Thyroid Peroxidase: Experimental and Clinical Integration

Endocrine Journal 1996, 43 (1), 1-14

Review

SACHIYAOHTAKI, HIDEHIKO NAKAGAWA, MASAO NAKAMURA*, AND ToMlo KOTANI

Department of Laboratory Medicine, Miyazaki Medical College, Miyazaki 889-16, and *Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060, Japan

tyrosine residues and the oxidative coupling of two Introduction iodotyrosine residues on thyroglobulin (Tg) [1-4]. TPO requires both iodide and H2O2 to initiate This article aims to review the recent advances hormone synthesis. Iodine is a trace element that in knowledge concerning thyroid peroxidase (TPO), is incorporated as inorganic iodide across the thy- focusing on experimental and clinical integration. roid cell membrane through the Na+-I--cotransport This is a propitious time to examine the topic, mechanism [5-7]. The NADPH-dependent O2 gen- because recent advances in the mechanisms of thy- erating system is isolated from the thyroid plasma roid hormone biosynthesis and increased membrane, where thyroid hormone synthesis main- understanding of the molecular biology of TPO ly occurs [8-10]. The resulting superoxide anions have provided a basis for obtaining much impor- dismutate rapidly to H2O2 through the action of tant knowledge of various thyroid diseases such superoxide dismutase, which is present in cytosol as autoimmune thyroiditis. and cell fragments. Pommier and coworkers [11, These developments, which we discuss and em- 12] have isolated a different H202-generating sys- phasize, are: first of all, mechanisms of thyroid tem, which produces H2O2 directly at the expense hormone synthesis catalyzed by this enzyme, in- of NADPH. cluding its structure, enzyme activity, and In addition to the stimulation of iodide trans- regulation of synthesis; secondly, gene structure port and H2O2 generation in the thyroid, the protein and transcription mechanisms; thirdly, mutation synthesis of Tg and TPO in response to TSH has of the TPO gene in hitherto reported cases, and been reported [3, 5]. The reaction mechanism of finally, the critical implications of TPO as an au- TPO and the regulation of thyroid hormone syn- toantigen causing experimental focal thyroiditis. thesis in the TPO reaction will be described in the next section.

I. Thyroid Hormone Synthesis B. Properties of TPO Catalyzed by TPO TPO, which is a membrane-bound hemoprotein, A. Iodide transport and H2O2 generation has been purified from thyroid microsomes by pro- cedures involving trypsin digestion and detergent TPO is involved in two different reactions in the treatment [13-17]. The enzyme thus purified has biosynthesis of thyroid hormone: the iodination of been used for studies of its mechanism of reaction. By monoclonal antibody-assisted immunoaffinity This review was written as a memorial article for the chromatography, Nakagawa et al. [16] have ob- Miyake Prize of Japan, which was awarded to one of the tained a native-type enzyme from hog thyroid authors, Dr. S. Ohtaki, on October 20, 1994. Correspondence to: Dr. Sachiya OHTAKI, Department of microsomes with a molecular weight of 100 kDa. Laboratory Medicine, Miyazaki Medical College, 5200 The molecular weight of the enzyme is similar to Kihara, Kiyotakecho, Miyazaki 889-16, Japan that deduced from cDNA [18, 19], and no differ- 2 OHTAKI et al.

ence is observed in the specific activities estimated from the ratio of activities to the heme concentration of TPO [20]. The visible and pyridine hemochrome spectra of TPO are very similar to those of lactoperoxidase (LPO) [17]. The heme prosthetic group of LPO is concluded to be an iron protoporphyrin thiol which is bound to the cysteine residue of the apoprotein via a disulfide bridge [21]. The similarities in nucleotide and amino acid sequences between TPO and myeloperoxidase (MPO) in animal peroxidase have been presented, and candidates for the fifth and sixth iron ligand histidines in these heme peroxidases have been suggested [19]. Recently, the same similarity of LPO to TPO and MPO and the positions of half cystine in the amino acid sequence of LPO binding to heme thiol have been suggested by Cals et al. [22]. It is not surprising, therefore, that TPO and LPO catalyze both iodinating and coupling reactions.

