gland (30). Additionally, other studies showed that TPO exiting ER does not necessarily acquire endoglycosidase H resistance (31). Human TPO produced in MDCK (Madin-Darby Canine Kidney) cells was Endo H-sensitive inside the cell and at the cell membrane. The same protein transfected to PC Cl3 (rat thyrocytes) was predominantly sensitive (about 75%) to Endo H digestion (being integrated to the plasma membrane) indicating the lack of conversion to complex carbohydrates upon passage through the Golgi complex (31). Nevertheless, in the same study it was observed that TPO ectodomain secreted to the medium by CHO cells was resistant to the endoglycosidase H digestion. The phenomenon of not acquiring Endo H resistance upon trafficking through the Golgi was earlier observed and it may be explained by the inaccessibility of high mannose oligosaccharides in the tertiary and quaternary structure (32). However, it needs explanation why in some studies the modification of these inaccessible high mannose N-glycans is possible.

N-linked oligosaccharide moieties serve various functions, e.g., stabilize the proteins against denaturation and proteolysis, improve solubility, modulate immune response, regulate protein turnover, stabilize the structure of proteins, and promote their adequate sorting and quality control (reviewed in Ref. 22, 33). The role of these units in TPO was examined. After Endo H treatment, the specific activity of TPO purified from human thyroid, measured by guaiacol and iodide oxidation assays, was reduced about 4 times in comparison to non-deglycosylated protein (30). The authors confirmed that the aggregated state of the molecule, that could be induced by the glycans removal, was not responsible for this decrease (30). They assigned this reduction to the modification of the molecule’s conformation at the enzymatic site (30). Kiso et al. (23) also demonstrated that enzymatic activity of native TPO treated with endoglycosidase H was significantly lower, when measured with guaiacol assay, but no effect was observed in the iodide oxidation assay. In further studies, the inhibition of N-glycosylation using tunicamycin (Tu) reduced by half enzymatic activity of human TPO at the cell surface in CHO cells (26). When the cells were treated with deoxymannojirimycin (dMM) which leads only to high mannose-type structures formation, the activity was not (or only slightly) reduced (26). The cell surface expression of newly synthesized TPO, after treatment with Tu, was almost totally blocked (by 95%) what may explain the decrease in enzymatic activity. All in all, it seems that N-glycans play an essential role in the intracellular trafficking and enzymatic activity of TPO. Additionally, the reactivity of unglycosylated TPO with anti-TPO antibodies, namely human autoimmune antibodies (23, 29, 30), mouse monoclonal antibodies (26, 30), and rabbit polyclonal antibodies (30), was determined. The conclusion was that the tertiary structure of epitopes recognized by anti-TPO antibodies is not markedly changed (23, 29, 30), however, the opposite observations were made by Fayadat et al. (26). Finally, as will be discussed later, N-linked oligosaccharides mediate the interaction between TPO and molecular chaperones (calnexin, calreticulin, and BiP) (34, 35).

The incorporation of O-glycans to human TPO was also analyzed. TPO purified from human thyroid (23), full-length human TPO expressed both in insect (25) and CHO cells (26) undergoes O-glycosylation. Opposite conclusions were drawn from the experiments conducted on full-length human TPO (29) and human TPO ectodomain (28), both expressed in CHO cells. In more detailed study, after separation of intracellular and cell-surface TPO, it was shown that only TPO localized in the latter fraction bears O-glycans (26). This finding was in agreement with the fact that O-glycosylation process occurs in the Golgi apparatus (reviewed in Ref. 33). O-glycans removal from native TPO, isolated from human thyroid gland, probably has no impact on enzymatic activity and interaction with autoantibodies (23). The treatment of the TPO-expressing CHO cells with phenyl-α-GalNAc, a specific inhibitor of O-glycosylation, only slightly altered TPO traffic to the cell surface and activity of this protein, nevertheless, the role of O-linked carbohydrates in the protection of TPO against proteolysis cannot be excluded (26).

The nature of the heme prosthetic group of TPO is still unclear. It was postulated that hog TPO contains other heme group than ferriprotoporphirin IX (36, 37). In other works, it was suggested that ferriprotoporphirin IX or other closely related porphyrin might be incorporated to the molecule of bovine (38) and hog TPO (39, 40). The probable localization in the TPO structure of proximal histidine (linked to the iron centre of the heme) and distal histidine (situated close to the peroxide binding pocket) was firstly proposed by Kimura and Ikeda-Saito (41). They indicated His407 or His414 as the candidates for the proximal and His494 or His586 as the probable distal ligand. More recently, His494 (42, 43) and His239 (43) were pointed out as possible proximal and distal histidine residues, respectively. It is hypothesized that Glu399 and Asn238 are bound covalently to the heme prosthetic group through ester linkage (43, reviewed in Ref. 44).