C. Iodinating activity of TPO

Scheme 1. Mechanism of reactions catalyzed by thyroid Peroxidase reactions catalyzed by heme peroxi- peroxidase. Values in parenthesis denote dases can be studied with horseradish peroxidase second order rate constants (M-'s-'). Path 1 and Path 2 indicate the coupling and iodinating (HRP) and LPO. Inorganic iodide in the thyroid cycles, respectively. Tyr, tyrosine; MIT, monoiodotyrosine; DIT, diiodotyrosine; 0.2% I 7.5 x 106M-'s-' TPO + H2O2 >Compound I ()1 Tg, 0.2% iodine-content thyroglobulin. 1-5 x 104M-'s~' C ompound I + Tyrosine - TPO + Tyrosine (2) radical reactions: iodotyrosine is formed at the active site of TPO through the reaction of iodide 2 x 10'M-'s-' C ompound I + I- TPO•I+ (3) radicals with tyrosine radicals. Conclusive evidence that TPO catalyzes two-electron oxidation 2.1 x 106M-'s-' TPO •I+ + Tyrosine 'TPO + MIT (4) of tyrosine has been reported [20, 23, 26] (equation 2). One can therefore rule out the possibility that gland must be first oxidized to a higher oxidation the iodinating cycle is a radical reaction. Bjorksten form as an iodinating agent. TPO is a catalyst [27] has concluded that HRP Compound I is re- which oxidizes iodide in the presence of H202. The duced directly to the ferric form with iodide. The reactions of TPO with H2O2 and iodide are very two-electron oxidation of iodide by LPO and TPO, similar to those reported with LPO [20, 23]. Rest- resulting in the formation of iodinium cation, has ing TPO (ferric) reacts with H2O2 to form a catalytic been confirmed in later studies [20, 23] (equation intermediate referred to as Compound I, which has 3). The rate of reaction of TPO Compound I with two oxidizing equivalents above the ferric form iodide is 100 fold higher than that with tyrosine (equation 1). The hypervalent enzyme mostly cat- [28]. Iodinating reaction (Path 2) therefore pro- alyzes consecutive one-electron oxidation of organic ceeds predominantly even in the presence of a low substrates through Compound II, forming substrate concentration of iodide [20, 28]. The iodinating radicals. The mechanism of the peroxidase reac- intermediate, TPO•I+, is assumed to be present from tion has been shown as Path 1 (Scheme 1). Early the overall kinetics [28]. By kinetically analyzing reports [1, 24, 25] have shown that iodinating reac- the iodinating reaction of tyrosine catalyzed by tions catalyzed by peroxidases are presented by TPO, the rate constant for the reaction of the io- REVIEW: THYROID PEROXIDASE 3 dinium cation transfer from the enzyme to tyrosine photometric analysis of the catalytic intermediates is calculated to be 2.1 x 106 M-1s-1 [28] (equation 4). of enzyme in the oxidation of various phenols, it These results indicate that the rate-determining step can be concluded that the mechanism of oxidation in the iodinating reaction is the iodinium cation catalyzed by TPO changes from a two-electron to transfer to tyrosine (Path 2). The rate constants for a one-electron type as the substituents at the l- the iodinium cation transfer to Tg have also been and 6-positions of phenol become bulky or heavy determined from overall kinetic experiments on the [20]. iodination of Tg [29]. Questions will arise as to whether the same The rate constants decrease as the iodine con- mechanism can be applied to the oxidation of Tgs tent of Tg increases. This iodine-transferring that have different iodine contents. The enzyme reaction is increased in the presence of iodothy- intermediate in the steady state of oxidation of Tg ronines in the order of diiodothyronine (T2) containing 0.2% iodine is Compound I, whereas >triiodothyronine (T3)>thyroxine (T4), similarly to Compound II is observed in the oxidation of Tg the reaction of tyrosine [28]. containing 0.7% iodine [32]. The number of DIT The mechanism of action of thioureylene anti- residues in Tg changes from 1.1 to 8.7 as the io- thyroid compounds has been examined by many dine content increases from 0.2 to 0.7% [33]. The workers [1, 3], and has been explained on the basis results indicate that TPO catalyzes two-electron of the iodinating cycle of TPO. Methylmercap- oxidation of Tg with 0.2% iodine and one-electron toimidazole (MMI), a typical goitrogen, inhibits the oxidation of 0.7% iodine-containing Tg (Scheme 1). iodinating activity of TPO in two ways. First, MMI ESR detection of 0.7% iodine-containing Tg radi- competes with Tg for the reaction with TPO•I+ [30, cals is unsuccessful, but 0.7% iodine-containing 31]. The spectrum of iodinated MMI is optically Tg-mediated ascorbate radical and GSH radical for- discernible with that of MMI. Second, MMI reacts mation catalyzed by TPO is observed [32]. rapidly with Compound I to form Compound II, Phenoxyl radicals are unstable but are readily scav- which subsequently reacts with MMI, forming the enged by ascorbate and GSH, leading to the sulf form [30]. The formation of either Compound corresponding radicals [34]. These results indicate II or the sulf form causes TPO to deviate from the that DIT residues in 0.7% iodine-containing Tg are iodinating cycle (Path 2). oxidized mainly to radicals and imply that the oxidative coupling cycle is a radical reaction (Path D. Oxidative coupling by TPO 1). Neither significant oxidation of Tg nor 0.7% iodine-containing Tg-mediated radical formation > 105M-1 s 1 C by LPO is observed [32]. Thus it seems that, un- ompound I + DIT -- - like iodination, the oxidation of Tg occurs through -~ Compound 11+ DIT • (5) a more specific interaction with TPO. This might be a physiologically important difference in the Compound II +DIT 1'7_x 104M-1s-1 catalytic activity of LPO and TPO. -• TPO + DIT• (6) Iodinating and coupling cycles catalyzed by TPO are summarized in Scheme 1. It has been confirmed that TPO catalyzes two- electron oxidation of iodide to form iodinated E. Regulation of thyroid hormone synthesis by TPO tyrosines and one-electron oxidation of diiodotyrosine (DIT) to form dimerized products through TPO catalyzes the two-electron oxidation of ty- a formation of DIT radicals (equations 5 and 6). rosine and MIT, and the one-electron oxidation of These two reactions are not necessarily specific for DIT. This difference in reaction with tyrosine and TPO, and are also catalyzed by HRP, LPO and DIT is also observed in the reaction of TPO with MPO. The mechanism of oxidation of iodide and 0.2 and 0.7% iodine-containing Tg. The inhibition DIT appears to be essentially the same for all of of the iodinating activity of TPO by DIT has been these peroxidases, whereas there is a marked dif- explained in terms of Compound II accumulation ference between TPO and the other peroxidases in during the reaction [28, 35]. The results support the mechanism of oxidation of tyrosine and the hypothesis that the increase in the DIT residue monoiodotyrosine (MIT) [23, 26]. From spectro- inhibits further iodination by changing the catalyt- 4 OHTAKI et al.

is cycle of TPO from the two-electron to the radi- second human TPO cDNA, designated hTPO-2, is cal reaction (Scheme 1). almost identical to hTPO-1 beginning 605 base pairs From the kinetic data on the iodinating and cou- downstream, except that it contains a one-base- pling reactions of free tyrosines catalyzed by TPO, pair difference and lacks 171 base pairs in the Nakamura et al. [20, 28] have concluded that the middle of the sequence [18]. This 171-nucleotide mechanism of the enzyme action fits the preferen- sequence has CT and AG at its 5' and 3' bound- tial formation of T4, even though the formation aries, respectively, in good agreement with donor started from free tyrosine. The results, which can and acceptor splice site consensus sequences. Two be summarized as follows, are in agreement with mRNAs generated through alternative splicing of the mechanism proposed by Nunez [1] and Tau- the same gene are expressed in various human thy- rog [3]:1) the iodination rate for MIT catalyzed by roid tissues with different levels of expression. TPO is faster than that for tyrosine; 2) the activa- Although these two mRNAs have been expected tion effect on iodination is greater in the order to correspond to mature doublet TPO protein [50- T2>T3>T4;3) DIT is oxidized by Compound II fast- 52], no report has yet been presented. Far from er than MIT and tyrosine, and 4) DIT inhibits the that, when the cDNA compatible with hTPO-1 was iodinating activity during its coupling reaction. inserted into an expression vector and transfected It is evident that the native structure of Tg plays into CHO cells, doublet TPO protein was produced. a major role in the formation of T4 [1-3, 36-38]. hTPO-2 encoding protein expressed in HepG2 cells The sites of T4 synthesis are located near the ami- by the vaccine virus system did not have TPO ac- no and carboxyl ends of the Tg monomer [39]. A tivity (our unpublished observation). Some relationship between iodine content and the additional forms of alternative splicing have been amount of iodothyronine and iodotyrosine in Tg reported: splicing of 130 nucleotides correspond- has been reported [33]. The iodine distribution ing to exon 16 [53] and abnormal splicing of exon has also been analyzed for various Tgs iodinated 5 or exon 6 [54]. chemically and in vivo [40, 41]. That T4 is formed The human TPO amino acid sequence deduced at an early stage of iodination suggests that the from cDNA has 44% homology to human MPO, native structure of Tg plays an important role in indicating that TPO and MPO share a common the "preferential formation of thyroxine". This is ancestor [18, 19, 55]. Further homology analyses supported by the fact that a specific peptide struc- have revealed that C4b-F'2 glycoprotein and EGF- ture of Tg is involved in the biosynthesis of thyroid LDL receptor modules are contained at the hormone [42]: the hormonogenic tyrosine residues COOH-end of TPO protein and that the heme-bind- are iodinated in rigid sequential order, resulting in ing region module of cytochrome c oxidase subunit the formation of DIT derivatives via MIT, which I is located at the middle portion of TPO protein subsequently undergo one-electron oxidation to [19, 55, 56]. form T4. It is important to note that thyroid hor- Somatic cell hybrid mapping to determine hu- mones formed in Tg are prevented from further man TPO gene localization assigned it to the short oxidation by TPO. Because free iodothyronines arm of chromosome 2 [18]. By a similar analysis are readily oxidized by TPO [43]. of DNA from somatic cell hybrids, the human TPO gene was mapped to 2pter-p12 [57], but our recent analysis with fluorescence in situ hybridization II. Gene Structure of TPO (FISH) indicates that the human TPO gene locates at 2p25 [58]. A. Complementary DNA for TPO B. Genomic DNA for human TPO The cDNAs encoding TPO have been isolated in man [18, 44, 45], pig [46], rat [47, 48] and mouse The human TPO gene contains 17 exons and cov- [49]. It has been revealed that the complete hu- ers at least 150 kilobase pairs of chromosome 2 man cDNA consists of 3,042 to 3,060 nucleotides (Fig. 1) [56]. A typical TATA box is located 25 and encodes 933 amino acids. Five potential as- base pairs upstream of the putative starting site. paragine-linked glycosylation sites are found in the Histidine residues important for TPO activity are deduced amino acid sequence of human TPO. The contained in the latter half of the 8th exon, and 9th REVIEW: THYROID PEROXIDASE 5

Fig. 1. Structure of human TPO gene. Seventeen exons of the human TPO gene are scattered in the region of about 150 kilobase pairs of chromosome 2. Histidine residue contained in the latter half of the 8th exon will work as proximal histidine, and either that of the 9th or 10th exon as distal histidine. Sequencing of genomic DNA for TPO revealed that the 3rd to 11th exon-intron junctions in TPO correspond exactly to those of the 2nd to 6th and the 8th to 11th in MPO [56]. From the homology search, exons 13,14 and 15 have significant similarities to C4b-f2 glycoprotein, EGF-LDL receptor and a typical transmembrane domain, respectively.

and 10th exons. Histidine residue of the former will work as proximal hstidine, and either that of the 9th or 10th exon as distal hstidine [19]. In comparison with the human MPO gene [59], the positions of the 3rd through 11th exon-intron junctions in TPO coincide exactly with those of the 2nd through 11th exon-intron junctions in MPO except for the 7th MPO junction, which does not have any counterpart in TPO. Four exons of the human TPO gene (exons 13-16) encompass a different protein module. Three of these modules, representing exons 13,14 and 15, bear significant similarities to C4b-N2 glycoprotein, EGF-LDL re- Fig. 2. Structure of human TPO promoter. In the promoter region of human TPO, TTF-1 binds to three sites (A, ceptor and a typical transmembrane domain, B and C), TTF-2 to one site (0), and Pax-8 to one respectively. The 10th exon corresponds complete- site (A). The thyroid-specific enhancer element has ly to 171 nucleotides deleted in hTPO-2 cDNA. two TTF-1 CAE S (E2 and E3).

C. Structure of human TPO promoter and TAFs-binding sites in the human TPO pro- To understand the mechanisms of the thyroid- moter which is based on the results of DNase I specific expression of TPO and Tg, useful analyses footprinting and mobility shift assay [60, 61]. The of cis-activating elements (CAEs) and trans-acti- structure of human TPO promoter is very similar vating factors (TAFs) have been developed during to those of rat TPO and human Tg promoters, and the past several years. Figure 2 summarizes CAEs thyroid-specific expression of the TPO gene is de- 6 OHTAKI et al.

termined by the nucleotide sequence from -170 through protein kinase A [65]. The structure of to +1 [62]. Thyroid transcription factor-1 (TTF-1), genomic DNA for TTF-1 was sequenced last year TTF-2 and Pax-8 bind to 5 sites. And ubiquitous [66, 67], revealing that (i) Hox B3 gene is a regula- factor B (UFB) binds to the immediate upstream tory gene for the TTF-1 promoter region [66], and region of TTF-1A. Differences between TPO and (ii) since the CTCAAGC nucleotide sequence ex- Tg promoters can be summarized as follows: ists at -175 to -169 of the TTF-1 promoter region, i) The TATA box element of the Tg promoter is TTF-1 gene expression may be self-regulated [67]. more important in expression than that of the TPO Pax-8 mRNA 3.1 kilobases in size is expressed promoter. in both thyroid gland and kidney, and appears in ii) In the rat TPO promoter, the binding of TTF- the 10.5 day rat embryo along with TTF-1 mRNA. 2 has functional importance, so that the Pax-8 protein, consisting of 457 amino acids, con- introduction of mutations into CAE decreases tran- tains the paired domain built of 127 amino acids at scriptional activity about five fold. its NH2-end. Pax-8 is a member of the series Pax-1 iii) Although TTF-1C CAE is not important in to -9, which are thought to be important for mam- TPO expression, the CAE for Pax-8 is essential [63]. malian ontogeny [68]. This protein binds to the iv) The TPO promoter lacks the cyclic AMP re- characteristic nucleotide sequence and regulates sponsive element (CRE), and TPO expression by gene transcription as well as other proteins with a insulin or cyclic AMP is regulated by TTF-2 [60]. homeodomain. It is known that six mRNA iso- In other words, the hormonal regulation of TPO forms are generated by alternative splicing and that expression mainly depends on TTF-2. Furthermore, the longest Pax-8a has the strongest transcription TTF-2 plays a more pivotal role in the TPO pro- activity. moter than in the Tg promoter. TTF-2 protein is an essential factor in the thy- v) Thyroid-specific enhancer element localizes roid-specific transcription by hormones. TSH to a 230 base-pair region approximately 5.5 kilo- stimulation leads to synthesis of proteins through base pairs upstream of the human TPO gene cyclic AMP, and TTF-2 protein binds to the site of transcription starting site. DNase I footprinting the -117 to -103 nucleotide sequence (cyclic protects three regions within the 230 base-pair frag- AMP-modulating binding region, CMB) (Fig. 2). ment, two of which have TTF-1 CAE [61]. It is In other words, the binding of TTF-2 is positively generally considered that TTF-1 binding is impor- regulated by TSH stimulation through cyclic AMP tant for Tg gene expression while Pax-8 binding is and is negatively regulated by A23187 or TPA. The involved in TPO gene expression. molecular weight of TTF-2 protein is 60 kDa, and Zn2+ is needed for its binding to DNA. Civitareale D. Transactivating factors for TPO promoter et al. [69] have recently reported the following observations: i) TTF-2 is a thyroid-specific DNA- As described above, three factors, TTF-1, Pax-8 binding protein; ii) TTF-2 is a zink-finger and TTF-2, are characterized as major TAFs. TTF- DNA-binding protein that binds in the form of a 1 protein consists of 371 amino acids and contains dimer; iii) the binding to DNA occurs in a reduced a homeodomain, which binds to the specific nu- state of TTF-2 but not in the oxidized state. cleotide-sequence and regulates gene transcription. As three kinds of TAFs having homeodomain, The homeodomain of TTF-1 is very similar to that paired domain and zink-finger have been charac- of drosophila NK2. And NKX-2.2 to NKX-2.6 are terized, the mechanism involved in the overall identified in mammalians as NK2 homologues. regulation of the TPO gene will be examined in TTF-1 homeodomain recognizes a CAAG motif the near future. unlike the TAAT motif recognized by other known homeodomains [64]. TTF-1 mRNA is localized in thyroid and lung, and its size ranges from 2.4 to III. Mutation of TPO Gene 2.7 kilobases. In rat fetus, this mRNA appears on day 10.5, and it is suggested that TTF-1 protein Defective organification of iodine is due to ab- embryologically contributes to determining the normalities of Tg synthesis, TPO synthesis or H2O2 development of thyrocyte as well as Pax-8 protein. production. Organification defect in iodine brought The activity of TTF-1 is regulated by cyclic AMP about by abnormal H2O2 production is rare, so that REVIEW: THYROID PEROXIDASE 7

abnormal Tg and TPO syntheses are thought to be been found to be hypothyroid at the age of 4 the major causes of defective organification of io- months and thereafter was treated with dessicated dine. The rate of neonatal hypothyroidism is about thyroid or T3 supplements. Despite daily thyroid 1/4,000, 1 /5 of which is caused by genetically de- hormone replacement therapy, the patient devel- termined thyroid dyshormonogenesis. As defective oped a large nodular goiter, and thyroidectomy TPO activity is one of the two major causes of de- was performed. In the thyroid tissue, TPO activi- fective organification of iodine, about one in 40,000 ty and iodination of Tg were decreased to below newborns has hypothyroidism due to defective the limits of detection, and no TPO mRNA was TPO. But the results of clinical observations sug- detected by Northern blot analysis. A homozy- gest that compensatory hyperplasia of the thyroid gous mutation in the 2nd exon was observed by tissue masks some hypothyroidisms when TPO denaturing gel electrophoretic analysis. A 20 base- activity is on the borderline between normal and pair duplication at 47 base-pairs downstream of hypothyroidism. Therefore, more than one in the ATG start codon was shown by sequence anal- 40,000 newborns may have hypothyroidism caused ysis. This duplication generated a frame shift, by defective TPO [70, 71]. resulting in a termination signal in the 3rd exon, Defective TPO is mainly divided into quantita- which was compatible with the complete absence tive and qualitative types. The following of TPO protein. observations have been reported with regard to As other mutations of the TPO gene, various the latter: numbers of tandem repeats at the positions of - i) TPO cannot bind to heme. 799 and - 706 nucleotide sequences [74] and point ii) TPO cannot bind to Tg or iodine as substrates. mutations in thyroid cancer [75] have been report- iii) TPO is abnormally localized in thyrocyte. ed, but these mutations are thought to have little iv) TPO activity is abnormally inhibited. to do with thyroid diseases. Up to 1992, 118 cases of hereditary defective organification of iodine ascribable to defective TPO had been reported, and the hereditary form was IV. TPO as Autoantigen autosomal recessive. Detailed investigation of the pedigree maps of these families found consanguin- Since it was discovered that TPO is a major mi- eous marriages in many cases. crosomal antigen corresponding to microsomal Only two cases of congenital goitrous hypothy- autoantibodies [50, 52, 76, 77], much effort has been roidism due to TPO gene mutation have been made to elaborate antigenic epitopes for B- and T- clarified [72, 73]. The first case reported by cells relating to thyroid autoimmune diseases. Abramowicz et al. [72] was a 12 year-old boy hav- TPO, Tg and TSH receptor are major thyroid au- ing congenital goiter with hypothyroidism. In this toantigens [78] and have been examined for their case, 4 base pairs (GGCC) were inserted to the 8th relation to the developmental mechanisms of au- exon of the TPO gene, resulting in a frame shift. toimmune thyroid diseases (AITD). Needless to The frame shift generated a shorter protein, Gns, say, the measurement of autoantibodies to these and another shorter protein, Gas, deleted only 124 antigens is very useful in diagnosing AITD and nucleotides of the 9th exon by aid of a cryptic ac- determining therapeutic efficacy. Advances in receptor splice site. Since the insertion site of 4 base combinant DNA technology have supported recent pairs was located immediately upstream of the studies on epitopes, paratopes and the develop- codon of proximal histidine, both Gns and Gas ment of an assay system for autoantibodies [79-87]. lacked the proximal histidine. But the Gas protein conserved the distal histidine by alternative splic- A. Autoantibodies to TPO ing. These results corresponded closely to the fact that the patient's thyroid tissue had TPO activity, In the sera of patients with AITD, anti-TPO, -Tg although it was low. That is, it was thought that and -TSH receptor autoantibodies are detected at the existence of the distal histidine is capable of very high frequencies. Anti-TSH receptor autoan- ensuring enzyme activity against a complete loss tibodies are important in diagnosing Graves' of binding to heme. disease and obtaining information about therapeu- In the second case, the 26-year-old patient had tic efficacy [88]. For the following reasons, 8 OHTAKI et a!.

anti-TPO autoantibodies are considered more im- nonidentical VH regions [107, 108]. portant than anti-Tg autoantibodies. Patients with Hashimoto's thyroiditis have anti-microsomal au- B. T-cell epitope toantibodies but may lack anti-Tg autoantibodies [89]. The level of anti-microsomal autoantibodies In general, T-cell proliferative responses are rel- correlates with the AITD condition [90, 91]. Anti- atively weak in primary cultures of peripheral microsomal autoantibodies may damage thyrocytes blood lymphocytes as well as in lymphocytes iso- via activation of the complement system and anti- lated from Hashimoto or Graves' thyroid tissue. body-dependent cell-mediated cytolysis (ADCC) These weak responses may be due to the limited [92, 93]. The level of anti-TPO autoantibody pro- number of TPO-specific T-cells, and they make it duction in women is inheritable in autosomal difficult to study T-cell proliferation to TPO. An- dominant fashion [94, 95], although the gene re- other potential problem to be resolved is whether sponsible for this phenomenon has not been thyrocytes can present TPO to autologous T-cells. characterized. Almost all anti-TPO autoantibodies Under the influence of interferon y, thyrocytes ex- are IgG1 or IgG4 [97]. The IgG subclass distribu- press class II MHC molecules [109]. tion of autoantibodies varies from case to case. Results of primary lymphocyte cultures with syn- Anti-Tg autoantibodies cross-react with TPO [98, thetic peptides have produced a degree of 99], and these antibodies are generally detected in consensus on the TPO T-cell epitopes recognized patients with Hashimoto's thyroiditis, although by patients [82,110-112]. The following three re- they are not always present [100]. gions of TPO protein probably contain T-cell Epitopes recognized by autoantibodies have been epitopes: amino acid residues 110-250, 414-589 and extensively studied by using synthetic peptides, 841-901. T-cell lines and clones generated from recombinant DNA molecules and recombinant Fabs intrathyroidal T-cells by interleukin 2 and anti-CD3 specific for TPO. B-cell epitopes are generally con- give slightly different results [78]. These T-cells formational [101, 102], whereas T-cell epitopes are respond well to intact TPO as well as to two pep- short, linear peptide fragments. This rule applies tides, NP-7 (amino acid residues 535-551) and B6 to TPO. Since reduced TPO decreases its antige- (amino acid residues 632-645). In terms of the nicity to autoantibodies, almost all epitopes on TPO magnitude of the proliferative responses, the re- are thought to be conformational [103]. TPO is sults obtained by using T-cell lines and clones are located on the cell surface as a disulfide-linked more reliable than those of primary cultures. Fur- dimer, which is more antigenic to autoantibodies thermore, amino acid residues 119-126 of TPO have than monomer [104]. been predicted from algorithms as a T-cell epitope In studies on B-cell epitopes with recombinant common to both TPO and Tg [113], and have been TPO, it has been found that many B-cell epitopes proved to aid in the activation of thyroiditogenic are located on the latter half of the COOH-end of cells [114]. TPO molecule [105] and that the region between In an interesting approach to the study of TPO- amino acids 590 and 767 contains an epitopic reactive T-cells, the EBV-transformed B-cell line hotspot. Within this region are two linear epitopes from a patient with Graves' disease was transfect- located between amino acids 713 and 721 [103, 105] ed with the expression vector encoding TPO to and between 590 and 622 [105]. Several B-cell stimulate TPO-specific T-cells cloned from autolo- epitopes may be present on the TPO molecule, al- gous thyroid tissue [86, 87]. though it is very difficult to determine the exact number of epitopes. From a different point of view, C. Thyroiditis about 80% of anti-TPO autoantibodies recognize two conformational epitopes [106]. In this study, Experimental autoimmune thyroiditis as a mod- 30 anti-TPO IgG autoantibodies were cloned and el of Hashimoto's thyroiditis has been induced by their H and L chains were analyzed, showing two the immunization of Tg, but both porcine TPO and closely associated major domains, A and B. Fur- human TSH receptor can induce thyroiditis in mice, thermore, it is suggested that the L chain is critical although the antigen donor and recipient are het- in defining paratope specificity, even in the pres- erologous [115-118]. These findings may well ence of completely different D regions and explain why Hashimoto's thyroiditis varies from REVIEW: THYROID PEROXIDASE 9

case to case in light-microscopic morphology, au- other thyroid antigens. It will be possible to clari- toantibodies to three major thyroid antigens, Tg, fy the autoantigenicity of murine TPO, including TPO and TSH receptor, and the clinical course. The thyroiditogenicity, by using recombinant murine relationship between the genetic background of the TPO. patient and these three major thyroid antigens may define various pathological types of Hashimoto's thyroiditis. Summary In the murine thyroiditis induced by porcine TPO, murine strains with a high incidence of thy- Since TPO plays a cardinal role in regulating roiditis are quite different from those of Tg-induced many cellular processes of thyroid hormone bio- thyroiditis. Although the high responder strains synthesis in thyrocytes, it is important to present a in Tg-induced thyroiditis are those with H-2k, H-2S summary of the impact that TPO research on the and H-2q [119], C57BL/6 and C57BL/10, both with basis of molecular structure has had on our under- H-2b, are high responders in porcine TPO-induced standing of thyroid diseases. This review has thyroiditis [115]. No correlation is observed be- therefore been written to highlight the biochemi- tween the incidence of thyroiditis and the amount cal, molecular biological, immunological and of anti-porcine TPO antibody. Genetic linkage anal- clinical aspects of TPO in thyroid follicular cells. yses with congenic murine strains and B10.A As for hormonogenesis, further details of the mutation lines showed that at least two genes are properties of the superoxide generating system re- related to the induction of thyroid lesions, one of main unknown, but based on decisive evidence which links to H-2 and another to non-H-2. The showing what active iodinating species are at ini- porcine TPO-specific T-cell line derived from tial and principal stages, a novel concept of the C57BL/6 mice lymph nodes also could successful- mechanism of two electron oxidation has been put ly transfer thyroiditis to naive C57BL / 6 mice. The forth. thyroidiogenic epitope on porcine TPO has been Recent progress in studies of analyses of TPO defined for C57BL/6 mice [116], but its counter- gene structure has also provided stimuli for re- part on murine TPO has not been identified. It is newed investigation into a much wider field of not yet clear whether the epitope on porcine TPO thyroidology, including thyroiditogenesis. cross-reacts with an epitope on murine TPO or on

References

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